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1
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Raza A, Chattopadhayay S, Mondal D, Talukdar P. Carbonohydrazonoyl dicyanide-linked indole carboxamides as a new scaffold for transmembrane H +/Cl - transport. Chem Commun (Camb) 2025. [PMID: 39968883 DOI: 10.1039/d4cc06371f] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/20/2025]
Abstract
Herein, we introduced a series of carbonohydrazonoyl dicyanide-linked indole carboxamide 1a-1c transporters. Detailed mechanistic insight into the transport manifested that superior transporter 1a facilitates the movement of Cl- and H+/Cl- in a non-specific manner via the carrier mode. Geometrical optimization with the [1a + Cl-] complex provided an understanding of the Cl- ion binding process with the receptor 1a.
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Affiliation(s)
- Akram Raza
- Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr Homi Bhabha Road, Pashan, Pune 411008, Maharashtra, India.
| | - Sandip Chattopadhayay
- Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr Homi Bhabha Road, Pashan, Pune 411008, Maharashtra, India.
| | - Debashis Mondal
- Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr Homi Bhabha Road, Pashan, Pune 411008, Maharashtra, India.
| | - Pinaki Talukdar
- Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr Homi Bhabha Road, Pashan, Pune 411008, Maharashtra, India.
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2
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Zhang S, Wang L, Yi S, Tsai YT, Cheng YH, Lin YT, Lin CC, Lee YH, Wang H, Li S, Wang R, Liu Y, Yan W, Liu C, He KW, Ho MS. Drosophila aux orchestrates the phosphorylation-dependent assembly of the lysosomal V-ATPase in glia and contributes to SNCA/α-synuclein degradation. Autophagy 2025:1-20. [PMID: 39878136 DOI: 10.1080/15548627.2024.2442858] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2024] [Revised: 12/07/2024] [Accepted: 12/11/2024] [Indexed: 01/31/2025] Open
Abstract
Glia contribute to the neuropathology of Parkinson disease (PD), but how they react opposingly to be beneficial or detrimental under pathological conditions, like promoting or eliminating SNCA/α-syn (synuclein alpha) inclusions, remains elusive. Here we present evidence that aux (auxilin), the Drosophila homolog of the PD risk factor GAK (cyclin G associated kinase), regulates the lysosomal degradation of SNCA/α-syn in glia. Lack of glial GAK/aux increases the lysosome number and size, regulates lysosomal acidification and hydrolase activity, and ultimately blocks the degradation of substrates including SNCA/α-syn. Whereas SNCA/α-syn accumulates prominently in lysosomes devoid of glial aux, levels of injected SNCA/α-syn preformed fibrils are further enhanced in the absence of microglial GAK. Mechanistically, aux mediates phosphorylation at the serine 543 of Vha44, the V1 C subunit of the vacuolar-type H+-translocating ATPase (V-ATPase), and regulates its assembly to control proper acidification of the lysosomal milieu. Expression of Vha44, but not the Vha44 variant lacking S543 phosphorylation, restores lysosome acidity, locomotor deficits, and DA neurodegeneration upon glial aux depletion, linking this pathway to PD. Our findings identify a phosphorylation-dependent switch controlling V-ATPase assembly for lysosomal SNCA/α-syn degradation in glia. Targeting the clearance of glial SNCA/α-syn inclusions via this lysosomal pathway could potentially be a therapeutic approach to ameliorate the disease progression in PD.Abbreviation: aux: auxilin; GAK: cyclin G associated kinase; LTG: LysoTracker Green; LTR: LysoTracker Red; MR: Magic Red; PD: Parkinson disease; SNCA/a-syn: synuclein alpha; V-ATPase: vacuolar-type H+-translocating ATPase.
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Affiliation(s)
- Shiping Zhang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, School of Life Sciences, Fudan University, Shanghai, China
| | - Linfang Wang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- The Institute of Seed Industry, Xianghu Laboratory, Qiantang River International Innovation Belt of the Xiaoshan Economic and Technological Development Zone, Hangzhou, China
| | - Shuanglong Yi
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- Shanghai Institute of Thoracic Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yu-Ting Tsai
- Institute of Neuroscience, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yi-Hsuan Cheng
- Institute of Neuroscience, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yu-Tung Lin
- Institute of Neuroscience, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Chia-Ching Lin
- Institute of Neuroscience, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Yi-Hua Lee
- Institute of Neuroscience, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Honglei Wang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- The Key Laboratory of Developmental Genes and Human Disease, School of Life Science and Technology, Southeast University, Nanjing, China
| | - Shuhua Li
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Ruiqi Wang
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- School of Brain Science and Brain Medicine, Zhejiang University School of Medicine, Hangzhou, China
| | - Yang Liu
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China
| | - Wei Yan
- Guangdong Provincial Key Laboratory of Brain Connectome and Behavior, CAS Key Laboratory of Brain Connectome and Manipulation, the Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, China
| | - Chang Liu
- Guangdong Provincial Key Laboratory of Brain Connectome and Behavior, CAS Key Laboratory of Brain Connectome and Manipulation, the Brain Cognition and Brain Disease Institute (BCBDI), Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, China
| | - Kai-Wen He
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China
| | - Margaret S Ho
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
- Institute of Neuroscience, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Brain Research Center, National Yang Ming Chiao Tung University, Taipei, Taiwan
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3
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Knight K, Park JB, Oot RA, Khan MM, Roh SH, Wilkens S. Monoclonal nanobodies alter the activity and assembly of the yeast vacuolar H +-ATPase. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.10.632502. [PMID: 39829782 PMCID: PMC11741422 DOI: 10.1101/2025.01.10.632502] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
The vacuolar ATPase (V-ATPase; V1Vo) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of higher organisms. Evidence suggests that metastatic breast cancers mislocalize V-ATPase to the plasma membrane to promote cell survival and facilitate metastasis, making the V-ATPase a potential drug target. We have generated a library of camelid single-domain antibodies (Nanobodies; Nbs) against lipid-nanodisc reconstituted yeast V-ATPase Vo proton channel subcomplex. Here, we present an in-depth characterization of three anti-Vo Nbs using biochemical and biophysical in vitro experiments. We find that the Nbs bind Vo with high affinity, with one Nb inhibiting holoenzyme activity and another one preventing enzyme assembly. Using cryoEM, we find that two of the Nbs bind the c subunit ring of the Vo on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast Vo can pull down human V-ATPase (HsV1Vo). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.
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Affiliation(s)
- Kassidy Knight
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
| | - Jun Bae Park
- Department of Biological Sciences, Seoul National University, Seoul, Korea
- Present address: Department of Cancer Biology, Lerner research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Rebecca A. Oot
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
| | - Md. Murad Khan
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
- Present address: Howard Hughes Medical Institute, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
| | - Soung-Hun Roh
- Department of Biological Sciences, Seoul National University, Seoul, Korea
| | - Stephan Wilkens
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
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4
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Romano R, Cordella P, Bucci C. The Type III Intermediate Filament Protein Peripherin Regulates Lysosomal Degradation Activity and Autophagy. Int J Mol Sci 2025; 26:549. [PMID: 39859265 PMCID: PMC11766092 DOI: 10.3390/ijms26020549] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2024] [Revised: 01/03/2025] [Accepted: 01/08/2025] [Indexed: 01/27/2025] Open
Abstract
Peripherin belongs to heterogeneous class III of intermediate filaments, and it is the only intermediate filament protein selectively expressed in the neurons of the peripheral nervous system. It has been previously discovered that peripherin interacts with proteins important for the endo-lysosomal system and for the transport to late endosomes and lysosomes, such as RAB7A and AP-3, although little is known about its role in the endocytic pathway. Here, we show that peripherin silencing affects lysosomal abundance but also positioning, causing the redistribution of lysosomes from the perinuclear area to the cell periphery. Moreover, peripherin silencing affects lysosomal activity, inhibiting EGFR degradation and the degradation of a fluorogenic substrate for proteases. Furthermore, we demonstrate that peripherin silencing affects lysosomal biogenesis by reducing the TFEB and TFE3 contents. Finally, in peripherin-depleted cells, the autophagic flux is strongly inhibited. Therefore, these data indicate that peripherin has an important role in regulating lysosomal biogenesis, and positioning and functions of lysosomes, affecting both the endocytic and autophagic pathways. Considering that peripherin is the most abundant intermediate filament protein of peripheral neurons, its dysregulation, affecting its functions, could be involved in the onset of several neurodegenerative diseases of the peripheral nervous system characterized by alterations in the endocytic and/or autophagic pathways.
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Affiliation(s)
| | | | - Cecilia Bucci
- Department of Experimental Medicine, University of Salento, Via Provinciale Lecce-Monteroni n. 165, 73100 Lecce, Italy; (R.R.); (P.C.)
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5
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D'Souza-Schorey C, Stahl PD. Resolving the two-body problem: A postulated role for the V0 sector of the V0V1-ATPase in exosome biogenesis and multivesicular body fate. Mol Biol Cell 2025; 36:pe1. [PMID: 39705591 PMCID: PMC11742106 DOI: 10.1091/mbc.e24-09-0412] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2024] [Revised: 11/04/2024] [Accepted: 11/22/2024] [Indexed: 12/22/2024] Open
Abstract
Because the discovery of the multivesicular body (MVB) as the origin of secreted vesicles or exosomes, the question arose and still looms-what distinguishes an MVB destined for fusion with the plasma membrane (EXO-MVB) facilitating exosome release from an MVB involved in transport of content to the lysosome (LYSO-MVB). Do they have independent origins? Hence, the two-body problem. We hypothesize that a key to this conundrum is the membrane spanning V0 sector of the proton pump, V0V1-ATPase. The V0V1-ATPase participates in the acidification of intracellular compartments, although V0 can function separately from V1 and different V0 isoforms are endowed with membrane binding capabilities that allow the V0V1-ATPase to selectively localize to different endocytic compartments including early and late endosomes and lysosomes. We propose that V0, in collaboration with cholesterol and phosphoinositides, plays a central role in the early endosome as a nucleation center to direct the de novo assembly of an EXO-MVB scaffold. The EXO-MVB scaffold may play multiple roles-operating as an assembly platform, participating in membrane fission as well as providing downstream navigational queues necessary for exosome secretion. Thus, V0 may represent an influential nexus, a starting point, in exosome biogenesis.
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Affiliation(s)
| | - Philip D. Stahl
- Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO
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6
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Mançano ASF, Pina JG, Froes BR, Sciani JM. Autophagy-lysosomal pathway impairment and cathepsin dysregulation in Alzheimer's disease. Front Mol Biosci 2024; 11:1490275. [PMID: 39544403 PMCID: PMC11560772 DOI: 10.3389/fmolb.2024.1490275] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Accepted: 10/15/2024] [Indexed: 11/17/2024] Open
Abstract
Alzheimer's disease (AD) is characterized by neuronal loss, attributed to amyloid-beta (Aβ) aggregation and accumulation. The autophagy-lysosomal pathway, including cathepsins B and D, is crucial for protein degradation and clearance, but it is impaired in some diseases. This review summarizes current knowledge on the dysregulation of this pathway in AD. Accumulating evidence suggests that Aβ overload impairs autophagy-lysosomal function and cathepsin activity, exacerbating Aβ accumulation and neurodegeneration. However, the precise mechanisms underlying these interactions remain elusive. Despite these challenges, targeting the lysosomal pathway emerges as a promising therapeutic strategy, and a comprehensive understanding of the autophagy-lysosomal system is essential to develop effective interventions for AD.
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Affiliation(s)
| | | | | | - Juliana Mozer Sciani
- Laboratório de Produtos Naturais, Universidade São Francisco, Bragança Paulista, São Paulo, Brazil
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7
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Chen Q, Kou H, Demy DL, Liu W, Li J, Wen Z, Herbomel P, Huang Z, Zhang W, Xu J. The different roles of V-ATPase a subunits in phagocytosis/endocytosis and autophagy. Autophagy 2024; 20:2297-2313. [PMID: 38873931 PMCID: PMC11423658 DOI: 10.1080/15548627.2024.2366748] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2023] [Revised: 04/27/2024] [Accepted: 06/07/2024] [Indexed: 06/15/2024] Open
Abstract
Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species.Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.
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Affiliation(s)
- Qi Chen
- School of Biology and Biological Engineering, South China University of Technology, Guangzhou, PRChina
| | - Hanjing Kou
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
| | - Doris Lou Demy
- Institut Pasteur, Department of Developmental & Stem Cell Biology, Paris, France
- CNRS, UMR 3738, Paris, France
| | - Wei Liu
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
| | - Jianchao Li
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
| | - Zilong Wen
- Division of Life Science, State Key Laboratory of Molecular Neuroscience and Center of Systems Biology and Human Health, The Hong Kong University of Science and Technology, Hong Kong, PRChina
| | - Philippe Herbomel
- Institut Pasteur, Department of Developmental & Stem Cell Biology, Paris, France
- CNRS, UMR 3738, Paris, France
| | - Zhibin Huang
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
| | - Wenqing Zhang
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
- Greater Bay Biomedical Innocenter, Shenzhen Bay Laboratory, Shenzhen, China
| | - Jin Xu
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
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8
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Jiang J, Ren R, Fang W, Miao J, Wen Z, Wang X, Xu J, Jin H. Lysosomal biogenesis and function in osteoclasts: a comprehensive review. Front Cell Dev Biol 2024; 12:1431566. [PMID: 39170917 PMCID: PMC11335558 DOI: 10.3389/fcell.2024.1431566] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2024] [Accepted: 07/19/2024] [Indexed: 08/23/2024] Open
Abstract
Lysosomes serve as catabolic centers and signaling hubs in cells, regulating a multitude of cellular processes such as intracellular environment homeostasis, macromolecule degradation, intracellular vesicle trafficking and autophagy. Alterations in lysosomal level and function are crucial for cellular adaptation to external stimuli, with lysosome dysfunction being implicated in the pathogenesis of numerous diseases. Osteoclasts (OCs), as multinucleated cells responsible for bone resorption and maintaining bone homeostasis, have a complex relationship with lysosomes that is not fully understood. Dysregulated function of OCs can disrupt bone homeostasis leading to the development of various bone disorders. The regulation of OC differentiation and bone resorption for the treatment of bone disease have received considerable attention in recent years, yet the role and regulation of lysosomes in OCs, as well as the potential therapeutic implications of intervening in lysosomal biologic behavior for the treatment of bone diseases, remain relatively understudied. This review aims to elucidate the mechanisms involved in lysosomal biogenesis and to discuss the functions of lysosomes in OCs, specifically in relation to differentiation, bone resorption, and autophagy. Finally, we explore the potential therapeutic implication of targeting lysosomes in the treatment of bone metabolic disorders.
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Affiliation(s)
- Junchen Jiang
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Rufeng Ren
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Weiyuan Fang
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Jiansen Miao
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Zijun Wen
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Xiangyang Wang
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
| | - Jiake Xu
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
| | - Haiming Jin
- Department of Orthopaedics, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, China
- The Second School of Medicine, Wenzhou Medical University, Wenzhou, China
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9
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Tümen D, Heumann P, Huber J, Hahn N, Macek C, Ernst M, Kandulski A, Kunst C, Gülow K. Unraveling Cancer's Wnt Signaling: Dynamic Control through Protein Kinase Regulation. Cancers (Basel) 2024; 16:2686. [PMID: 39123414 PMCID: PMC11312265 DOI: 10.3390/cancers16152686] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 07/25/2024] [Accepted: 07/25/2024] [Indexed: 08/12/2024] Open
Abstract
Since the initial identification of oncogenic Wnt in mice and Drosophila, the Wnt signaling pathway has been subjected to thorough and extensive investigation. Persistent activation of Wnt signaling exerts diverse cancer characteristics, encompassing tumor initiation, tumor growth, cell senescence, cell death, differentiation, and metastasis. Here we review the principal signaling mechanisms and the regulatory influence of pathway-intrinsic and extrinsic kinases on cancer progression. Additionally, we underscore the divergences and intricate interplays of the canonical and non-canonical Wnt signaling pathways and their critical influence in cancer pathophysiology, exhibiting both growth-promoting and growth-suppressing roles across diverse cancer types.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Karsten Gülow
- Department of Internal Medicine I Gastroenterology, Hepatology, Endocrinology, Rheumatology, Immunology, and Infectious Diseases, University Hospital Regensburg, 93053 Regensburg, Germany; (D.T.); (N.H.)
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10
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Oot RA, Wilkens S. Human V-ATPase function is positively and negatively regulated by TLDc proteins. Structure 2024; 32:989-1000.e6. [PMID: 38593795 PMCID: PMC11246223 DOI: 10.1016/j.str.2024.03.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2023] [Revised: 02/23/2024] [Accepted: 03/13/2024] [Indexed: 04/11/2024]
Abstract
Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear. Here we examine the effects of five human TLDc proteins, TLDC2, NCOA7, OXR1, TBC1D24, and mEAK7 on the activity of the human V-ATPase. We find that while TLDC2, TBC1D24, and the TLDc domains of OXR1 and NCOA7 inhibit V-ATPase by inducing enzyme disassembly, mEAK7 activates the pump. The data thus shed new light both on mammalian TLDc protein function and V-ATPase regulation.
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Affiliation(s)
- Rebecca A Oot
- Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
| | - Stephan Wilkens
- Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA
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11
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Ai Y, Guo C, Garcia-Contreras M, Sánchez B. LS, Saftics A, Shodubi O, Raghunandan S, Xu J, Tsai SJ, Dong Y, Li R, Jovanovic-Talisman T, Gould SJ. Endocytosis blocks the vesicular secretion of exosome marker proteins. SCIENCE ADVANCES 2024; 10:eadi9156. [PMID: 38718108 PMCID: PMC11078179 DOI: 10.1126/sciadv.adi9156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Accepted: 04/04/2024] [Indexed: 05/12/2024]
Abstract
Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.
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Affiliation(s)
- Yiwei Ai
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Chenxu Guo
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Marta Garcia-Contreras
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Laura S. Sánchez B.
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Andras Saftics
- Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA
| | - Oluwapelumi Shodubi
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Shankar Raghunandan
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Junhao Xu
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Shang Jui Tsai
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Yi Dong
- Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Rong Li
- Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
- Mechanobiology Institute and Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore
| | - Tijana Jovanovic-Talisman
- Department of Cancer Biology and Molecular Medicine, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA
| | - Stephen J. Gould
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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12
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Khan MM, Wilkens S. Molecular mechanism of Oxr1p mediated disassembly of yeast V-ATPase. EMBO Rep 2024; 25:2323-2347. [PMID: 38565737 PMCID: PMC11094088 DOI: 10.1038/s44319-024-00126-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2023] [Revised: 03/07/2024] [Accepted: 03/09/2024] [Indexed: 04/04/2024] Open
Abstract
The eukaryotic vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes. We recently reported that the TLDc protein Oxr1p induces V-ATPase disassembly in vitro. Whether and how Oxr1p is involved in enzyme disassembly in vivo, however, is not known. Here, using yeast genetics and fluorescence microscopy, we show that Oxr1p is essential for efficient V-ATPase disassembly in the cell. Supporting biochemical and biophysical in vitro experiments show that whereas Oxr1p-driven holoenzyme disassembly can occur in the absence of nucleotides, the presence of ATP greatly accelerates the process. ATP hydrolysis is needed, however, for subsequent release of Oxr1p so that the free V1 can adopt the autoinhibited conformation. Overall, our study unravels the molecular mechanism of Oxr1p-induced disassembly that occurs in vivo as part of the canonical V-ATPase regulation by reversible disassembly.
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Affiliation(s)
- Md Murad Khan
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, 13210, USA
| | - Stephan Wilkens
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, 13210, USA.
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13
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Govindan G, Harini P, Alphonse V, Parani M. From swamp to field: how genes from mangroves and its associates can enhance crop salinity tolerance. Mol Biol Rep 2024; 51:598. [PMID: 38683409 DOI: 10.1007/s11033-024-09539-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2024] [Accepted: 04/09/2024] [Indexed: 05/01/2024]
Abstract
Salinity stress is a critical challenge in crop production and requires innovative strategies to enhance the salt tolerance of plants. Insights from mangrove species, which are renowned for their adaptability to high-salinity environments, provides valuable genetic targets and resources for improving crops. A significant hurdle in salinity stress is the excessive uptake of sodium ions (Na+) by plant roots, causing disruptions in cellular balance, nutrient deficiencies, and hampered growth. Specific ion transporters and channels play crucial roles in maintaining a low Na+/K+ ratio in root cells which is pivotal for salt tolerance. The family of high-affinity potassium transporters, recently characterized in Avicennia officinalis, contributes to K+ homeostasis in transgenic Arabidopsis plants even under high-salt conditions. The salt overly sensitive pathway and genes related to vacuolar-type H+-ATPases hold promise for expelling cytosolic Na+ and sequestering Na+ in transgenic plants, respectively. Aquaporins contribute to mangroves' adaptation to saline environments by regulating water uptake, transpiration, and osmotic balance. Antioxidant enzymes mitigate oxidative damage, whereas genes regulating osmolytes, such as glycine betaine and proline, provide osmoprotection. Mangroves exhibit increased expression of stress-responsive transcription factors such as MYB, NAC, and CBFs under high salinity. Moreover, genes involved in various metabolic pathways, including jasmonate synthesis, triterpenoid production, and protein stability under salt stress, have been identified. This review highlights the potential of mangrove genes to enhance salt tolerance of crops. Further research is imperative to fully comprehend and apply these genes to crop breeding to improve salinity resilience.
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Affiliation(s)
- Ganesan Govindan
- Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, TN, 603203, India
| | - Prakash Harini
- Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, TN, 603203, India
| | - Vinoth Alphonse
- Department of Botany, St. Xavier's College (Autonomous), Palayamkottai, TN, 627 002, India
| | - Madasamy Parani
- Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, TN, 603203, India.
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14
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Choi E, Song J, Lee Y, Jeong Y, Jang W. Prioritizing susceptibility genes for the prognosis of male-pattern baldness with transcriptome-wide association study. Hum Genomics 2024; 18:34. [PMID: 38566255 PMCID: PMC10985920 DOI: 10.1186/s40246-024-00591-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2024] [Accepted: 02/27/2024] [Indexed: 04/04/2024] Open
Abstract
BACKGROUND Male-pattern baldness (MPB) is the most common cause of hair loss in men. It can be categorized into three types: type 2 (T2), type 3 (T3), and type 4 (T4), with type 1 (T1) being considered normal. Although various MPB-associated genetic variants have been suggested, a comprehensive study for linking these variants to gene expression regulation has not been performed to the best of our knowledge. RESULTS In this study, we prioritized MPB-related tissue panels using tissue-specific enrichment analysis and utilized single-tissue panels from genotype-tissue expression version 8, as well as cross-tissue panels from context-specific genetics. Through a transcriptome-wide association study and colocalization analysis, we identified 52, 75, and 144 MPB associations for T2, T3, and T4, respectively. To assess the causality of MPB genes, we performed a conditional and joint analysis, which revealed 10, 11, and 54 putative causality genes for T2, T3, and T4, respectively. Finally, we conducted drug repositioning and identified potential drug candidates that are connected to MPB-associated genes. CONCLUSIONS Overall, through an integrative analysis of gene expression and genotype data, we have identified robust MPB susceptibility genes that may help uncover the underlying molecular mechanisms and the novel drug candidates that may alleviate MPB.
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Affiliation(s)
- Eunyoung Choi
- Department of Life Sciences, Dongguk University, Seoul, 04620, Republic of Korea
| | - Jaeseung Song
- Department of Life Sciences, Dongguk University, Seoul, 04620, Republic of Korea
| | - Yubin Lee
- Department of Life Sciences, Dongguk University, Seoul, 04620, Republic of Korea
| | - Yeonbin Jeong
- Department of Life Sciences, Dongguk University, Seoul, 04620, Republic of Korea
| | - Wonhee Jang
- Department of Life Sciences, Dongguk University, Seoul, 04620, Republic of Korea.
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15
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Li B, Lan S, Liu XR, Ji JJ, He YY, Zhang DM, Xu J, Sun H, Shi Z, Wang J, Tian Y. ATP6V1A variants are associated with childhood epilepsy with favorable outcome. Seizure 2024; 116:81-86. [PMID: 37574426 DOI: 10.1016/j.seizure.2023.08.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2023] [Revised: 08/03/2023] [Accepted: 08/06/2023] [Indexed: 08/15/2023] Open
Abstract
PURPOSE ATP6V1A variants have been identified in patients with highly variable phenotypes such as autosomal dominant epileptic encephalopathy and autosomal recessive cutis laxa. However, the mechanism underlying phenotype variation is unknown. We screened ATP6V1A variants in patients with epilepsy and analyzed the genotype-phenotype correlation to explain the mechanism underlying phenotypic variations. METHODS We performed trio-based whole-exome sequencing in people with epilepsy without acquired causes. All previously reported ATP6V1A variants were systematically retrieved from the HGMD and PubMed databases. RESULTS Three novel de novo ATP6V1A variants, including c.749G>C/p.Gly250Ala, c.782A>G/p.Gln261Arg, and c.1103T>C/p.Met368Thr, were identified in three unrelated cases with childhood focal (partial) epilepsy. None of the variants were listed in any public population database and evaluated as likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics (ACMG). All persons showed good responses to anti-seizure medication and psychomotor development was normal. Further analysis showed that monoallelic missense variants were associated with epilepsy with variable severity, whereas biallelic variants resulted in developmental abnormalities of multisystem that may result in early lethality. CONCLUSION Childhood focal epilepsy with favorable outcome was probably a novel phenotype of ATP6V1A. ATP6V1A variants are associated with a range of phenotypes that correlate with genotypes. The relationship between phenotype severity and the genotype (genetic impairment) of ATP6V1A variants helps explain the phenotypic variations.
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Affiliation(s)
- Bin Li
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China
| | - Song Lan
- Department of Neurology, Maoming People's Hospital, Maoming, China
| | - Xiao-Rong Liu
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China
| | - Jing-Jing Ji
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China
| | - Yun-Yan He
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China
| | - Dong-Ming Zhang
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China
| | - Jie Xu
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China
| | - Hui Sun
- Department of Neurology, the First Affiliated Hospital of Guangzhou Medical University
| | - Zhen Shi
- Department of Neurology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, China
| | - Jie Wang
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China.
| | - Yang Tian
- Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou, China; Department of Neurology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, China.
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16
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Shariq M, Khan MF, Raj R, Ahsan N, Kumar P. PRKAA2, MTOR, and TFEB in the regulation of lysosomal damage response and autophagy. J Mol Med (Berl) 2024; 102:287-311. [PMID: 38183492 DOI: 10.1007/s00109-023-02411-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Revised: 12/07/2023] [Accepted: 12/18/2023] [Indexed: 01/08/2024]
Abstract
Lysosomes function as critical signaling hubs that govern essential enzyme complexes. LGALS proteins (LGALS3, LGALS8, and LGALS9) are integral to the endomembrane damage response. If ESCRT fails to rectify damage, LGALS-mediated ubiquitination occurs, recruiting autophagy receptors (CALCOCO2, TRIM16, and SQSTM1) and VCP/p97 complex containing UBXN6, PLAA, and YOD1, initiating selective autophagy. Lysosome replenishment through biogenesis is regulated by TFEB. LGALS3 interacts with TFRC and TRIM16, aiding ESCRT-mediated repair and autophagy-mediated removal of damaged lysosomes. LGALS8 inhibits MTOR and activates TFEB for ATG and lysosomal gene transcription. LGALS9 inhibits USP9X, activates PRKAA2, MAP3K7, ubiquitination, and autophagy. Conjugation of ATG8 to single membranes (CASM) initiates damage repair mediated by ATP6V1A, ATG16L1, ATG12, ATG5, ATG3, and TECPR1. ATG8ylation or CASM activates the MERIT system (ESCRT-mediated repair, autophagy-mediated clearance, MCOLN1 activation, Ca2+ release, RRAG-GTPase regulation, MTOR modulation, TFEB activation, and activation of GTPase IRGM). Annexins ANAX1 and ANAX2 aid damage repair. Stress granules stabilize damaged membranes, recruiting FLCN-FNIP1/2, G3BP1, and NUFIP1 to inhibit MTOR and activate TFEB. Lysosomes coordinate the synergistic response to endomembrane damage and are vital for innate and adaptive immunity. Future research should unveil the collaborative actions of ATG proteins, LGALSs, TRIMs, autophagy receptors, and lysosomal proteins in lysosomal damage response.
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Affiliation(s)
- Mohd Shariq
- Quantlase Imaging Laboratory, Quantlase Lab LLC, Unit 1-8, Masdar City, Abu Dhabi, UAE.
| | - Mohammad Firoz Khan
- Quantlase Imaging Laboratory, Quantlase Lab LLC, Unit 1-8, Masdar City, Abu Dhabi, UAE.
| | - Reshmi Raj
- Quantlase Imaging Laboratory, Quantlase Lab LLC, Unit 1-8, Masdar City, Abu Dhabi, UAE
| | - Nuzhat Ahsan
- Quantlase Imaging Laboratory, Quantlase Lab LLC, Unit 1-8, Masdar City, Abu Dhabi, UAE
| | - Pramod Kumar
- Quantlase Imaging Laboratory, Quantlase Lab LLC, Unit 1-8, Masdar City, Abu Dhabi, UAE
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17
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Chu A, Yao Y, Glibowicka M, Deber CM, Manolson MF. The Human Mutation K237_V238del in a Putative Lipid Binding Motif within the V-ATPase a2 Isoform Suggests a Molecular Mechanism Underlying Cutis Laxa. Int J Mol Sci 2024; 25:2170. [PMID: 38396846 PMCID: PMC10889665 DOI: 10.3390/ijms25042170] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2023] [Revised: 01/19/2024] [Accepted: 02/01/2024] [Indexed: 02/25/2024] Open
Abstract
Vacuolar ATPases (V-ATPases), proton pumps composed of 16 subunits, are necessary for a variety of cellular functions. Subunit "a" has four isoforms, a1-a4, each with a distinct cellular location. We identified a phosphoinositide (PIP) interaction motif, KXnK(R)IK(R), conserved in all four isoforms, and hypothesize that a/PIP interactions regulate V-ATPase recruitment/retention to different organelles. Among the four isoforms, a2 is enriched on Golgi with a2 mutations in the PIP motif resulting in cutis laxa. We hypothesize that the hydrophilic N-terminal (NT) domain of a2 contains a lipid-binding domain, and mutations in this domain prevent interaction with Golgi-enriched PIPs, resulting in cutis laxa. We recreated the cutis laxa-causing mutation K237_V238del, and a double mutation in the PIP-binding motif, K237A/V238A. Circular dichroism confirmed that there were no protein structure alterations. Pull-down assays with PIP-enriched liposomes revealed that wildtype a2NT preferentially binds phosphatidylinositol 4-phosphate (PI(4)P), while mutants decreased binding to PI(4)P. In HEK293 cells, wildtype a2NT was localized to Golgi and co-purified with microsomal membranes. Mutants reduced Golgi localization and membrane association. Rapamycin depletion of PI(4)P diminished a2NT-Golgi localization. We conclude that a2NT is sufficient for Golgi retention, suggesting the lipid-binding motif is involved in V-ATPase targeting and/or retention. Mutational analyses suggest a molecular mechanism underlying how a2 mutations result in cutis laxa.
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Affiliation(s)
- Anh Chu
- Faculty of Dentistry, University of Toronto, Toronto M5G 1G6, ON, Canada; (A.C.); (Y.Y.)
| | - Yeqi Yao
- Faculty of Dentistry, University of Toronto, Toronto M5G 1G6, ON, Canada; (A.C.); (Y.Y.)
| | - Miroslawa Glibowicka
- Division of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, ON, Canada; (M.G.); (C.M.D.)
| | - Charles M. Deber
- Division of Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, ON, Canada; (M.G.); (C.M.D.)
- Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto M5S 1A8, ON, Canada
| | - Morris F. Manolson
- Faculty of Dentistry, University of Toronto, Toronto M5G 1G6, ON, Canada; (A.C.); (Y.Y.)
- Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto M5S 1A8, ON, Canada
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18
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Bychkova S, Bychkov M, Dordevic D, Vítězová M, Rittmann SKMR, Kushkevych I. Bafilomycin A1 Molecular Effect on ATPase Activity of Subcellular Fraction of Human Colorectal Cancer and Rat Liver. Int J Mol Sci 2024; 25:1657. [PMID: 38338935 PMCID: PMC10855383 DOI: 10.3390/ijms25031657] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2024] [Revised: 01/24/2024] [Accepted: 01/25/2024] [Indexed: 02/12/2024] Open
Abstract
Bafilomycin A1 inhibits V-type H+ ATPases on the molecular level, which acidifies endo-lysosomes. The main objective of the study was to assess the effect of bafilomycin A1 on Ca2+ content, NAADP-induced Ca2+ release, and ATPase activity in rat hepatocytes and human colon cancer samples. Chlortetracycline (CTC) was used for a quantitative measure of stored calcium in permeabilized rat hepatocytes. ATPase activity was determined by orthophosphate content released after ATP hydrolysis in subcellular post-mitochondrial fraction obtained from rat liver as well as from patients' samples of colon mucosa and colorectal cancer samples. In rat hepatocytes, bafilomycin A1 decreased stored Ca2+ and prevented the effect of NAADP on stored Ca2+. This effect was dependent on EGTA-Ca2+ buffers in the medium. Bafilomycin A1 significantly increased the activity of Ca2+ ATPases of endoplasmic reticulum (EPR), but not plasma membrane (PM) Ca2+ ATPases in rat liver. Bafilomycin A1 also prevented the effect of NAADP on these pumps. In addition, bafilomycin A1 reduced Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in the subcellular fraction of rat liver. Concomitant administration of bafilomycin A1 and NAADP enhanced these effects. Bafilomycin A1 increased the activity of the Ca2+ ATPase of EPR in the subcellular fraction of normal human colon mucosa and also in colon cancer tissue samples. In contrast, it decreased Ca2+ ATPase PM activity in samples of normal human colon mucosa and caused no changes in colon cancer. Bafilomycin A1 decreased Na+/K+ ATPase activity and increased basal Mg2+ ATPase activity in normal colon mucosa samples and in human colon cancer samples. It can be concluded that bafilomycin A1 targets NAADP-sensitive acidic Ca2+ stores, effectively modulates ATPase activity, and assumes the link between acidic stores and EPR. Bafilomycin A1 may be useful for cancer therapy.
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Affiliation(s)
- Solomiia Bychkova
- Department of Human and Animal Physiology, Faculty of Biology, Ivan Franko National University of Lviv, 79005 Lviv, Ukraine;
| | - Mykola Bychkov
- Department of Therapy No. 1, Medical Diagnostic and Hematology and Transfusiology of Faculty of Postgraduate Education, Danylo Halytsky Lviv National Medical University, 79010 Lviv, Ukraine;
| | - Dani Dordevic
- Department of Plant Origin Food Sciences, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, 612 42 Brno, Czech Republic;
| | - Monika Vítězová
- Department of Experimental Biology, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic;
| | - Simon K.-M. R. Rittmann
- Department of Functional and Evolutionary Ecology, Archaea Physiology & Biotechnology Group, Universität Wien, 1030 Wien, Austria
| | - Ivan Kushkevych
- Department of Experimental Biology, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic;
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19
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Zhao Z, Wang X, Ma Y, Duan X. Atp6v1h Deficiency Blocks Bone Loss in Simulated Microgravity Mice through the Fos-Jun-Src-Integrin Pathway. Int J Mol Sci 2024; 25:637. [PMID: 38203808 PMCID: PMC10779874 DOI: 10.3390/ijms25010637] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2023] [Revised: 12/05/2023] [Accepted: 12/29/2023] [Indexed: 01/12/2024] Open
Abstract
The microgravity conditions in outer space are widely acknowledged to induce significant bone loss. Recent studies have implicated the close relationship between Atp6v1h gene and bone loss. Despite this, the role of Atp6v1h in bone remodeling and its molecular mechanisms in microgravity have not been fully elucidated. To address this, we used a mouse tail suspension model to simulate microgravity. We categorized both wild-type and Atp6v1h knockout (Atp6v1h+/-) mice into two groups: regular feeding and tail-suspension feeding, ensuring uniform feeding conditions across all cohorts. Analysis via micro-CT scanning, hematoxylin-eosin staining, and tartrate-resistant acid phosphatase assays indicated that wild-type mice underwent bone loss under simulated microgravity. Atp6v1h+/- mice exhibited bone loss due to Atp6v1h deficiency but did not present aggravated bone loss under the same simulated microgravity. Transcriptomic sequencing revealed the upregulation of genes, such as Fos, Src, Jun, and various integrin subunits in the context of simulated microgravity and Atp6v1h knockout. Real-time quantitative polymerase chain reaction (RT-qPCR) further validated the modulation of downstream osteoclast-related genes in response to interactions with ATP6V1H overexpression cell lines. Co-immunoprecipitation indicated potential interactions between ATP6V1H and integrin beta 1, beta 3, beta 5, alpha 2b, and alpha 5. Our results indicate that Atp6v1h level influences bone loss in simulated microgravity by modulating the Fos-Jun-Src-Integrin pathway, which, in turn, affects osteoclast activity and bone resorption, with implications for osteoporosis. Therefore, modulating Atp6v1h expression could mitigate bone loss in microgravity conditions. This study elucidates the molecular mechanism of Atp6v1h's role in osteoporosis and positions it as a potential therapeutic target against environmental bone loss. These findings open new possibilities for the treatment of multifactorial osteoporosis.
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Affiliation(s)
| | | | - Yu Ma
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Key Laboratory of Stomatology, Department of Oral Biology, Clinic of Oral Rare and Genetic Diseases, School of Stomatology, The Fourth Military Medical University, Xi’an 710032, China; (Z.Z.); (X.W.); (Y.M.)
| | - Xiaohong Duan
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Key Laboratory of Stomatology, Department of Oral Biology, Clinic of Oral Rare and Genetic Diseases, School of Stomatology, The Fourth Military Medical University, Xi’an 710032, China; (Z.Z.); (X.W.); (Y.M.)
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20
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Nakanishi-Matsui M, Matsumoto N, Sun-Wada GH, Wada Y. Role of the Cytosolic Domain of the a3 Subunit of V-ATPase in the Interaction with Rab7 and Secretory Lysosome Trafficking in Osteoclasts. Biol Pharm Bull 2024; 47:339-344. [PMID: 38296463 DOI: 10.1248/bpb.b23-00833] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2024]
Abstract
We previously reported that the a3 subunit of proton-pumping vacuolar-type ATPase (V-ATPase) interacts with Rab7 and its guanine nucleotide exchange factor, Mon1a-Ccz1, and recruits them to secretory lysosomes in osteoclasts, which is essential for anterograde trafficking of secretory lysosomes. The a3 subunit interacts with Mon1a-Ccz1 through its cytosolic N-terminal domain. Here, we examined the roles of this domain in the interaction with Rab7 and trafficking of secretory lysosomes. Immunoprecipitation experiments showed that a3 interacted with Rab7 through its cytosolic domain, similar to the interaction with Mon1a-Ccz1. We connected this domain with a lysosome localization signal and expressed it in a3-knockout (a3KO) osteoclasts. Although the signal connected to the cytosolic domain was mainly detected in lysosomes, impaired lysosome trafficking in a3KO osteoclasts was not rescued. These results indicate that the cytosolic domain of a3 can interact with trafficking regulators, but is insufficient to induce secretory lysosome trafficking. The C-terminal domain of a3 and other subunits of V-ATPase are likely required to form a fully functional complex for secretory lysosome trafficking.
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Affiliation(s)
| | - Naomi Matsumoto
- Division of Biochemistry, School of Pharmacy, Iwate Medical University
- Center for Basic Medical Research, International University of Health and Welfare
| | - Ge-Hong Sun-Wada
- Department of Biochemistry, Faculty of Pharmaceutical Sciences, Doshisha Women's College
| | - Yoh Wada
- Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University
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21
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Bestion E, Rachid M, Tijeras-Raballand A, Roth G, Decaens T, Ansaldi C, Mezouar S, Raymond E, Halfon P. Ezurpimtrostat, A Palmitoyl-Protein Thioesterase-1 Inhibitor, Combined with PD-1 Inhibition Provides CD8 + Lymphocyte Repopulation in Hepatocellular Carcinoma. Target Oncol 2024; 19:95-106. [PMID: 38133710 DOI: 10.1007/s11523-023-01019-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/20/2023] [Indexed: 12/23/2023]
Abstract
BACKGROUND Palmitoyl-protein thioesterase-1 (PPT1) is a clinical stage druggable target for inhibiting autophagy in cancer. OBJECTIVE We aimed to determine the cellular and molecular activity of targeting PPT1 using ezurpimtrostat, in combination with an anti-PD-1 antibody. METHODS In this study we used a transgenic immunocompetent mouse model of hepatocellular carcinoma. RESULTS Herein, we revealed that inhibition of PPT1 using ezurpimtrostat decreased the liver tumor burden in a mouse model of hepatocellular carcinoma by inducing the penetration of lymphocytes into tumors when combined with anti-programmed death-1 (PD-1). Inhibition of PPT1 potentiates the effects of anti-PD-1 immunotherapy by increasing the expression of major histocompatibility complex (MHC)-I at the surface of liver cancer cells and modulates immunity through recolonization and activation of cytotoxic CD8+ lymphocytes. CONCLUSIONS Ezurpimtrostat turns cold tumors into hot tumors and, thus, could improve T cell-mediated immunotherapies in liver cancer.
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Affiliation(s)
- Eloïne Bestion
- Genoscience Pharma, 10, Rue d'Iéna, 13006, Marseille, France
| | - Madani Rachid
- Genoscience Pharma, 10, Rue d'Iéna, 13006, Marseille, France
| | | | - Gael Roth
- Centre hospitalouniversitaire Grenoble Alpes/Institute for Advanced Biosciences, Centre national de la recherché scienti-fique, Unité mixte de recherche 5309-Institut national de la santé et de la recherche médicale U1209, University Grenoble Alpes/Hepato-Gastroenterology and Digestive Oncology Department, 38043, Grenoble, France
| | - Thomas Decaens
- Centre hospitalouniversitaire Grenoble Alpes/Institute for Advanced Biosciences, Centre national de la recherché scienti-fique, Unité mixte de recherche 5309-Institut national de la santé et de la recherche médicale U1209, University Grenoble Alpes/Hepato-Gastroenterology and Digestive Oncology Department, 38043, Grenoble, France
| | | | - Soraya Mezouar
- Genoscience Pharma, 10, Rue d'Iéna, 13006, Marseille, France
- Etablissement français du sang, Centre national de la recherche scientifique, Anthropologie bio-culturelle, droit, éthique et santé, "Biologie des Groupes Sanguins", Aix-Marseille University, Marseille, France
| | - Eric Raymond
- Genoscience Pharma, 10, Rue d'Iéna, 13006, Marseille, France
- Oncology Department, Groupe Hospitalier Paris Saint Joseph, Paris, France
| | - Philippe Halfon
- Genoscience Pharma, 10, Rue d'Iéna, 13006, Marseille, France.
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22
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Paquet Luzy C, Doppler E, Polasek TM, Giorgino R. First-in-human single-dose study of nizubaglustat, a dual inhibitor of ceramide glucosyltransferase and non-lysosomal glucosylceramidase: Safety, tolerability, pharmacokinetics, and pharmacodynamics of single ascending and multiple doses in healthy adults. Mol Genet Metab 2024; 141:108113. [PMID: 38113551 DOI: 10.1016/j.ymgme.2023.108113] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/31/2023] [Revised: 11/27/2023] [Accepted: 12/06/2023] [Indexed: 12/21/2023]
Abstract
Nizubaglustat is a novel, orally available, brain penetrant, potent, and selective dual inhibitor of ceramide glucosyltranferase and non-lysosomal neutral glucosylceramidase (NLGase), which is currently under development for the treatment of subjects with neurological manifestations in primary and secondary gangliosidoses. The objectives of this first-in-human study were to evaluate the safety and tolerability, pharmacokinetics, and pharmacodynamics (PD) of single oral doses of nizubaglustat after single (1, 3, and 9 mg) and multiple oral doses (9 mg once per day (QD) over 14 days) in healthy adults. Nizubaglustat was rapidly absorbed and systemic exposure was dose-proportional. Steady-state was achieved after three days of QD multiple dosing with minimal accumulation. Renal clearance accounted for around 15% of nizubaglustat elimination. Following multiple dosing, plasma concentrations of glucosylceramide (GlcCer), lactosylceramide (LacCer), and monosialodihexosylganglioside (GM3) decreased to a nadir at Day 10. PD target engagement of GCS inhibition was shown by a median decrease from baseline of plasma concentrations of GlcCer, LacCer, and GM3 ganglioside by 70%, 50%, and 48%, respectively. NLGase inhibition was also manifested by increased concentrations of GlcCer in cerebrospinal fluid from Day 1 to Day 14. Nizubaglustat was safe and well-tolerated at all doses tested. Consistent with the high selectivity, and the absence of intestinal disaccharidases inhibition, no cases of diarrhea were reported. No decreased appetite or weight loss was noted. Only treatment-emergent adverse events with preferred terms belonging to the system organ class skin and subcutaneous disorders of mild intensity were reported as drug-related in the nizubaglustat arm, in line with the pharmacological mechanism targeting glucosylceramide metabolism. Taken together, these data support QD dosing of nizubaglustat and its ongoing development in patients with primary and secondary forms of gangliosidoses.
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Affiliation(s)
| | | | - Thomas M Polasek
- Principal Investigator, CMAX Research Phase 1 Unit, Ground Floor 21-24 North Terrace, Adelaide, 5000, SA, Australia; Department of Clinical Pharmacology, Royal Adelaide Hospital, Port Rd, Adelaide, SA 5000, Australia
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23
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Li W, Kawaguchi K, Tanaka S, He C, Maeshima Y, Suzuki E, Toi M. Cellular senescence triggers intracellular acidification and lysosomal pH alkalinized via ATP6AP2 attenuation in breast cancer cells. Commun Biol 2023; 6:1147. [PMID: 37993606 PMCID: PMC10665353 DOI: 10.1038/s42003-023-05433-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Accepted: 10/09/2023] [Indexed: 11/24/2023] Open
Abstract
Several chemotherapeutic drugs induce senescence in cancer cells; however, the mechanisms underlying intracellular pH dysregulation in senescent cells remain unclear. Adenosine triphosphatase H+ transporting accessory protein 2 (ATP6AP2) plays a critical role in maintaining pH homeostasis in cellular compartments. Herein, we report the regulatory role of ATP6AP2 in senescent breast cancer cells treated with doxorubicin (Doxo) and abemaciclib (Abe). A decline in ATP6AP2 triggers aberrant pH levels that impair lysosomal function and cause immune profile changes in senescent breast cancer cells. Doxo and Abe elicited a stable senescent phenotype and altered the expression of senescence-related genes. Additionally, senescent cells show altered inflammatory and immune transcriptional profiles due to reprogramming of the senescence-associated secretory phenotype. These findings elucidate ATP6AP2-mediated cellular pH regulation and suggest a potential link in immune profile alteration during therapy-induced senescence in breast cancer cells, providing insights into the mechanisms involved in the senescence response to anticancer therapy.
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Affiliation(s)
- Wei Li
- Department of Breast Surgery, Kyoto University Graduate School of Medicine, 54 Shogoin-kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Kosuke Kawaguchi
- Department of Breast Surgery, Kyoto University Graduate School of Medicine, 54 Shogoin-kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan.
| | - Sunao Tanaka
- Department of Breast Surgery, Kyoto University Graduate School of Medicine, 54 Shogoin-kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Chenfeng He
- Department of Breast Surgery, Kyoto University Graduate School of Medicine, 54 Shogoin-kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Yurina Maeshima
- Department of Breast Surgery, Kyoto University Graduate School of Medicine, 54 Shogoin-kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Eiji Suzuki
- Kobe City Medical Center General Hospital, 2-1-1 Minatojimaminami-cho, Chuo-ku, Kobe, 650-0047, Japan
| | - Masakazu Toi
- Department of Breast Surgery, Kyoto University Graduate School of Medicine, 54 Shogoin-kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan
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24
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Hou J, Liu J, Huang Z, Wang Y, Yao H, Hu Z, Shi C, Xu J, Wang Q. Structure and function of the membrane microdomains in osteoclasts. Bone Res 2023; 11:61. [PMID: 37989999 PMCID: PMC10663511 DOI: 10.1038/s41413-023-00294-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 09/07/2023] [Accepted: 09/18/2023] [Indexed: 11/23/2023] Open
Abstract
The cell membrane structure is closely related to the occurrence and progression of many metabolic bone diseases observed in the clinic and is an important target to the development of therapeutic strategies for these diseases. Strong experimental evidence supports the existence of membrane microdomains in osteoclasts (OCs). However, the potential membrane microdomains and the crucial mechanisms underlying their roles in OCs have not been fully characterized. Membrane microdomain components, such as scaffolding proteins and the actin cytoskeleton, as well as the roles of individual membrane proteins, need to be elucidated. In this review, we discuss the compositions and critical functions of membrane microdomains that determine the biological behavior of OCs through the three main stages of the OC life cycle.
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Affiliation(s)
- Jialong Hou
- Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Department of Orthopaedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Jian Liu
- Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Department of Orthopaedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Zhixian Huang
- Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Department of Orthopaedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Yining Wang
- Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Department of Orthopaedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Hanbing Yao
- Department of Orthopaedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Zhenxin Hu
- Department of Spine Surgery, Peking University Fourth School of Clinical Medicine, Beijing, China
| | - Chengge Shi
- Department of Orthopaedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
| | - Jiake Xu
- School of Biomedical Sciences, The University of Western Australia, Perth, WA, Australia.
- Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
| | - Qingqing Wang
- Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.
- Department of Orthopaedics, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
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25
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Wang H, Zhu Y, Liu H, Liang T, Wei Y. Advances in Drug Discovery Targeting Lysosomal Membrane Proteins. Pharmaceuticals (Basel) 2023; 16:ph16040601. [PMID: 37111358 PMCID: PMC10145713 DOI: 10.3390/ph16040601] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Revised: 03/10/2023] [Accepted: 03/14/2023] [Indexed: 04/29/2023] Open
Abstract
Lysosomes are essential organelles of eukaryotic cells and are responsible for various cellular functions, including endocytic degradation, extracellular secretion, and signal transduction. There are dozens of proteins localized to the lysosomal membrane that control the transport of ions and substances across the membrane and are integral to lysosomal function. Mutations or aberrant expression of these proteins trigger a variety of disorders, making them attractive targets for drug development for lysosomal disorder-related diseases. However, breakthroughs in R&D still await a deeper understanding of the underlying mechanisms and processes of how abnormalities in these membrane proteins induce related diseases. In this article, we summarize the current progress, challenges, and prospects for developing therapeutics targeting lysosomal membrane proteins for the treatment of lysosomal-associated diseases.
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Affiliation(s)
- Hongna Wang
- Affiliated Cancer Hospital, Institute of Guangzhou Medical University, Guangzhou 510095, China
- Key Laboratory for Cell Homeostasis, Cancer Research of Guangdong Higher Education Institutes, Guangzhou 510095, China
| | - Yidong Zhu
- Affiliated Cancer Hospital, Institute of Guangzhou Medical University, Guangzhou 510095, China
- Key Laboratory for Cell Homeostasis, Cancer Research of Guangdong Higher Education Institutes, Guangzhou 510095, China
| | - Huiyan Liu
- Affiliated Cancer Hospital, Institute of Guangzhou Medical University, Guangzhou 510095, China
- Key Laboratory for Cell Homeostasis, Cancer Research of Guangdong Higher Education Institutes, Guangzhou 510095, China
| | - Tianxiang Liang
- Affiliated Cancer Hospital, Institute of Guangzhou Medical University, Guangzhou 510095, China
- Key Laboratory for Cell Homeostasis, Cancer Research of Guangdong Higher Education Institutes, Guangzhou 510095, China
| | - Yongjie Wei
- Affiliated Cancer Hospital, Institute of Guangzhou Medical University, Guangzhou 510095, China
- Key Laboratory for Cell Homeostasis, Cancer Research of Guangdong Higher Education Institutes, Guangzhou 510095, China
- State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, Guangzhou 510095, China
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26
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Regulation of mTORC1 by the Rag GTPases. Biochem Soc Trans 2023; 51:655-664. [PMID: 36929165 DOI: 10.1042/bst20210038] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2022] [Revised: 02/21/2023] [Accepted: 02/23/2023] [Indexed: 03/18/2023]
Abstract
The Rag GTPases are an evolutionarily conserved family that play a crucial role in amino acid sensing by the mammalian target of rapamycin complex 1 (mTORC1). mTORC1 is often referred to as the master regulator of cell growth. mTORC1 hyperactivation is observed in multiple diseases such as cancer, obesity, metabolic disorders, and neurodegeneration. The Rag GTPases sense amino acid levels and form heterodimers, where RagA or RagB binds to RagC or RagD, to recruit mTORC1 to the lysosome where it becomes activated. Here, we review amino acid signaling to mTORC1 through the Rag GTPases.
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27
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Characterization of a PIP Binding Site in the N-Terminal Domain of V-ATPase a4 and Its Role in Plasma Membrane Association. Int J Mol Sci 2023; 24:ijms24054867. [PMID: 36902293 PMCID: PMC10002524 DOI: 10.3390/ijms24054867] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 02/13/2023] [Accepted: 02/21/2023] [Indexed: 03/06/2023] Open
Abstract
Vacuolar ATPases (V-ATPases) are multi-subunit ATP-dependent proton pumps necessary for cellular functions, including pH regulation and membrane fusion. The evidence suggests that the V-ATPase a-subunit's interaction with the membrane signaling lipid phosphatidylinositol (PIPs) regulates the recruitment of V-ATPase complexes to specific membranes. We generated a homology model of the N-terminal domain of the human a4 isoform (a4NT) using Phyre2.0 and propose a lipid binding domain within the distal lobe of the a4NT. We identified a basic motif, K234IKK237, critical for interaction with phosphoinositides (PIP), and found similar basic residue motifs in all four mammalian and both yeast a-isoforms. We tested PIP binding of wildtype and mutant a4NT in vitro. In protein lipid overlay assays, the double mutation K234A/K237A and the autosomal recessive distal renal tubular-causing mutation K237del reduced both PIP binding and association with liposomes enriched with PI(4,5)P2, a PIP enriched within plasma membranes. Circular dichroism spectra of the mutant protein were comparable to wildtype, indicating that mutations affected lipid binding, not protein structure. When expressed in HEK293, wildtype a4NT localized to the plasma membrane in fluorescence microscopy and co-purified with the microsomal membrane fraction in cellular fractionation experiments. a4NT mutants showed reduced membrane association and decreased plasma membrane localization. Depletion of PI(4,5)P2 by ionomycin caused reduced membrane association of the WT a4NT protein. Our data suggest that information contained within the soluble a4NT is sufficient for membrane association and that PI(4,5)P2 binding capacity is involved in a4 V-ATPase plasma membrane retention.
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28
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Bowman BJ. The structure of prevacuolar compartments in Neurospora crassa as observed with super-resolution microscopy. PLoS One 2023; 18:e0282989. [PMID: 37093794 PMCID: PMC10124863 DOI: 10.1371/journal.pone.0282989] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Accepted: 02/28/2023] [Indexed: 04/25/2023] Open
Abstract
The hyphal tips of Neurospora crassa have prevacuolar compartments (PVCs) of unusual size and shape. They appear to function as late endosomes/multivesicular bodies. PVCs are highly variable in size (1-3 microns) and exhibit rapid changes in structure. When visualized with tagged integral membrane proteins of the vacuole the PVCs appear as ring or horseshoe-shaped structures. Some soluble molecules that fill the lumen of mature spherical vacuoles do not appear in the lumen of the PVC but are seen in the ring or horseshoe-shaped structures. By using super-resolution microscopy I have achieved a better understanding of the structure of the PVCs. The PVC appears to form a pouch with an open end. The walls of the pouch are composed of small vesicles or tubules, approximately 250 nm in diameter. The shape of the PVC can change in a few seconds, caused by the apparent movement of the vesicles/tubules. In approximately 85% of the PVCs dynein and dynactin were observed as poorly defined lumps inside the pouch-shaped PVCs. Within the PVCs they were not attached to microtubules nor did they appear to be in direct contact with the vesicles and tubules that formed the PVCs. In the future, the structure and relatively large size of the Neurospora PVC may allow us to visualize protein-sorting events that occur in the formation of vacuoles.
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Affiliation(s)
- Barry J Bowman
- Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, California, United States of America
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29
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Li Z, Hadlich F, Wimmers K, Murani E. Glucocorticoid receptor hypersensitivity enhances inflammatory signaling and inhibits cell cycle progression in porcine PBMCs. Front Immunol 2022; 13:976454. [PMID: 36505401 PMCID: PMC9730246 DOI: 10.3389/fimmu.2022.976454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Accepted: 11/10/2022] [Indexed: 11/27/2022] Open
Abstract
The consequences of glucocorticoid receptor (GR) hypersensitivity during infection have so far received little attention. We previously discovered that a natural gain-of-function Ala610Val substitution in the porcine GR aggravates response of pigs to lipopolysaccharide (LPS)-induced endotoxemia, which can be alleviated by dexamethasone (DEX) pretreatment. In this work, we investigated the relevant molecular basis of these phenotypes by transcriptomic profiling of porcine peripheral blood mononuclear cells (PBMCs) carrying different GR genotypes, in unstimulated conditions or in response to DEX and/or LPS in vitro. The Val allele differentially regulated abunda+nt genes in an additive-genetic manner. A subset of more than 200 genes was consistently affected by the substitution across treatments. This was associated with upregulation of genes related i.a. to endo-lysosomal system, lipid and protein catabolism, and immune terms including platelet activation, and antigen presentation, while downregulated genes were mainly involved in cell cycle regulation. Most importantly, the set of genes constitutively upregulated by Val includes members of the TLR4/LPS signaling pathway, such as LY96. Consequently, when exposing PBMCs to LPS treatment, the Val variant upregulated a panel of additional genes related to TLR4 and several other pattern recognition receptors, as well as cell death and lymphocyte signaling, ultimately amplifying the inflammatory responses. In contrast, when stimulated by DEX treatment, the Val allele orchestrated several genes involved in anti-inflammatory responses during infection. This study provides novel insights into the impact of GR hypersensitivity on the fate and function of immune cells, which may be useful for endotoxemia therapy.
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Affiliation(s)
- Zhiwei Li
- Institute of Genome Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany
| | - Frieder Hadlich
- Institute of Genome Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany
| | - Klaus Wimmers
- Institute of Genome Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany,Faculty of Agricultural and Environmental Sciences, University Rostock, Rostock, Germany
| | - Eduard Murani
- Institute of Genome Biology, Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany,*Correspondence: Eduard Murani,
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30
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Sekiya M. Proton Pumping ATPases: Rotational Catalysis, Physiological Roles in Oral Pathogenic Bacteria, and Inhibitors. Biol Pharm Bull 2022; 45:1404-1411. [PMID: 36184496 DOI: 10.1248/bpb.b22-00396] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Proton pumping ATPases, both F-type and V/A-type ATPases, generate ATP using electrochemical energy or pump protons/sodium ions by hydrolyzing ATP. The enzymatic reaction and proton transport are coupled through subunit rotation, and this unique rotational mechanism (rotational catalysis) has been intensively studied. Single-molecule and thermodynamic analyses have revealed the detailed rotational mechanism, including the catalytically inhibited state and the roles of subunit interactions. In mammals, F- and V-ATPases are involved in ATP synthesis and organelle acidification, respectively. Most bacteria, including anaerobes, have F- and/or A-ATPases in the inner membrane. However, these ATPases are not believed to be essential in anaerobic bacteria since anaerobes generate sufficient ATP without oxidative phosphorylation. Recent studies suggest that F- and A-ATPases perform indispensable functions beyond ATP synthesis in oral pathogenic anaerobes; F-ATPase is involved in acid tolerance in Streptococcus mutans, and A-ATPase mediates nutrient import in Porphyromonas gingivalis. Consistently, inhibitors of oral bacterial F- and A-ATPases, such as phytopolyphenols and bedaquiline, strongly diminish growth and survival. Herein, we discuss rotational catalysis of bacterial F- and A-ATPases, and discuss their physiological roles, focusing on oral bacteria. We also review the effects of ATPase inhibitors on the growth and survival of oral pathogenic bacteria. The features of the catalytic mechanism and unique physiological roles in oral bacteria highlight the potential for proton pumping ATPases to serve as targets for oral antimicrobial agents.
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Affiliation(s)
- Mizuki Sekiya
- Division of Biochemistry, School of Pharmacy, Iwate Medical University
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31
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Nakanishi-Matsui M, Matsumoto N. V-ATPase a3 Subunit in Secretory Lysosome Trafficking in Osteoclasts. Biol Pharm Bull 2022; 45:1426-1431. [PMID: 36184499 DOI: 10.1248/bpb.b22-00371] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Vacuolar-type ATPase (V-ATPase) shares its structure and rotational catalysis with F-type ATPase (F-ATPase, ATP synthase). However, unlike subunits of F-ATPase, those of V-ATPase have tissue- and/or organelle-specific isoforms. Structural diversity of V-ATPase generated by different combinations of subunit isoforms enables it to play diverse physiological roles in mammalian cells. Among these various roles, this review focuses on the functions of lysosome-specific V-ATPase in bone resorption by osteoclasts. Lysosomes remain in the cytoplasm in most cell types, but in osteoclasts, secretory lysosomes move toward and fuse with the plasma membrane to secrete lysosomal enzymes, which is essential for bone resorption. Through this process, lysosomal V-ATPase harboring the a3 isoform of the a subunit is relocated to the plasma membrane, where it transports protons from the cytosol to the cell exterior to generate the acidic extracellular conditions required for secreted lysosomal enzymes. In addition to this role as a proton pump, we recently found that the lysosomal a3 subunit of V-ATPase is essential for anterograde trafficking of secretory lysosomes. Specifically, a3 interacts with Rab7, a member of the Rab guanosine 5'-triphosphatase (GTPase) family that regulates organelle trafficking, and recruits it to the lysosomal membrane. These findings revealed the multifunctionality of lysosomal V-ATPase in osteoclasts; V-ATPase is responsible not only for the formation of the acidic environment by transporting protons, but also for intracellular trafficking of secretory lysosomes by recruiting organelle trafficking factors. Herein, we summarize the molecular mechanism underlying secretory lysosome trafficking in osteoclasts, and discuss the possible regulatory role of V-ATPase in organelle trafficking.
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Affiliation(s)
| | - Naomi Matsumoto
- Division of Biochemistry, School of Pharmacy, Iwate Medical University
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32
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Sun-Wada GH, Wada Y. Exploring the Link between Vacuolar-Type Proton ATPase and Epithelial Cell Polarity. Biol Pharm Bull 2022; 45:1419-1425. [PMID: 36184498 DOI: 10.1248/bpb.b22-00205] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Vacuolar-type H+-ATPase (V-ATPase) was first identified as an electrogenic proton pump that acidifies the lumen of intracellular organelles. Subsequently, it was observed that the proton pump also participates in the acidification of extracellular compartments. V-ATPase plays important roles in a wide range of cell biological processes and physiological functions by generating an acidic pH; therefore, it has attracted much attention not only in basic research but also in pathological and clinical aspects. Emerging evidence indicates that the luminal acidic endocytic organelles and their trafficking may function as important hubs that connect and coordinate various signaling pathways. Various pharmacological analyses have suggested that acidic endocytic organelles are important for the maintenance of cell polarity. Recently, several studies using genetic approaches have revealed the involvement of V-ATPase in the establishment and maintenance of apico-basal polarity. This review provides a brief overview of the relationship between the polarity of epithelial cells and V-ATPase as well as V-ATPase driven luminal acidification.
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Affiliation(s)
- Ge-Hong Sun-Wada
- Faculty of Pharmaceutical Sciences, Doshisha Women's College of Liberal Arts
| | - Yoh Wada
- Division of Biological Science, Institute of Scientific and Industrial Research, Osaka University
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33
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Qiu J, Ghaffari S. Mitochondrial Deep Dive into Hematopoietic Stem Cell Dormancy: Not Much Glycolysis but Plenty of Sluggish Lysosomes. Exp Hematol 2022; 114:1-8. [PMID: 35908627 PMCID: PMC9949493 DOI: 10.1016/j.exphem.2022.07.299] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2022] [Revised: 07/20/2022] [Accepted: 07/21/2022] [Indexed: 01/16/2023]
Abstract
The discovery of hematopoietic stem cells (HSCs) heterogeneity has had major implications for investigations of hematopoietic stem cell disorders, clonal hematopoiesis, and HSC aging. More recent studies of the heterogeneity of HSCs' organelles have begun to provide additional insights into HSCs' behavior with far-reaching ramifications for the mechanistic understanding of aging of HSCs and stem cell-derived diseases. Mitochondrial heterogeneity has been explored to expose HSC subsets with distinct properties and functions. Here we review some of the recent advances in these lines of studies that challenged the classic view of glycolysis in HSCs and led to the identification of lysosomes as dynamic pivotal switches in controlling HSC quiescence versus activation beyond their function in autophagy.
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Affiliation(s)
- Jiajing Qiu
- Department of Cell, Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY
| | - Saghi Ghaffari
- Department of Cell, Developmental & Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY; Developmental and Stem Cell Biology Multidisciplinary Training, Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY; Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY; Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY.
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34
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Proteomic Changes in Paspalum fasciculatum Leaves Exposed to Cd Stress. PLANTS 2022; 11:plants11192455. [PMID: 36235321 PMCID: PMC9573290 DOI: 10.3390/plants11192455] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/11/2022] [Revised: 09/03/2022] [Accepted: 09/05/2022] [Indexed: 11/17/2022]
Abstract
(1) Background: Cadmium is a toxic heavy metal that is widely distributed in water, soil, and air. It is present in agrochemicals, wastewater, battery waste, and volcanic eruptions. Thus, it can be absorbed by plants and enter the trophic chain. P. fasciculatum is a plant with phytoremediation capacity that can tolerate Cd stress, but changes in its proteome related to this tolerance have not yet been identified. (2) Methods: We conducted a quantitative analysis of the proteins present in P. fasciculatum leaves cultivated under greenhouse conditions in mining soils doped with 0 mg kg−1 (control), 30 mg kg−1, or 50 mg kg−1. This was carried out using the label-free shotgun proteomics technique. In this way, we determined the changes in the proteomes of the leaves of these plants, which allowed us to propose some tolerance mechanisms involved in the response to Cd stress. (3) Results: In total, 329 variable proteins were identified between treatments, which were classified into those associated with carbohydrate and energy metabolism; photosynthesis; structure, transport, and metabolism of proteins; antioxidant stress and defense; RNA and DNA processing; and signal transduction. (4) Conclusions: Based on changes in the differences in the leaf protein profiles between treatments, we hypothesize that some proteins associated with signal transduction (Ras-related protein RABA1e), HSPs (heat shock cognate 70 kDa protein 2), growth (actin-7), and cellular development (actin-1) are part of the tolerance response to Cd stress.
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35
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Francescutti CM, Martin A, Hanly JJ. Knockdowns of red Malphigian tubules reveal pigmentation roles in the milkweed bug. JOURNAL OF EXPERIMENTAL ZOOLOGY. PART B, MOLECULAR AND DEVELOPMENTAL EVOLUTION 2022; 338:382-387. [PMID: 35189035 DOI: 10.1002/jez.b.23123] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Revised: 01/25/2022] [Accepted: 02/03/2022] [Indexed: 06/14/2023]
Abstract
Classical Drosophila eye color mutations have unearthed a toolkit of genes that have permitted candidate gene studies of the outstanding diversity of coloration patterns in other insects. The gene underlying the eye color phenotypes of the red Malphigian tubules (red) fly mutant was mapped to a LysM domain gene of unknown molecular function. Here, we used RNAi to test the role of a red ortholog in the pigmentation of the milkweed bug Oncopeltus fasciatus, and contrast its effect with the ommochrome biosynthetic pathway gene vermilion (ver). Pigmentation was reduced in the cuticle of embryonic legs and first instar abdomens following parental RNAi against red, but not against ver, likely reflecting an effect on pterin biogenesis. Nymphal RNAi of red and ver both resulted in adult eye depigmentation, consistent with an effect on ommochrome content. These results suggest red loss-of-function impacts biochemically distinct types of pigments, and we discuss its putative role in the biogenesis of lysosome-related organelles such as ommochromasomes and pterinosomes.
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Affiliation(s)
- Caroline M Francescutti
- Department of Biological Sciences, The George Washington University, Washington, District of Columbia, USA
| | - Arnaud Martin
- Department of Biological Sciences, The George Washington University, Washington, District of Columbia, USA
| | - Joseph J Hanly
- Department of Biological Sciences, The George Washington University, Washington, District of Columbia, USA
- Smithsonian Tropical Research Institute, Gamboa, Panama
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36
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The a subunit isoforms of vacuolar-type proton ATPase exhibit differential distribution in mouse perigastrulation embryos. Sci Rep 2022; 12:13590. [PMID: 35948619 PMCID: PMC9365772 DOI: 10.1038/s41598-022-18002-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Accepted: 08/03/2022] [Indexed: 11/25/2022] Open
Abstract
Vacuolar-type H+-ATPases (V-ATPases) are large multi-subunit complexes that play critical roles in the acidification of a variety of intracellular or extracellular compartments. Mammalian cells contain four isoforms of the membrane integral subunit a (a1–a4); these isoforms contain the information necessary to target the enzyme to different cellular destinations. They are also involved in regulating the efficiency of ATP hydrolysis and proton transport. Previously, we showed that early embryogenesis requires V-ATPase function, and the luminal acidic endocytic and lysosomal compartments in the visceral endoderm of mouse embryos at the pre-gastrulation stage (E6.5) are essential for both nutrition and signal transduction during early embryogenesis. In this study, we examined the expression and distribution of a subunit isoforms in mouse embryos at E6.5. We found that all four isoforms expressed and exhibited differential distribution in the E6.5 embryo. At this developmental stage, the embryos establish highly elaborate endocytic compartments called apical vacuoles, on which the a3 isoform specifically accumulated.
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Marshansky V. Discovery and Study of Transmembrane Rotary Ion-Translocating Nano-Motors: F-ATPase/Synthase of Mitochondria/Bacteria and V-ATPase of Eukaryotic Cells. BIOCHEMISTRY. BIOKHIMIIA 2022; 87:702-719. [PMID: 36171652 DOI: 10.1134/s000629792208003x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/19/2022] [Revised: 07/08/2022] [Accepted: 07/08/2022] [Indexed: 06/16/2023]
Abstract
This review discusses the history of discovery and study of the operation of the two rotary ion-translocating ATPase nano-motors: (i) F-ATPase/synthase (holocomplex F1FO) of mitochondria/bacteria and (ii) eukaryotic V-ATPase (holocomplex V1VO). Vacuolar adenosine triphosphatase (V-ATPase) is a transmembrane multisubunit complex found in all eukaryotes from yeast to humans. It is structurally and functionally similar to the F-ATPase/synthase of mitochondria/bacteria and the A-ATPase/synthase of archaebacteria, which indicates a common evolutionary origin of the rotary ion-translocating nano-motors built into cell membranes and invented by Nature billions of years ago. Previously we have published several reviews on this topic with appropriate citations of our original research. This review is focused on the historical analysis of the discovery and study of transmembrane rotary ion-translocating ATPase nano-motors functioning in bacteria, eukaryotic cells and mitochondria of animals.
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38
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Shi L, Jian Y, Li M, Hao T, Yang C, Wang X. Filamin FLN-2 promotes MVB biogenesis by mediating vesicle docking on the actin cytoskeleton. J Cell Biol 2022; 221:e202201020. [PMID: 35575797 PMCID: PMC9115679 DOI: 10.1083/jcb.202201020] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2022] [Revised: 04/21/2022] [Accepted: 04/28/2022] [Indexed: 01/07/2023] Open
Abstract
Multivesicular bodies (MVBs) contain intralumenal vesicles that are delivered to lysosomes for degradation or released extracellularly for intercellular signaling. Here, we identified Caenorhabditis elegans filamin FLN-2 as a novel regulator of MVB biogenesis. FLN-2 co-localizes with V-ATPase subunits on MVBs, and the loss of FLN-2 affects MVB biogenesis, reducing the number of MVBs in C. elegans hypodermis. FLN-2 associates with actin filaments and is required for F-actin organization. Like fln-2(lf) mutation, inactivation of the V0 or V1 sector of V-ATPase or inhibition of actin polymerization impairs MVB biogenesis. Super-resolution imaging shows that FLN-2 docks V-ATPase-decorated MVBs onto actin filaments. FLN-2 interacts via its calponin-homology domains with F-actin and the V1-E subunit, VHA-8. Our data suggest that FLN-2 mediates the docking of MVBs on the actin cytoskeleton, which is required for MVB biogenesis.
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Affiliation(s)
- Leiling Shi
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- School of Life Sciences, Tsinghua University, Beijing, China
- National Institute of Biological Sciences, Beijing, China
| | - Youli Jian
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Meijiao Li
- State Key Laboratory of Conservation and Utilization of Bio-Resources in Yunnan, and Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
| | - Tianchao Hao
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Chonglin Yang
- State Key Laboratory of Conservation and Utilization of Bio-Resources in Yunnan, and Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, China
| | - Xiaochen Wang
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, China
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39
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Zeziulia M, Blin S, Schmitt FW, Lehmann M, Jentsch TJ. Proton-gated anion transport governs macropinosome shrinkage. Nat Cell Biol 2022; 24:885-895. [PMID: 35590106 PMCID: PMC9203271 DOI: 10.1038/s41556-022-00912-0] [Citation(s) in RCA: 34] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 04/05/2022] [Indexed: 01/25/2023]
Abstract
Intracellular organelles change their size during trafficking and maturation. This requires the transport of ions and water across their membranes. Macropinocytosis, a ubiquitous form of endocytosis of particular importance for immune and cancer cells, generates large vacuoles that can be followed optically. Shrinkage of macrophage macropinosomes depends on TPC-mediated Na+ efflux and Cl- exit through unknown channels. Relieving osmotic pressure facilitates vesicle budding, positioning osmotic shrinkage upstream of vesicular sorting and trafficking. Here we identify the missing macrophage Cl- channel as the proton-activated Cl- channel ASOR/TMEM206. ASOR activation requires Na+-mediated depolarization and luminal acidification by redundant transporters including H+-ATPases and CLC 2Cl-/H+ exchangers. As corroborated by mathematical modelling, feedback loops requiring the steep voltage and pH dependencies of ASOR and CLCs render vacuole resolution resilient towards transporter copy numbers. TMEM206 disruption increased albumin-dependent survival of cancer cells. Our work suggests a function for the voltage and pH dependence of ASOR and CLCs, provides a comprehensive model for ion-transport-dependent vacuole maturation and reveals biological roles of ASOR.
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Affiliation(s)
- Mariia Zeziulia
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
- Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany
- Graduate Program of the Freie Universität Berlin, Berlin, Germany
| | - Sandy Blin
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
- Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany
| | - Franziska W Schmitt
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
- Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany
- Graduate Program of the Humboldt Universität Berlin, Berlin, Germany
| | - Martin Lehmann
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
| | - Thomas J Jentsch
- Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany.
- Max-Delbrück-Centrum für Molekulare Medizin (MDC), Berlin, Germany.
- NeuroCure Cluster of Excellence, Charité Universitätsmedizin, Berlin, Germany.
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Matsumoto N, Sekiya M, Sun-Wada GH, Wada Y, Nakanishi-Matsui M. The lysosomal V-ATPase a3 subunit is involved in localization of Mon1-Ccz1, the GEF for Rab7, to secretory lysosomes in osteoclasts. Sci Rep 2022; 12:8455. [PMID: 35589873 PMCID: PMC9120031 DOI: 10.1038/s41598-022-12397-w] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2021] [Accepted: 05/10/2022] [Indexed: 11/16/2022] Open
Abstract
We have shown previously that the lysosomal a3 isoform of the a subunit of vacuolar-type ATPase (V-ATPase) interacts with inactive (GDP-bound form) Rab7, a small GTPase that regulates late endosome/lysosome trafficking, and that a3 recruits Rab7 to secretory lysosomes in mouse osteoclasts. This is essential for outward trafficking of secretory lysosomes and thus for bone resorption. However, the molecular mechanism underlying the recruitment of Rab7 by a3 remains to be fully elucidated. Here, we showed that a3 interacts with the Mon1A-Ccz1 complex, a guanine nucleotide exchange factor (GEF) for Rab7, using HEK293T cells. The interaction was mediated by the amino-terminal half domain of a3 and the longin motifs of Mon1A and Ccz1. Exogenous expression of the GEF promoted the interaction between a3 and Rab7. Mon1A mutants that interact inefficiently with Rab7 interacted with a3 at a similar level to wild-type Mon1A. Lysosomal localization of endogenous Ccz1 was abolished in osteoclasts lacking a3. These results suggest that the lysosomal a3 isoform of V-ATPase interacts with Mon1A-Ccz1, and that a3 is important for Mon1A-Ccz1 localization to secretory lysosomes, which mediates Rab7 recruitment to the organelle.
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Affiliation(s)
- Naomi Matsumoto
- Division of Biochemistry, School of Pharmacy, Iwate Medical University, Idaidori 1-1-1, Shiwa, Yahaba, Iwate, 028-3694, Japan
| | - Mizuki Sekiya
- Division of Biochemistry, School of Pharmacy, Iwate Medical University, Idaidori 1-1-1, Shiwa, Yahaba, Iwate, 028-3694, Japan
| | - Ge-Hong Sun-Wada
- Department of Biochemistry, Faculty of Pharmaceutical Sciences, Doshisha Women's College, Kyotanabe, Kyoto, 610-0395, Japan
| | - Yoh Wada
- Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, 567-0047, Japan
| | - Mayumi Nakanishi-Matsui
- Division of Biochemistry, School of Pharmacy, Iwate Medical University, Idaidori 1-1-1, Shiwa, Yahaba, Iwate, 028-3694, Japan.
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Schvartzman C, Fresia P, Murchio S, Mujica MV, Dalla-Rizza M. RNAi in Piezodorus guildinii (Hemiptera: Pentatomidae): Transcriptome Assembly for the Development of Pest Control Strategies. FRONTIERS IN PLANT SCIENCE 2022; 13:804839. [PMID: 35432425 PMCID: PMC9011191 DOI: 10.3389/fpls.2022.804839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Accepted: 02/28/2022] [Indexed: 06/14/2023]
Abstract
Red-banded stink bug Piezodorus guildinii (P. guildinii) has been described as the most damaging stink bug regarding soybean crops, leading to seed injury, low germination percentages, and foliar retention, at low population densities. In recent years, RNA interference (RNAi), a conserved eukaryote silencing mechanism has been explored to develop species-selective pesticides. In this work, we evaluated RNAi in P. guildinii to develop new pest-control strategies. For this, we assembled and annotated a P. guildinii transcriptome from a pool of all developmental stages. Analysis of this transcriptome led to the identification of 56 genes related to the silencing process encompassing siRNA, miRNA, and piRNA pathways. To evaluate the functionality of RNAi machinery, P. guildinii adults were injected with 28 ng/mg of body weight of double stranded RNA (dsRNA) targeting vATPase A. A mortality of 35 and 51.6% was observed after 7 and 14 days, respectively, and a downregulation of vATPase A gene of 84% 72 h post-injection. In addition, Dicer-2 and Argonaute-2 genes, core RNAi proteins, were upregulated 1.8-fold 48 h after injection. These findings showed for the first time that RNAi is functional in P. guildinii and the silencing of essential genes has a significant effect in adult viability. Taken together, the work reported here shows that RNAi could be an interesting approach for the development of red-banded stink bug control strategies.
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Affiliation(s)
- Claudia Schvartzman
- Unidad de Biotecnología, Instituto Nacional de Investigación Agropecuaria, Canelones, Uruguay
| | - Pablo Fresia
- Unidad Mixta Pasteur + INIA (UMPI), Institut Pasteur de Montevideo, Montevideo, Uruguay
| | - Sara Murchio
- Unidad de Biotecnología, Instituto Nacional de Investigación Agropecuaria, Canelones, Uruguay
| | - María Valentina Mujica
- Unidad de Protección Vegetal, Instituto Nacional de Investigación Agropecuaria, Canelones, Uruguay
| | - Marco Dalla-Rizza
- Unidad de Biotecnología, Instituto Nacional de Investigación Agropecuaria, Canelones, Uruguay
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42
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A review on lactoferrin as a proton pump inhibitor. Int J Biol Macromol 2022; 202:309-317. [PMID: 35038474 DOI: 10.1016/j.ijbiomac.2022.01.075] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Revised: 01/11/2022] [Accepted: 01/11/2022] [Indexed: 12/12/2022]
Abstract
Lactoferrin (Lf) is a versatile natural milk-derived protein that exhibits multiple interesting biological activities. Since it is safe for human administration and currently manufactured using low cost and well-established large-scale processes, the Lf scientific community has been devoted at dissecting its mechanisms of action towards its more rational and efficient use for various applications. Emerging literature has identified proton pumping ATPases as molecular targets of Lf in different cellular models linked to distinct activities of this natural protein. Information on this subject has not been systematically analysed before, hence herein we review the current state of art on the effect of Lf on proton pumping ATPases. Though structurally different, we propose that Lf holds a proton pump inhibitor (PPI)-like activity based on the functional resemblance with the classical inhibitors of the stomach H+/K+-ATPase. The downstream events and outcomes of the PPI-like activity of Lf, as well as its impact for the development of improved Lf applications are also discussed.
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43
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Maxson ME, Abbas YM, Wu JZ, Plumb JD, Grinstein S, Rubinstein JL. Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK. J Biophys Biochem Cytol 2022; 221:212963. [PMID: 35024770 PMCID: PMC8763849 DOI: 10.1083/jcb.202107174] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Revised: 12/14/2021] [Accepted: 12/21/2021] [Indexed: 12/11/2022] Open
Abstract
Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar H+ ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive or specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with Alexa Fluor 568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on their subcellular localization.
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Affiliation(s)
- Michelle E Maxson
- Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada
| | - Yazan M Abbas
- Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, Canada
| | - Jing Ze Wu
- Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada.,Department of Biochemistry, University of Toronto, Toronto, Canada
| | - Jonathan D Plumb
- Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada
| | - Sergio Grinstein
- Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada.,Department of Biochemistry, University of Toronto, Toronto, Canada
| | - John L Rubinstein
- Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, Canada.,Department of Medical Biophysics, University of Toronto, Toronto, Canada.,Department of Biochemistry, University of Toronto, Toronto, Canada
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44
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Lu J, Ma J, Hao Z, Li W. HPS6 Regulates the Biogenesis of Weibel–Palade Body in Endothelial Cells Through Trafficking v-ATPase to Its Limiting Membrane. Front Cell Dev Biol 2022; 9:743124. [PMID: 35252216 PMCID: PMC8891752 DOI: 10.3389/fcell.2021.743124] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2021] [Accepted: 12/22/2021] [Indexed: 11/13/2022] Open
Abstract
The Weibel–Palade body (WPB) is one of the lysosome-related organelles (LROs) in endothelial cells, whose main content is von Willebrand factor (vWF). The biogenesis of LROs is regulated by the Hermansky–Pudlak syndrome (HPS) protein-associated complexes through transporting cargo proteins to WPBs. Our previous studies have shown that HPS6, a subunit of BLOC-2 complex, is likely involved in the maturation of WPBs. However, the underlying mechanism remains unknown. In this study, we found that the knockdown of HPS6 in human umbilical vein endothelial cells (HUVECs) resulted in misshaped WPBs, decreased WPB number, and impaired vWF tubulation, which are similar to the characteristics of HPS6-deficient mouse endothelial cells. We observed similar morphological changes of WPBs in HUVECs after the knockdown of ATP6V0D1 (a subunit of v-ATPase). Furthermore, we found that HPS6 interacted with ATP6V0D1, suggesting that HPS6 transports ATP6V0D1 to the WPB limiting membrane for the assembly of the v-ATPase complex to maintain its acidic luminal pH, which is critical for the formation of vWF tubules during WPB maturation. In conclusion, HPS6 likely regulates the biogenesis of WPBs by participating in the trafficking of v-ATPase to the WPB membrane.
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45
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Khan MM, Lee S, Couoh‐Cardel S, Oot RA, Kim H, Wilkens S, Roh S. Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner. EMBO J 2022; 41:e109360. [PMID: 34918374 PMCID: PMC8804929 DOI: 10.15252/embj.2021109360] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2021] [Revised: 10/30/2021] [Accepted: 11/15/2021] [Indexed: 11/09/2022] Open
Abstract
The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V1 -ATPase and Vo proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted Vo and mutant V1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V1 to Vo provides sufficient free energy to overcome Vo autoinhibition. Moreover, the structures suggest that the unequal spacing of Vo 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V1 and Vo motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.
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Affiliation(s)
- Md. Murad Khan
- Department of Biochemistry and Molecular BiologySUNY Upstate Medical UniversitySyracuseNYUSA
| | - Seowon Lee
- School of Biological ScienceInstitute of Molecular Biology and GeneticsSeoul National UniversitySeoulSouth Korea
| | - Sergio Couoh‐Cardel
- Department of Biochemistry and Molecular BiologySUNY Upstate Medical UniversitySyracuseNYUSA
- Present address:
Department of Molecular and Cellular PhysiologyStanford UniversityStanfordCAUSA
| | - Rebecca A Oot
- Department of Biochemistry and Molecular BiologySUNY Upstate Medical UniversitySyracuseNYUSA
| | - Hyunmin Kim
- School of Biological ScienceInstitute of Molecular Biology and GeneticsSeoul National UniversitySeoulSouth Korea
| | - Stephan Wilkens
- Department of Biochemistry and Molecular BiologySUNY Upstate Medical UniversitySyracuseNYUSA
| | - Soung‐Hun Roh
- School of Biological ScienceInstitute of Molecular Biology and GeneticsSeoul National UniversitySeoulSouth Korea
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Portilla Y, Mulens-Arias V, Paradela A, Ramos-Fernández A, Pérez-Yagüe S, Morales MP, Barber DF. The surface coating of iron oxide nanoparticles drives their intracellular trafficking and degradation in endolysosomes differently depending on the cell type. Biomaterials 2022; 281:121365. [PMID: 35038611 DOI: 10.1016/j.biomaterials.2022.121365] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2021] [Revised: 12/15/2021] [Accepted: 01/04/2022] [Indexed: 12/13/2022]
Abstract
Magnetic nanoparticles (MNPs) are potential theranostic tools that are biodegraded through different endocytic pathways. However, little is known about the endolysosomal network through which MNPs transit and the influence of the surface coating in this process. Here, we studied the intracellular transit of two MNPs with identical iron oxide core size but with two distinct coatings: 3-aminopropyl-trietoxysilane (APS) and dimercaptosuccinic acid (DMSA). Using endolysosomal markers and a high throughput analysis of the associated proteome, we tracked the MNPs intracellularly in two different mouse cell lines, RAW264.7 (macrophages) and Pan02 (tumor cells). We did not detect differences in the MNP trafficking kinetics nor in the MNP-containing endolysosome phenotype in Pan02 cells. Nonetheless, DMSA-MNPs transited at slower rate than APS-MNPs in macrophages as measured by MNP accumulation in Rab7+ endolysosomes. Macrophage DMSA-MNP-containing endolysosomes had a higher percentage of lytic enzymes and catalytic proteins than their APS-MNP counterparts, concomitantly with a V-type ATPase enrichment, suggesting an acidic nature. Consequently, more autophagic vesicles are induced by DMSA-MNPs in macrophages, enhancing the expression of iron metabolism-related genes and proteins. Therefore, unlike Pan02 cells, the MNP coating appears to influence the intracellular trafficking rate and the endolysosome nature in macrophages. These results highlight how the MNP coating can determine the nanoparticle intracellular fate and biodegradation in a cell-type bias.
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Affiliation(s)
- Yadileiny Portilla
- Department of Immunology and Oncology and Nanobiomedicine Initiative, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049, Madrid, Spain
| | - Vladimir Mulens-Arias
- Department of Immunology and Oncology and Nanobiomedicine Initiative, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049, Madrid, Spain; Current address: Integrative Biomedical Materials and Nanomedicine Lab, Department of Experimental and Health Sciences (DCEXS), Pompeu Fabra University, PRBB, Carrer Doctor Aiguader 88, 08003, Barcelona, Spain
| | - Alberto Paradela
- Proteomics Facility, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049, Madrid, Spain
| | - Antonio Ramos-Fernández
- Proteomics Facility, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049, Madrid, Spain
| | - Sonia Pérez-Yagüe
- Department of Immunology and Oncology and Nanobiomedicine Initiative, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049, Madrid, Spain
| | - M Puerto Morales
- Department of Energy, Environment and Health, Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Sor Juana Inés de la Cruz 3, 28049, Madrid, Spain
| | - Domingo F Barber
- Department of Immunology and Oncology and Nanobiomedicine Initiative, Centro Nacional de Biotecnología (CNB-CSIC), Darwin 3, 28049, Madrid, Spain.
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Chen F, Kang R, Liu J, Tang D. The V-ATPases in cancer and cell death. Cancer Gene Ther 2022; 29:1529-1541. [PMID: 35504950 PMCID: PMC9063253 DOI: 10.1038/s41417-022-00477-y] [Citation(s) in RCA: 53] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Revised: 04/07/2022] [Accepted: 04/21/2022] [Indexed: 02/04/2023]
Abstract
Transmembrane ATPases are membrane-bound enzyme complexes and ion transporters that can be divided into F-, V-, and A-ATPases according to their structure. The V-ATPases, also known as H+-ATPases, are large multi-subunit protein complexes composed of a peripheral domain (V1) responsible for the hydrolysis of ATP and a membrane-integrated domain (V0) that transports protons across plasma membrane or organelle membrane. V-ATPases play a fundamental role in maintaining pH homeostasis through lysosomal acidification and are involved in modulating various physiological and pathological processes, such as macropinocytosis, autophagy, cell invasion, and cell death (e.g., apoptosis, anoikis, alkaliptosis, ferroptosis, and lysosome-dependent cell death). In addition to participating in embryonic development, V-ATPase pathways, when dysfunctional, are implicated in human diseases, such as neurodegenerative diseases, osteopetrosis, distal renal tubular acidosis, and cancer. In this review, we summarize the structure and regulation of isoforms of V-ATPase subunits and discuss their context-dependent roles in cancer biology and cell death. Updated knowledge about V-ATPases may enable us to design new anticancer drugs or strategies.
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Affiliation(s)
- Fangquan Chen
- grid.417009.b0000 0004 1758 4591DAMP Laboratory, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510120 China
| | - Rui Kang
- grid.267313.20000 0000 9482 7121Department of Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75390 USA
| | - Jiao Liu
- grid.417009.b0000 0004 1758 4591DAMP Laboratory, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510120 China
| | - Daolin Tang
- grid.267313.20000 0000 9482 7121Department of Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75390 USA
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48
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Yan T, Liu S, Xu J, Sun H, Yu S, Liu J. Unimolecular Helix-Based Transmembrane Nanochannel with a Smallest Luminal Cavity of 1 Å Expressing High Proton Selectivity and Transport Activity. NANO LETTERS 2021; 21:10462-10468. [PMID: 34860025 DOI: 10.1021/acs.nanolett.1c03858] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Natural protein channels have evolved with exquisite structures to transport ions selectively and rapidly. Learning from nature to construct biomimetic artificial channels is always challenging. Herein we present a unimolecular transmembrane proton channel by quinoline-derived helix, which exhibited highly selective and ultrafast proton transport behaviors. This helix-based channel possesses a small luminal cavity of 1 Å in diameter, which could efficiently reject the permeation of cations, anions or water molecules but only permits the translocation of protons owing to the size effect. The proton flow rate exceeded 107 H+ s-1 channel-1 and reached the same magnitude with gramicidin A. Mechanism investigation revealed that the directionally arrayed NH-chain inside the synthetic channel played a pivotal role during the proton flux. This work not only presented a helix-based channel with the smallest observable nanopore, but also unveiled an unexplored pathway for realizing efficient transport of protons via the consecutive NH-chain.
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Affiliation(s)
- Tengfei Yan
- College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, China
| | - Shengda Liu
- College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, China
| | - Jiayun Xu
- College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, China
| | - Hongcheng Sun
- College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, China
| | - Shuangjiang Yu
- College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, China
| | - Junqiu Liu
- College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, Hangzhou 311121, China
- College of Chemistry, State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130012, China
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An Uninvited Seat at the Dinner Table: How Apicomplexan Parasites Scavenge Nutrients from the Host. Microorganisms 2021; 9:microorganisms9122592. [PMID: 34946193 PMCID: PMC8707601 DOI: 10.3390/microorganisms9122592] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 12/10/2021] [Accepted: 12/13/2021] [Indexed: 12/24/2022] Open
Abstract
Obligate intracellular parasites have evolved a remarkable assortment of strategies to scavenge nutrients from the host cells they parasitize. Most apicomplexans form a parasitophorous vacuole (PV) within the invaded cell, a replicative niche within which they survive and multiply. As well as providing a physical barrier against host cell defense mechanisms, the PV membrane (PVM) is also an important site of nutrient uptake that is essential for the parasites to sustain their metabolism. This means nutrients in the extracellular milieu are separated from parasite metabolic machinery by three different membranes, the host plasma membrane, the PVM, and the parasite plasma membrane (PPM). In order to facilitate nutrient transport from the extracellular environment into the parasite itself, transporters on the host cell membrane of invaded cells can be modified by secreted and exported parasite proteins to maximize uptake of key substrates to meet their metabolic demand. To overcome the second barrier, the PVM, apicomplexan parasites secrete proteins contained in the dense granules that remodel the vacuole and make the membrane permissive to important nutrients. This bulk flow of host nutrients is followed by a more selective uptake of substrates at the PPM that is operated by specific transporters of this third barrier. In this review, we recapitulate and compare the strategies developed by Apicomplexa to scavenge nutrients from their hosts, with particular emphasis on transporters at the parasite plasma membrane and vacuolar solute transporters on the parasite intracellular digestive organelle.
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Niemuth NJ, Curtis BJ, Laudadio ED, Sostare E, Bennett EA, Neureuther NJ, Mohaimani AA, Schmoldt A, Ostovich ED, Viant MR, Hamers RJ, Klaper RD. Energy Starvation in Daphnia magna from Exposure to a Lithium Cobalt Oxide Nanomaterial. Chem Res Toxicol 2021; 34:2287-2297. [PMID: 34724609 DOI: 10.1021/acs.chemrestox.1c00189] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Growing evidence across organisms points to altered energy metabolism as an adverse outcome of metal oxide nanomaterial toxicity, with a mechanism of toxicity potentially related to the redox chemistry of processes involved in energy production. Despite this evidence, the significance of this mechanism has gone unrecognized in nanotoxicology due to the field's focus on oxidative stress as a universal─but nonspecific─nanotoxicity mechanism. To further explore metabolic impacts, we determined lithium cobalt oxide's (LCO's) effects on these pathways in the model organism Daphnia magna through global gene-expression analysis using RNA-Seq and untargeted metabolomics by direct-injection mass spectrometry. Our results show that a sublethal 1 mg/L 48 h exposure of D. magna to LCO nanosheets causes significant impacts on metabolic pathways versus untreated controls, while exposure to ions released over 48 h does not. Specifically, transcriptomic analysis using DAVID indicated significant enrichment (Benjamini-adjusted p ≤0.0.5) in LCO-exposed animals for changes in pathways involved in the cellular response to starvation (25 genes), mitochondrial function (70 genes), ATP-binding (70 genes), oxidative phosphorylation (53 genes), NADH dehydrogenase activity (12 genes), and protein biosynthesis (40 genes). Metabolomic analysis using MetaboAnalyst indicated significant enrichment (γ-adjusted p <0.1) for changes in amino acid metabolism (19 metabolites) and starch, sucrose, and galactose metabolism (7 metabolites). Overlap of significantly impacted pathways by RNA-Seq and metabolomics suggests amino acid breakdown and increased sugar import for energy production. Results indicate that LCO-exposed Daphnia respond to energy starvation by altering metabolic pathways, both at the gene expression and metabolite levels. These results support altered energy production as a sensitive nanotoxicity adverse outcome for LCO exposure and suggest negative impacts on energy metabolism as an important avenue for future studies of nanotoxicity, including for other biological systems and for metal oxide nanomaterials more broadly.
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Affiliation(s)
- Nicholas J Niemuth
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
| | - Becky J Curtis
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
| | - Elizabeth D Laudadio
- Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, Wisconsin 53706, United States
| | - Elena Sostare
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K
| | - Evan A Bennett
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
| | - Nicklaus J Neureuther
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
| | - Aurash A Mohaimani
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
| | - Angela Schmoldt
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
| | - Eric D Ostovich
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
| | - Mark R Viant
- School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K
| | - Robert J Hamers
- Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, Wisconsin 53706, United States
| | - Rebecca D Klaper
- School of Freshwater Sciences, University of Wisconsin-Milwaukee, 600 E Greenfield Ave., Milwaukee, Wisconsin 53204, United States
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