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1
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Pöschko P, Berrou CM, Pakari K, Ziegler MJ, Kern C, Koch B, Wittbrodt J, Wombacher R. Photoactivatable Plant Hormone-Based Chemical Inducers of Proximity for In Vivo Applications. ACS Chem Biol 2025; 20:332-339. [PMID: 39868662 PMCID: PMC11851429 DOI: 10.1021/acschembio.4c00592] [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: 09/06/2024] [Revised: 01/07/2025] [Accepted: 01/10/2025] [Indexed: 01/28/2025]
Abstract
Protein interactions play a crucial role in regulating cellular mechanisms, highlighting the need for effective methods to control these processes. In this regard, chemical inducers of proximity (CIPs) offer a promising approach to precisely manipulate protein-protein interactions in live cells and in vivo. In this study, we introduce pMandi, a photocaged version of the plant hormone-based CIP mandipropamid (Mandi), which allows the use of light as an external trigger to induce protein proximity in live mammalian cells. Furthermore, we present opabactin (OP) as a new plant hormone-based CIP that is effective in live mammalian cells at low nanomolar concentration and in live medaka embryos at submicromolar concentration. Its photocaged derivative, pOP, enables the induction of protein proximity upon light exposure in individual cells, enhancing spatiotemporal control to the level of single-cell resolution. Additionally, we explored the use of both photocaged CIPs to promote protein proximity in live medaka embryos.
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Affiliation(s)
- Philipp Pöschko
- Department
of Chemical Biology, Max Planck Institute
for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany
- Faculty
of Biosciences, Heidelberg University, Im Neuenheimer Feld 234, 69120 Heidelberg, Germany
| | - Caroline M. Berrou
- Department
of Chemical Biology, Max Planck Institute
for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany
| | - Kaisa Pakari
- Centre
for Organismal Studies Heidelberg (COS), Heidelberg University, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany
- Heidelberg
Biosciences International Graduate School (HBIGS), Heidelberg University, Im Neuenheimer Feld 501, 69120 Heidelberg, Germany
| | - Michael J. Ziegler
- Department
of Chemical Biology, Max Planck Institute
for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany
| | - Christoph Kern
- Department
of Chemical Biology, Max Planck Institute
for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany
| | - Birgit Koch
- Department
of Chemical Biology, Max Planck Institute
for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany
| | - Joachim Wittbrodt
- Centre
for Organismal Studies Heidelberg (COS), Heidelberg University, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany
| | - Richard Wombacher
- Department
of Chemical Biology, Max Planck Institute
for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany
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2
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Aldrete CA, An C, Call CC, Gao XJ, Vlahos AE. Perspectives on Synthetic Protein Circuits in Mammalian Cells. CURRENT OPINION IN BIOMEDICAL ENGINEERING 2024; 32:100555. [PMID: 39372446 PMCID: PMC11448451 DOI: 10.1016/j.cobme.2024.100555] [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] [Indexed: 10/08/2024]
Abstract
Mammalian synthetic biology aims to engineer cellular behaviors for therapeutic applications, such as enhancing immune cell efficacy against cancers or improving cell transplantation outcomes. Programming complex biological functions necessitates an understanding of molecular mechanisms governing cellular responses to stimuli. Traditionally, synthetic biology has focused on transcriptional circuits, but recent advances have led to the development of synthetic protein circuits, leveraging programmable binding, proteolysis, or phosphorylation to modulate protein interactions and cellular functions. These circuits offer advantages including robust performance, rapid functionality, and compact design, making them suitable for cellular engineering or gene therapies. This review outlines the post-translational toolkit, emphasizing synthetic protein components utilizing proteolysis or phosphorylation to program mammalian cell behaviors. Finally, we focus on key differences between rewiring native signaling pathways and creating orthogonal behaviors, alongside a proposed framework for translating synthetic protein circuits from tool development to pre-clinical applications in biomedicine.
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Affiliation(s)
- Carlos A. Aldrete
- Department of Chemical Engineering, Stanford University, CA, USA, 94305
| | - Connie An
- Department of Chemical Engineering, Stanford University, CA, USA, 94305
| | - Connor C. Call
- Department of Chemical Engineering, Stanford University, CA, USA, 94305
| | - Xiaojing J. Gao
- Department of Chemical Engineering, Stanford University, CA, USA, 94305
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3
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Tycko J, Van MV, Aradhana, DelRosso N, Ye H, Yao D, Valbuena R, Vaughan-Jackson A, Xu X, Ludwig C, Spees K, Liu K, Gu M, Khare V, Mukund AX, Suzuki PH, Arana S, Zhang C, Du PP, Ornstein TS, Hess GT, Kamber RA, Qi LS, Khalil AS, Bintu L, Bassik MC. Development of compact transcriptional effectors using high-throughput measurements in diverse contexts. Nat Biotechnol 2024:10.1038/s41587-024-02442-6. [PMID: 39487265 DOI: 10.1038/s41587-024-02442-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Accepted: 09/20/2024] [Indexed: 11/04/2024]
Abstract
Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.
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Affiliation(s)
- Josh Tycko
- Department of Genetics, Stanford University, Stanford, CA, USA
- Department of Neurobiology, Harvard Medical School, Boston, MA, USA
| | - Mike V Van
- Department of Biology, Stanford University, Stanford, CA, USA
| | - Aradhana
- Department of Genetics, Stanford University, Stanford, CA, USA
| | | | - Hanrong Ye
- Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA
| | - David Yao
- Department of Genetics, Stanford University, Stanford, CA, USA
| | | | - Alun Vaughan-Jackson
- Department of Genetics, Stanford University, Stanford, CA, USA
- Chan Zuckerberg Biohub-San Francisco, San Francisco, CA, USA
| | - Xiaoshu Xu
- Department of Bioengineering, Stanford University, Stanford, CA, USA
| | - Connor Ludwig
- Department of Bioengineering, Stanford University, Stanford, CA, USA
| | - Kaitlyn Spees
- Department of Genetics, Stanford University, Stanford, CA, USA
| | - Katherine Liu
- Department of Biology, Stanford University, Stanford, CA, USA
| | - Mingxin Gu
- Department of Genetics, Stanford University, Stanford, CA, USA
| | - Venya Khare
- Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA
| | | | - Peter H Suzuki
- Department of Bioengineering, Stanford University, Stanford, CA, USA
| | - Sophia Arana
- Department of Genetics, Stanford University, Stanford, CA, USA
| | - Catherine Zhang
- Department of Cancer Biology, Stanford University, Stanford, CA, USA
| | - Peter P Du
- Department of Cancer Biology, Stanford University, Stanford, CA, USA
| | - Thea S Ornstein
- Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA
| | - Gaelen T Hess
- Department of Biomolecular Chemistry and Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI, USA
| | - Roarke A Kamber
- Department of Genetics, Stanford University, Stanford, CA, USA
| | - Lei S Qi
- Chan Zuckerberg Biohub-San Francisco, San Francisco, CA, USA
- Department of Bioengineering, Stanford University, Stanford, CA, USA
- Sarafan ChEM-H, Stanford University, Stanford, CA, USA
| | - Ahmad S Khalil
- Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA
- Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA
| | - Lacramioara Bintu
- Department of Bioengineering, Stanford University, Stanford, CA, USA.
| | - Michael C Bassik
- Department of Genetics, Stanford University, Stanford, CA, USA.
- Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
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4
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Bansal A, Sharma M, Choudhury H. Generation of a new DiCre expressing parasite strain for functional characterization of Plasmodium falciparum genes in blood stages. Sci Rep 2024; 14:24076. [PMID: 39402380 PMCID: PMC11473785 DOI: 10.1038/s41598-024-75657-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 10/07/2024] [Indexed: 10/19/2024] Open
Abstract
Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria.
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Affiliation(s)
- Abhisheka Bansal
- Molecular Parasitology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India.
| | - Manish Sharma
- Molecular Parasitology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India
| | - Himashree Choudhury
- Molecular Parasitology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India
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5
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Iburg M, Anderson AP, Wong VT, Anton ED, He A, Lu GJ. Elucidating the assembly of gas vesicles by systematic protein-protein interaction analysis. EMBO J 2024; 43:4156-4172. [PMID: 39227754 PMCID: PMC11445434 DOI: 10.1038/s44318-024-00178-2] [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: 10/04/2023] [Revised: 05/31/2024] [Accepted: 06/19/2024] [Indexed: 09/05/2024] Open
Abstract
Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process of GV assembly remains elusive. To begin understanding this process, we have devised a high-throughput in vivo assay to determine the interactions of all 11 proteins in the pNL29 GV operon. Complete or partial deletions of the operon establish interdependent relationships among GV proteins during assembly. We also examine the tolerance of the GV assembly process to protein mutations and the cellular burdens caused by GV proteins. Clusters of GV protein interactions are revealed, proposing plausible protein complexes that are important for GV assembly. We anticipate our findings will set the stage for designing GVs that efficiently assemble in heterologous hosts during biomedical applications.
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Affiliation(s)
- Manuel Iburg
- Department of Bioengineering, Rice University, Houston, TX, 77005, USA
| | - Andrew P Anderson
- Department of Bioengineering, Rice University, Houston, TX, 77005, USA
| | - Vivian T Wong
- Department of Bioengineering, Rice University, Houston, TX, 77005, USA
| | - Erica D Anton
- Department of Bioengineering, Rice University, Houston, TX, 77005, USA
| | - Art He
- Department of Bioengineering, Rice University, Houston, TX, 77005, USA
| | - George J Lu
- Department of Bioengineering, Rice University, Houston, TX, 77005, USA.
- Department of BioSciences, Rice University, Houston, TX, 77005, USA.
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6
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Inobe T, Sakaguchi R, Obita T, Mukaiyama A, Koike S, Yokoyama T, Mizuguchi M, Akiyama S. Structural insights into rapamycin-induced oligomerization of a FRB-FKBP fusion protein. FEBS Lett 2024; 598:2292-2305. [PMID: 39031920 DOI: 10.1002/1873-3468.14986] [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: 03/07/2024] [Revised: 06/21/2024] [Accepted: 07/01/2024] [Indexed: 07/22/2024]
Abstract
Inducible dimerization systems, such as rapamycin-induced dimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain, are widely employed chemical biology tools to manipulate cellular functions. We previously advanced an inducible dimerization system into an inducible oligomerization system by developing a bivalent fusion protein, FRB-FKBP, which forms large oligomers upon rapamycin addition and can be used to manipulate cells. However, the oligomeric structure of FRB-FKBP remains unclear. Here, we report that FRB-FKBP forms a rotationally symmetric trimer in crystals, but a larger oligomer in solution, primarily tetramers and pentamers, which maintain similar inter-subunit contacts as in the crystal trimer. These findings expand the applications of the FRB-FKBP oligomerization system in diverse biological events.
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Affiliation(s)
- Tomonao Inobe
- Graduate School of Science and Engineering, University of Toyama, Japan
| | - Runa Sakaguchi
- Graduate School of Science and Engineering, University of Toyama, Japan
| | - Takayuki Obita
- Faculty of Pharmaceutical Sciences, University of Toyama, Japan
| | - Atushi Mukaiyama
- Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan
- Molecular Science Program, Graduate Institute for Advanced Studies, SOKENDAI, Okazaki, Japan
| | - Seiichi Koike
- Graduate School of Science and Engineering, University of Toyama, Japan
| | | | | | - Shuji Akiyama
- Research Center of Integrative Molecular Systems (CIMoS), Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan
- Molecular Science Program, Graduate Institute for Advanced Studies, SOKENDAI, Okazaki, Japan
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7
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Sun Y, Zhou R, Hu J, Feng S, Hu Q. Reversible control of kinase signaling through chemical-induced dephosphorylation. Commun Biol 2024; 7:1073. [PMID: 39217250 PMCID: PMC11366001 DOI: 10.1038/s42003-024-06771-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2023] [Accepted: 08/22/2024] [Indexed: 09/04/2024] Open
Abstract
The coordination between kinases and phosphatases is crucial for regulating the phosphorylation levels of essential signaling molecules. Methods enabling precise control of kinase activities are valuable for understanding the kinase functions and for developing targeted therapies. Here, we use the abscisic acid (ABA)-induced proximity system to reversibly control kinase signaling by recruiting phosphatases. Using this method, we found that the oncogenic tyrosine kinase BCR::ABL1 can be inhibited by recruiting various cytoplasmic phosphatases. We also discovered that the oncogenic serine/threonine kinase BRAF(V600E), which has been reported to bypass phosphorylation regulation, can be positively regulated by protein phosphatase 1 (PP1) and negatively regulated by PP5. Additionally, we observed that the dual-specificity kinase MEK1 can be inhibited by recruiting PP5. This suggests that bifunctional molecules capable of recruiting PP5 to MEK or RAF kinases could be promising anticancer drug candidates. Thus, the ABA-induced dephosphorylation method enables rapid screening of phosphatases to precisely control kinase signaling.
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Affiliation(s)
- Ying Sun
- Zhejiang University, Hangzhou, Zhejiang, China
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China
| | - Rihong Zhou
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China
| | - Jin Hu
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China
| | - Shan Feng
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China
| | - Qi Hu
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China.
- Westlake AI Therapeutics Lab, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China.
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8
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Dolberg TB, Gunnels TF, Ling T, Sarnese KA, Crispino JD, Leonard JN. Building Synthetic Biosensors Using Red Blood Cell Proteins. ACS Synth Biol 2024; 13:1273-1289. [PMID: 38536408 PMCID: PMC11536268 DOI: 10.1021/acssynbio.3c00754] [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] [Indexed: 04/09/2024]
Abstract
As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Toward addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC-protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including noninvasive monitoring of physiological signals for a range of diagnostic applications.
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Affiliation(s)
- Taylor B. Dolberg
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
| | - Taylor F. Gunnels
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
- Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA
| | - Te Ling
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Kelly A. Sarnese
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
| | - John D. Crispino
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Joshua N. Leonard
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
- Interdisciplinary Biological Sciences Training Program, Northwestern University, Evanston, IL, 60208, USA
- Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA
- Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208, USA
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9
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Wang X, Kang L, Kong D, Wu X, Zhou Y, Yu G, Dai D, Ye H. A programmable protease-based protein secretion platform for therapeutic applications. Nat Chem Biol 2024; 20:432-442. [PMID: 37872400 DOI: 10.1038/s41589-023-01433-z] [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: 01/19/2023] [Accepted: 09/02/2023] [Indexed: 10/25/2023]
Abstract
Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.
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Affiliation(s)
- Xinyi Wang
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Liping Kang
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Deqiang Kong
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Xin Wu
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Yang Zhou
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
- Wuhu Hospital, Health Science Center, East China Normal University, Wuhu City, China
| | - Guiling Yu
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Di Dai
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China
| | - Haifeng Ye
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Biomedical Synthetic Biology Research Center, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
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10
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Chakrabarty Y, Yang Z, Chen H, Chan DC. The HRI branch of the integrated stress response selectively triggers mitophagy. Mol Cell 2024; 84:1090-1100.e6. [PMID: 38340717 PMCID: PMC11062084 DOI: 10.1016/j.molcel.2024.01.016] [Citation(s) in RCA: 9] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Revised: 08/31/2023] [Accepted: 01/18/2024] [Indexed: 02/12/2024]
Abstract
To maintain mitochondrial homeostasis, damaged or excessive mitochondria are culled in coordination with the physiological state of the cell. The integrated stress response (ISR) is a signaling network that recognizes diverse cellular stresses, including mitochondrial dysfunction. Because the four ISR branches converge to common outputs, it is unclear whether mitochondrial stress detected by this network can regulate mitophagy, the autophagic degradation of mitochondria. Using a whole-genome screen, we show that the heme-regulated inhibitor (HRI) branch of the ISR selectively induces mitophagy. Activation of the HRI branch results in mitochondrial localization of phosphorylated eukaryotic initiation factor 2, which we show is sufficient to induce mitophagy. The HRI mitophagy pathway operates in parallel with the mitophagy pathway controlled by the Parkinson's disease related genes PINK1 and PARKIN and is mechanistically distinct. Therefore, HRI repurposes machinery that is normally used for translational initiation to trigger mitophagy in response to mitochondrial damage.
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Affiliation(s)
- Yogaditya Chakrabarty
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Zheng Yang
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Hsiuchen Chen
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - David C Chan
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
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11
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Shi Y, Bashian EE, Hou Y, Wu P. Chemical immunology: Recent advances in tool development and applications. Cell Chem Biol 2024; 31:S2451-9456(24)00080-1. [PMID: 38508196 PMCID: PMC11393185 DOI: 10.1016/j.chembiol.2024.02.006] [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: 09/01/2023] [Revised: 02/01/2024] [Accepted: 02/22/2024] [Indexed: 03/22/2024]
Abstract
Immunology was one of the first biological fields to embrace chemical approaches. The development of new chemical approaches and techniques has provided immunologists with an impressive arsenal of tools to address challenges once considered insurmountable. This review focuses on advances at the interface of chemistry and immunobiology over the past two decades that have not only opened new avenues in basic immunological research, but also revolutionized drug development for the treatment of cancer and autoimmune diseases. These include chemical approaches to understand and manipulate antigen presentation and the T cell priming process, to facilitate immune cell trafficking and regulate immune cell functions, and therapeutic applications of chemical approaches to disease control and treatment.
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Affiliation(s)
- Yujie Shi
- Department of Molecular and Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Eleanor E Bashian
- Department of Molecular and Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Yingqin Hou
- Department of Molecular and Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Peng Wu
- Department of Molecular and Cellular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
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12
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Bacsa B, Hopl V, Derler I. Synthetic Biology Meets Ca 2+ Release-Activated Ca 2+ Channel-Dependent Immunomodulation. Cells 2024; 13:468. [PMID: 38534312 PMCID: PMC10968988 DOI: 10.3390/cells13060468] [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: 02/05/2024] [Revised: 02/27/2024] [Accepted: 03/05/2024] [Indexed: 03/28/2024] Open
Abstract
Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor-ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.
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Affiliation(s)
- Bernadett Bacsa
- Division of Medical Physics und Biophysics, Medical University of Graz, A-8010 Graz, Austria;
| | - Valentina Hopl
- Institute of Biophysics, JKU Life Science Center, Johannes Kepler University Linz, A-4020 Linz, Austria;
| | - Isabella Derler
- Institute of Biophysics, JKU Life Science Center, Johannes Kepler University Linz, A-4020 Linz, Austria;
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13
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Cui M, Lee S, Ban SH, Ryu JR, Shen M, Yang SH, Kim JY, Choi SK, Han J, Kim Y, Han K, Lee D, Sun W, Kwon HB, Lee D. A single-component, light-assisted uncaging switch for endoproteolytic release. Nat Chem Biol 2024; 20:353-364. [PMID: 37973890 DOI: 10.1038/s41589-023-01480-6] [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: 05/06/2022] [Accepted: 10/12/2023] [Indexed: 11/19/2023]
Abstract
Proteases function as pivotal molecular switches, initiating numerous biological events. Notably, potyviral protease, derived from plant viruses, has emerged as a trusted proteolytic switch in synthetic biological circuits. To harness their capabilities, we have developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), by employing a circularly permutated tobacco etch virus protease and a blue-light-gated substrate, which are connected by fine-tuned intermodular linkers. As a single-component system, LAUNCHER exhibits a superior signal-to-noise ratio compared with multi-component systems, enabling precise and user-controllable release of payloads. This characteristic renders LAUNCHER highly suitable for diverse cellular applications, including transgene expression, tailored subcellular translocation and optochemogenetics. Additionally, the plug-and-play integration of LAUNCHER into existing synthetic circuits facilitates the enhancement of circuit performance. The demonstrated efficacy of LAUNCHER in improving existing circuitry underscores its significant potential for expanding its utilization in various applications.
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Affiliation(s)
- Mingguang Cui
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
| | - Seunghwan Lee
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
| | - Sung Hwan Ban
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
| | - Jae Ryun Ryu
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
| | - Meiying Shen
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
| | - Soo Hyun Yang
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
| | - Jin Young Kim
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
| | - Seul Ki Choi
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
| | - Jaemin Han
- Korea University College of Medicine, Seoul, Republic of Korea
| | - Yoonhee Kim
- Department of Neuroscience, Korea University College of Medicine, Seoul, Republic of Korea
| | - Kihoon Han
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
- Department of Neuroscience, Korea University College of Medicine, Seoul, Republic of Korea
| | - Donghun Lee
- Department of Physics, Korea University, Seoul, Republic of Korea
| | - Woong Sun
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea
| | - Hyung-Bae Kwon
- Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Dongmin Lee
- Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea.
- BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul, Republic of Korea.
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14
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Teixeira AP, Fussenegger M. Synthetic Gene Circuits for Regulation of Next-Generation Cell-Based Therapeutics. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2309088. [PMID: 38126677 PMCID: PMC10885662 DOI: 10.1002/advs.202309088] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/24/2023] [Indexed: 12/23/2023]
Abstract
Arming human cells with synthetic gene circuits enables to expand their capacity to execute superior sensing and response actions, offering tremendous potential for innovative cellular therapeutics. This can be achieved by assembling components from an ever-expanding molecular toolkit, incorporating switches based on transcriptional, translational, or post-translational control mechanisms. This review provides examples from the three classes of switches, and discusses their advantages and limitations to regulate the activity of therapeutic cells in vivo. Genetic switches designed to recognize internal disease-associated signals often encode intricate actuation programs that orchestrate a reduction in the sensed signal, establishing a closed-loop architecture. Conversely, switches engineered to detect external molecular or physical cues operate in an open-loop fashion, switching on or off upon signal exposure. The integration of such synthetic gene circuits into the next generation of chimeric antigen receptor T-cells is already enabling precise calibration of immune responses in terms of magnitude and timing, thereby improving the potency and safety of therapeutic cells. Furthermore, pre-clinical engineered cells targeting other chronic diseases are gathering increasing attention, and this review discusses the path forward for achieving clinical success. With synthetic biology at the forefront, cellular therapeutics holds great promise for groundbreaking treatments.
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Affiliation(s)
- Ana P. Teixeira
- Department of Biosystems Science and EngineeringETH ZurichKlingelbergstrasse 48BaselCH‐4056Switzerland
| | - Martin Fussenegger
- Department of Biosystems Science and EngineeringETH ZurichKlingelbergstrasse 48BaselCH‐4056Switzerland
- Faculty of ScienceUniversity of BaselKlingelbergstrasse 48BaselCH‐4056Switzerland
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15
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Beshay M, Deng Y, Janetopoulos C. Controlling the Subcellular Localization of Signaling Proteins Using Chemically Induced Dimerization and Optogenetics. Methods Mol Biol 2024; 2814:107-118. [PMID: 38954201 DOI: 10.1007/978-1-0716-3894-1_8] [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: 07/04/2024]
Abstract
A given protein can perform numerous roles in a cell with its participation in protein complexes and distinct localization within the cell playing a critical role in its diverse functions. Thus, the ability to artificially dimerize proteins and recruit proteins to specific locations in a cell has become a powerful tool for the investigation of protein function and the understanding of cell biology. Here, we discuss two systems that have been used to activate signal transduction pathways, a chemically inducible dimerization (CID) and a light-inducible (LI) system to control signaling and cytoskeletal regulation in a spatial and temporal manner.
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Affiliation(s)
- Mariam Beshay
- Department of Biological Sciences, University of the Sciences, Philadelphia, PA, USA
| | - Yu Deng
- Department of Cell Biology and Center for Cell Dynamics, School of Medicine, Johns Hopkins University, Baltimore, MD, USA
- Department of Chemical and Biomolecular Engineering, Whiting School of Engineering, Johns Hopkins University, Baltimore, MD, USA
| | - Chris Janetopoulos
- Department of Cell Biology, School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
- Total Experience Learning, Albright College, Reading, PA, USA.
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16
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Dolberg TB, Gunnels TF, Ling T, Sarnese KA, Crispino JD, Leonard JN. Building synthetic biosensors using red blood cell proteins. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.16.571988. [PMID: 38168174 PMCID: PMC10760168 DOI: 10.1101/2023.12.16.571988] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Towards addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including non-invasive monitoring of physiological signals for a range of diagnostic applications.
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Affiliation(s)
- Taylor B. Dolberg
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
| | - Taylor F. Gunnels
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
- Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA
| | - Te Ling
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Kelly A. Sarnese
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
| | - John D. Crispino
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN
| | - Joshua N. Leonard
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
- Interdisciplinary Biological Sciences Training Program, Northwestern University, Evanston, IL, 60208, USA
- Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA
- Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208, USA
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17
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Chin SE, Schindler C, Vinall L, Dodd RB, Bamber L, Legg S, Sigurdardottir A, Rees DG, Malcolm TIM, Spratley SJ, Granéli C, Sumner J, Tigue NJ. A simeprevir-inducible molecular switch for the control of cell and gene therapies. Nat Commun 2023; 14:7753. [PMID: 38012128 PMCID: PMC10682029 DOI: 10.1038/s41467-023-43484-9] [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: 11/15/2022] [Accepted: 11/09/2023] [Indexed: 11/29/2023] Open
Abstract
Chemical inducer of dimerization (CID) modules can be used effectively as molecular switches to control biological processes, and thus there is significant interest within the synthetic biology community in identifying novel CID systems. To date, CID modules have been used primarily in engineering cells for in vitro applications. To broaden their utility to the clinical setting, including the potential to control cell and gene therapies, the identification of novel CID modules should consider factors such as the safety and pharmacokinetic profile of the small molecule inducer, and the orthogonality and immunogenicity of the protein components. Here we describe a CID module based on the orally available, approved, small molecule simeprevir and its target, the NS3/4A protease from hepatitis C virus. We demonstrate the utility of this CID module as a molecular switch to control biological processes such as gene expression and apoptosis in vitro, and show that the CID system can be used to rapidly induce apoptosis in tumor cells in a xenograft mouse model, leading to complete tumor regression.
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Affiliation(s)
- Stacey E Chin
- Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
| | | | - Lisa Vinall
- Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
| | - Roger B Dodd
- Biologics Engineering, Oncology R&D, AstraZeneca, Cambridge, UK
| | - Lisa Bamber
- Biologics Engineering, Oncology R&D, AstraZeneca, Cambridge, UK
| | - Sandrine Legg
- Biologics Engineering, Oncology R&D, AstraZeneca, Cambridge, UK
| | | | - D Gareth Rees
- Biologics Engineering, Oncology R&D, AstraZeneca, Cambridge, UK
| | - Tim I M Malcolm
- Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
| | | | - Cecilia Granéli
- BioPharmaceuticals R&D Cell Therapy Department, Research and Early Development, Cardiovascular, Renal, and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - Jonathan Sumner
- Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK
| | - Natalie J Tigue
- Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, UK.
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18
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Komatsu S, Ohno H, Saito H. Target-dependent RNA polymerase as universal platform for gene expression control in response to intracellular molecules. Nat Commun 2023; 14:7256. [PMID: 37978180 PMCID: PMC10656481 DOI: 10.1038/s41467-023-42802-5] [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: 06/21/2023] [Accepted: 10/23/2023] [Indexed: 11/19/2023] Open
Abstract
Controlling gene expression in response to specific molecules is an essential technique for regulating cellular functions. However, current platforms with transcription and translation regulators have a limited number of detectable molecules to induce gene expression. Here to address these issues, we present a Target-dependent RNA polymerase (TdRNAP) that can induce RNA transcription in response to the intracellular target specifically recognized by single antibody. By substituting the fused antibody, we demonstrate that TdRNAPs respond to a wide variety of molecules, including peptides, proteins, RNA, and small molecules, and produce desired transcripts in human cells. Furthermore, we show that multiple TdRNAPs can construct orthogonal and multilayer genetic circuits. Finally, we apply TdRNAP to achieve cell-specific genome editing that is autonomously triggered by detecting the target gene product. TdRNAP can expand the molecular variety for controlling gene expression and provide the genetic toolbox for bioengineering and future therapeutic applications.
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Affiliation(s)
- Shodai Komatsu
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
- Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan
| | - Hirohisa Ohno
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Hirohide Saito
- Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
- Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
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19
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Seo KW, Kleiner RE. Profiling dynamic RNA-protein interactions using small-molecule-induced RNA editing. Nat Chem Biol 2023; 19:1361-1371. [PMID: 37349582 PMCID: PMC11048738 DOI: 10.1038/s41589-023-01372-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2022] [Accepted: 05/26/2023] [Indexed: 06/24/2023]
Abstract
RNA-binding proteins (RBPs) play an important role in biology, and characterizing dynamic RNA-protein interactions is essential for understanding RBP function. In this study, we developed targets of RBPs identified by editing induced through dimerization (TRIBE-ID), a facile strategy for quantifying state-specific RNA-protein interactions upon rapamycin-mediated chemically induced dimerization and RNA editing. We performed TRIBE-ID with G3BP1 and YBX1 to study RNA-protein interactions during normal conditions and upon oxidative stress-induced biomolecular condensate formation. We quantified editing kinetics to infer interaction persistence and show that stress granule formation strengthens pre-existing RNA-protein interactions and induces new RNA-protein binding events. Furthermore, we demonstrate that G3BP1 stabilizes its targets under normal and oxidative stress conditions independent of stress granule formation. Finally, we apply our method to characterize small-molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile dynamic RNA-protein interactions in cellular contexts with temporal control.
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Affiliation(s)
- Kyung W Seo
- Department of Chemistry, Princeton University, Princeton, NJ, USA
| | - Ralph E Kleiner
- Department of Chemistry, Princeton University, Princeton, NJ, USA.
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20
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Park R, Ongpipattanakul C, Nair SK, Bowers AA, Kuhlman B. Designer installation of a substrate recruitment domain to tailor enzyme specificity. Nat Chem Biol 2023; 19:460-467. [PMID: 36509904 PMCID: PMC10065947 DOI: 10.1038/s41589-022-01206-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2022] [Accepted: 10/10/2022] [Indexed: 12/14/2022]
Abstract
Promiscuous enzymes that modify peptides and proteins are powerful tools for labeling biomolecules; however, directing these modifications to desired substrates can be challenging. Here, we use computational interface design to install a substrate recognition domain adjacent to the active site of a promiscuous enzyme, catechol O-methyltransferase. This design approach effectively decouples substrate recognition from the site of catalysis and promotes modification of peptides recognized by the recruitment domain. We determined the crystal structure of this novel multidomain enzyme, SH3-588, which shows that it closely matches our design. SH3-588 methylates directed peptides with catalytic efficiencies exceeding the wild-type enzyme by over 1,000-fold, whereas peptides lacking the directing recognition sequence do not display enhanced efficiencies. In competition experiments, the designer enzyme preferentially modifies directed substrates over undirected substrates, suggesting that we can use designed recruitment domains to direct post-translational modifications to specific sequence motifs on target proteins in complex multisubstrate environments.
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Affiliation(s)
- Rodney Park
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA
| | - Chayanid Ongpipattanakul
- Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA
- School of Pharmacy, University of California San Francisco, San Francisco, CA, USA
| | - Satish K Nair
- Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA
- Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
- Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Albert A Bowers
- Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
| | - Brian Kuhlman
- Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA.
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
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21
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Bui S, Dancourt J, Lavieu G. Virus-Free Method to Control and Enhance Extracellular Vesicle Cargo Loading and Delivery. ACS APPLIED BIO MATERIALS 2023; 6:1081-1091. [PMID: 36781171 PMCID: PMC10031566 DOI: 10.1021/acsabm.2c00955] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/15/2023]
Abstract
Extracellular vesicles (EVs)─including exosomes and microvesicles─are involved in cell-cell communication. EVs encapsulate different types of molecules such as proteins or nucleotides and are long-lasting contenders for the establishment of personalized drug delivery systems. Recent studies suggest that the intrinsic capacities for uptake and cargo delivery of basic EVs might be too limited to serve as a potent delivery system. Here, we develop two synergistic methods to, respectively, control EV cargo loading and enhance EV cargo delivery through fusion without requirement for any viral fusogenic protein. Briefly, cargo loading is enabled through a reversible drug-inducible system that triggers the interaction between a cargo of interest and CD63, a well-established transmembrane EV marker. Enhanced cargo delivery is promoted by overexpressing Syncytin-1, an endogenous retrovirus envelop protein with fusogenic properties encoded by the human genome. We validate our bioengineered EVs in a qualitative and quantitative manner. Finally, we utilize this method to develop highly potent killer EVs, which contain a lethal toxin responsible for protein translation arrest and acceptor cell death. These advanced methods and future downstream applications may open promising doors in the manufacture of virus-free and EV-based delivery systems.
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Affiliation(s)
- Sheryl Bui
- INSERM U1316, CNRS UMR 7057, Université Paris Cité, 75006 Paris, France
| | - Julia Dancourt
- INSERM U1316, CNRS UMR 7057, Université Paris Cité, 75006 Paris, France
| | - Gregory Lavieu
- INSERM U1316, CNRS UMR 7057, Université Paris Cité, 75006 Paris, France
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22
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Characterization of an immune-evading doxycycline-inducible lentiviral vector for gene therapy in the spinal cord. Exp Neurol 2022; 355:114120. [DOI: 10.1016/j.expneurol.2022.114120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Revised: 05/12/2022] [Accepted: 05/17/2022] [Indexed: 11/18/2022]
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23
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Erdenee E, Ting AY. A Dual-Purpose Real-Time Indicator and Transcriptional Integrator for Calcium Detection in Living Cells. ACS Synth Biol 2022; 11:1086-1095. [PMID: 35254056 PMCID: PMC10395047 DOI: 10.1021/acssynbio.1c00597] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Calcium is a ubiquitous second messenger in eukaryotes, correlated with neuronal activity and T-cell activation among other processes. Real-time calcium indicators such as GCaMP have recently been complemented by newer calcium integrators that convert transient calcium activity into stable gene expression. Here we introduce LuCID, a dual-purpose real-time calcium indicator and transcriptional calcium integrator that combines the benefits of both calcium detection technologies. We show that the calcium-dependent split luciferase component of LuCID provides a real-time bioluminescence readout of calcium dynamics in cells, while the GI/FKF1 split GAL4 component of LuCID converts calcium-generated bioluminescence into stable gene expression. We also show that LuCID's modular design enables it to read out other cellular events such as protein-protein interactions. LuCID adds to the arsenal of tools for studying cells and cell populations that utilize calcium for signaling.
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Affiliation(s)
- Elbegduuren Erdenee
- Department of Biology, Stanford University, Stanford, California 94305, United States
| | - Alice Y. Ting
- Department of Biology, Stanford University, Stanford, California 94305, United States
- Department of Genetics, Stanford University, Stanford, California 94305, United States
- Department of Chemistry, Stanford University, Stanford, California 94305, United States
- Chan Zuckerberg Biohub, San Francisco, California 94158, United States
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24
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Singh AK, Saharan K, Baral S, Luan S, Vasudevan D. Crystal packing reveals rapamycin-mediated homodimerization of an FK506-binding domain. Int J Biol Macromol 2022; 206:670-680. [PMID: 35218805 DOI: 10.1016/j.ijbiomac.2022.02.107] [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: 12/01/2021] [Revised: 02/16/2022] [Accepted: 02/17/2022] [Indexed: 11/28/2022]
Abstract
Chemically induced dimerization (CID) is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes. The induced heterodimerization of FKBP fusion proteins by rapamycin and FK506 has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes. Here, we report the crystal structure of the AtFKBP53 FKBD in complex with rapamycin. The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD. The triene arm of rapamycin appears to play a significant role in forming this dimer. This forms the first structural report of rapamycin-mediated homodimerization of an FKBP. The structural information on the rapamycin-mediated FKBD dimerization may be employed to design and synthesize covalently linked dimeric rapamycin, which may subsequently serve as a chemically induced dimerization system for the regulation and characterization of cellular processes.
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Affiliation(s)
- Ajit Kumar Singh
- Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India
| | - Ketul Saharan
- Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India; Regional Centre for Biotechnology, Faridabad 121001, India
| | - Somanath Baral
- Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India; School of Biotechnology, KIIT University, Bhubaneswar 751024, India
| | - Sheng Luan
- Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720, USA
| | - Dileep Vasudevan
- Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India.
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25
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Zhu D, Johnson HJ, Chen J, Schaffer DV. Optogenetic Application to Investigating Cell Behavior and Neurological Disease. Front Cell Neurosci 2022; 16:811493. [PMID: 35273478 PMCID: PMC8902366 DOI: 10.3389/fncel.2022.811493] [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: 11/08/2021] [Accepted: 01/04/2022] [Indexed: 11/13/2022] Open
Abstract
Cells reside in a dynamic microenvironment that presents them with regulatory signals that vary in time, space, and amplitude. The cell, in turn, interprets these signals and accordingly initiates downstream processes including cell proliferation, differentiation, migration, and self-organization. Conventional approaches to perturb and investigate signaling pathways (e.g., agonist/antagonist addition, overexpression, silencing, knockouts) are often binary perturbations that do not offer precise control over signaling levels, and/or provide limited spatial or temporal control. In contrast, optogenetics leverages light-sensitive proteins to control cellular signaling dynamics and target gene expression and, by virtue of precise hardware control over illumination, offers the capacity to interrogate how spatiotemporally varying signals modulate gene regulatory networks and cellular behaviors. Recent studies have employed various optogenetic systems in stem cell, embryonic, and somatic cell patterning studies, which have addressed fundamental questions of how cell-cell communication, subcellular protein localization, and signal integration affect cell fate. Other efforts have explored how alteration of signaling dynamics may contribute to neurological diseases and have in the process created physiologically relevant models that could inform new therapeutic strategies. In this review, we focus on emerging applications within the expanding field of optogenetics to study gene regulation, cell signaling, neurodevelopment, and neurological disorders, and we comment on current limitations and future directions for the growth of the field.
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Affiliation(s)
- Danqing Zhu
- California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, United States
| | - Hunter J. Johnson
- Department of Bioengineering, University of California, Berkeley, Berkeley, CA, United States
- Graduate Program in Bioengineering, University of California, San Francisco, San Francisco, CA, United States
- Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, CA, United States
| | - Jun Chen
- California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, United States
| | - David V. Schaffer
- California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, United States
- Department of Bioengineering, University of California, Berkeley, Berkeley, CA, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, United States
- Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, United States
- Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, United States
- *Correspondence: David V. Schaffer
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26
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Dang DT. Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry. Front Chem 2022; 10:829312. [PMID: 35211456 PMCID: PMC8861298 DOI: 10.3389/fchem.2022.829312] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2021] [Accepted: 01/14/2022] [Indexed: 11/17/2022] Open
Abstract
Protein dimerization plays a key role in many biological processes. Most cellular events such as enzyme activation, transcriptional cofactor recruitment, signal transduction, and even pathogenic pathways are significantly regulated via protein-protein interactions. Understanding and controlling the molecular mechanisms that regulate protein dimerization is crucial for biomedical applications. The limitations of engineered protein dimerization provide an opportunity for molecular chemistry to induce dimerization of protein in biological events. In this review, molecular control over dimerization of protein and activation in this respect are discussed. The well known molecule glue-based approaches to induced protein dimerization provide powerful tools to modulate the functionality of dimerized proteins and are shortly highlighted. Subsequently metal ion, nucleic acid and host-guest chemistry are brought forward as novel approaches for orthogonal control over dimerization of protein. The specific focus of the review will be on host-guest systems as novel, robust and versatile supramolecular approaches to modulate the dimerization of proteins, using functional proteins as model systems.
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Affiliation(s)
- Dung Thanh Dang
- Faculty of Biotechnology, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam
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27
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Ziegler MJ, Yserentant K, Dunsing V, Middel V, Gralak AJ, Pakari K, Bargstedt J, Kern C, Petrich A, Chiantia S, Strähle U, Herten DP, Wombacher R. Mandipropamid as a chemical inducer of proximity for in vivo applications. Nat Chem Biol 2022; 18:64-69. [PMID: 34934192 PMCID: PMC8709788 DOI: 10.1038/s41589-021-00922-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2020] [Accepted: 10/13/2021] [Indexed: 11/26/2022]
Abstract
Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.
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Affiliation(s)
- Michael J Ziegler
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
- Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany
| | - Klaus Yserentant
- Institute of Physical Chemistry, Heidelberg University, Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
- Institute of Cardiovascular Sciences & School of Chemistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
| | - Valentin Dunsing
- Institute of Biology and Biochemistry, University of Potsdam, Potsdam, Germany
| | - Volker Middel
- Institute of Biological and Chemical Systems (IBCS)-Biological Information Processing (BIP), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany
| | - Antoni J Gralak
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
- Institute of Physical Chemistry, Heidelberg University, Heidelberg, Germany
| | - Kaisa Pakari
- Institute of Physical Chemistry, Heidelberg University, Heidelberg, Germany
| | - Jörn Bargstedt
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
| | - Christoph Kern
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
- Institute of Physical Chemistry, Heidelberg University, Heidelberg, Germany
| | - Annett Petrich
- Institute of Biology and Biochemistry, University of Potsdam, Potsdam, Germany
| | - Salvatore Chiantia
- Institute of Biology and Biochemistry, University of Potsdam, Potsdam, Germany
| | - Uwe Strähle
- Institute of Biological and Chemical Systems (IBCS)-Biological Information Processing (BIP), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany
| | - Dirk-Peter Herten
- Institute of Physical Chemistry, Heidelberg University, Heidelberg, Germany
- Institute of Cardiovascular Sciences & School of Chemistry, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
- Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, UK
| | - Richard Wombacher
- Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.
- Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany.
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28
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IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish. Commun Biol 2021; 4:1405. [PMID: 34916605 PMCID: PMC8677817 DOI: 10.1038/s42003-021-02923-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2021] [Accepted: 11/24/2021] [Indexed: 11/08/2022] Open
Abstract
Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.
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29
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Courtney TM, Hankinson CP, Horst TJ, Deiters A. Targeted protein oxidation using a chromophore-modified rapamycin analog. Chem Sci 2021; 12:13425-13433. [PMID: 34777761 PMCID: PMC8528027 DOI: 10.1039/d1sc04464h] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Accepted: 08/30/2021] [Indexed: 01/23/2023] Open
Abstract
Chemically induced dimerization of FKBP and FRB using rapamycin and rapamycin analogs has been utilized in a variety of biological applications. Formation of the FKBP-rapamycin-FRB ternary complex is typically used to activate a biological process and this interaction has proven to be essentially irreversible. In many cases, it would be beneficial to also have temporal control over deactivating a biological process once it has been initiated. Thus, we developed the first reactive oxygen species-generating rapamycin analog toward this goal. The BODIPY-rapamycin analog BORap is capable of dimerizing FKBP and FRB to form a ternary complex, and upon irradiation with 530 nm light, generates singlet oxygen to oxidize and inactivate proteins of interest fused to FKBP/FRB.
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Affiliation(s)
- Taylor M Courtney
- Department of Chemistry, University of Pittsburgh Pittsburgh PA 15260 USA
| | | | - Trevor J Horst
- Department of Chemistry, University of Pittsburgh Pittsburgh PA 15260 USA
| | - Alexander Deiters
- Department of Chemistry, University of Pittsburgh Pittsburgh PA 15260 USA
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30
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Sahillioglu AC, Schumacher TN. Safety switches for adoptive cell therapy. Curr Opin Immunol 2021; 74:190-198. [PMID: 34389174 DOI: 10.1016/j.coi.2021.07.002] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Revised: 07/01/2021] [Accepted: 07/05/2021] [Indexed: 12/13/2022]
Abstract
Adoptive transfer of allogeneic and genetically modified T cells, such as CAR-T and TCR-T cells, can induce profound immune reactivity against cancer tissue. At the same time, these therapies are associated with severe off-target and on-target toxicities. For this reason, the development of genetic safety switches that can be used to control the activity of T cells in vivo has become an active field of research. With the spectrum of technologies developed, reversible control of cell products either by supply or removal of small molecules, by supply of protein-based regulators, or by physical stimuli such as light, ultrasound or heat, has become feasible. In this review, we describe the mechanistic classes of genetic safety switches, such as transcription-based or protein-based control of antigen receptors, split receptors, small molecule responsive antibodies, as well as universal remote controls, and discuss their advantages and limitations.
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Affiliation(s)
- Ali Can Sahillioglu
- Division of Molecular Oncology & Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Ton N Schumacher
- Division of Molecular Oncology & Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
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31
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Stroppel AS, Lappalainen R, Stafforst T. Controlling Site-Directed RNA Editing by Chemically Induced Dimerization. Chemistry 2021; 27:12300-12304. [PMID: 34169589 PMCID: PMC8456898 DOI: 10.1002/chem.202101985] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2021] [Indexed: 11/24/2022]
Abstract
Various RNA‐targeting approaches have been engineered to modify specific sites on endogenous transcripts, breaking new ground for a variety of basic research tools and promising clinical applications in the future. Here, we combine site‐directed adenosine‐to‐inosine RNA editing with chemically induced dimerization. Specifically, we achieve tight and dose‐dependent control of the editing reaction with gibberellic acid, and obtain editing yields up to 20 % and 44 % in the endogenous STAT1 and GAPDH transcript in cell culture. Furthermore, the disease‐relevant MECP2 R106Q mutation was repaired with editing yields up to 42 %. The introduced principle will enable new applications where temporal or spatiotemporal control of an RNA‐targeting mechanism is desired.
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Affiliation(s)
- Anna S Stroppel
- Interfaculty Institute of Biochemistry, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany
| | - Ruth Lappalainen
- Interfaculty Institute of Biochemistry, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany
| | - Thorsten Stafforst
- Interfaculty Institute of Biochemistry, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany
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32
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33
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Wagner HJ, Mohsenin H, Weber W. Synthetic Biology-Empowered Hydrogels for Medical Diagnostics. ADVANCES IN BIOCHEMICAL ENGINEERING/BIOTECHNOLOGY 2021; 178:197-226. [PMID: 33582837 DOI: 10.1007/10_2020_158] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/10/2023]
Abstract
Synthetic biology is strongly inspired by concepts of engineering science and aims at the design and generation of artificial biological systems in different fields of research such as diagnostics, analytics, biomedicine, or chemistry. To this aim, synthetic biology uses an engineering approach relying on a toolbox of molecular sensors and switches that endows cellular hosts with non-natural computing functions and circuits. Importantly, this concept is not only limited to cellular approaches. Synthetic biological building blocks have also conferred sensing and switching capability to otherwise inactive materials. This principle has attracted high interest for the development of biohybrid materials capable of sensing and responding to specific molecular stimuli, such as disease biomarkers, antibiotics, or heavy metals. Moreover, the interconnection of individual sense-and-respond materials to complex materials systems has enabled the processing of, for example, multiple inputs or the amplification of signals using feedback topologies. Such systems holding high potential for applications in the analytical and diagnostic sectors will be described in this chapter.
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Affiliation(s)
- Hanna J Wagner
- Faculty of Biology, Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg im Breisgau, Germany.,Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland
| | - Hasti Mohsenin
- Faculty of Biology, Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg im Breisgau, Germany
| | - Wilfried Weber
- Faculty of Biology, Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg im Breisgau, Germany.
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34
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Hsieh TS, Lopez VA, Black MH, Osinski A, Pawłowski K, Tomchick DR, Liou J, Tagliabracci VS. Dynamic remodeling of host membranes by self-organizing bacterial effectors. Science 2021; 372:935-941. [PMID: 33927055 PMCID: PMC8543759 DOI: 10.1126/science.aay8118] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 02/17/2021] [Accepted: 04/14/2021] [Indexed: 01/09/2023]
Abstract
During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.
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Affiliation(s)
- Ting-Sung Hsieh
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Victor A Lopez
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Miles H Black
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Adam Osinski
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Krzysztof Pawłowski
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Department of Biochemistry and Microbiology, Institute of Biology, Warsaw University of Life Sciences, Warsaw 02-776, Poland
| | - Diana R Tomchick
- Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jen Liou
- Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Vincent S Tagliabracci
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
- Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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35
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Michalec-Wawiórka B, Czapiński J, Filipek K, Rulak P, Czerwonka A, Tchórzewski M, Rivero-Müller A. An Improved Vector System for Homogeneous and Stable Gene Regulation. Int J Mol Sci 2021; 22:ijms22105206. [PMID: 34069024 PMCID: PMC8157167 DOI: 10.3390/ijms22105206] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 04/24/2021] [Accepted: 05/08/2021] [Indexed: 11/16/2022] Open
Abstract
Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.
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Affiliation(s)
- Barbara Michalec-Wawiórka
- Department of Molecular Biology, Institute of Biological Sciences, Maria Curie-Skłodowska University, 20-033 Lublin, Poland; (K.F.); (P.R.); (M.T.)
- Correspondence:
| | - Jakub Czapiński
- Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093 Lublin, Poland; (J.C.); (A.C.); (A.R.-M.)
- Postgraduate School of Molecular Medicine, 02-091 Warsaw, Poland
| | - Kamil Filipek
- Department of Molecular Biology, Institute of Biological Sciences, Maria Curie-Skłodowska University, 20-033 Lublin, Poland; (K.F.); (P.R.); (M.T.)
| | - Patrycja Rulak
- Department of Molecular Biology, Institute of Biological Sciences, Maria Curie-Skłodowska University, 20-033 Lublin, Poland; (K.F.); (P.R.); (M.T.)
| | - Arkadiusz Czerwonka
- Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093 Lublin, Poland; (J.C.); (A.C.); (A.R.-M.)
| | - Marek Tchórzewski
- Department of Molecular Biology, Institute of Biological Sciences, Maria Curie-Skłodowska University, 20-033 Lublin, Poland; (K.F.); (P.R.); (M.T.)
| | - Adolfo Rivero-Müller
- Department of Biochemistry and Molecular Biology, Medical University of Lublin, 20-093 Lublin, Poland; (J.C.); (A.C.); (A.R.-M.)
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36
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Zárate RV, Arancibia D, Fernández A, Signorelli JR, Larrondo LF, Andrés ME, Zamorano P. Optimization of the Light-On system in a lentiviral platform to a light-controlled expression of genes in neurons. ELECTRON J BIOTECHN 2021. [DOI: 10.1016/j.ejbt.2021.03.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
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37
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Wagner HJ, Weber W, Fussenegger M. Synthetic Biology: Emerging Concepts to Design and Advance Adeno-Associated Viral Vectors for Gene Therapy. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:2004018. [PMID: 33977059 PMCID: PMC8097373 DOI: 10.1002/advs.202004018] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 12/18/2020] [Indexed: 05/28/2023]
Abstract
Three recent approvals and over 100 ongoing clinical trials make adeno-associated virus (AAV)-based vectors the leading gene delivery vehicles in gene therapy. Pharmaceutical companies are investing in this small and nonpathogenic gene shuttle to increase the therapeutic portfolios within the coming years. This prospect of marking a new era in gene therapy has fostered both investigations of the fundamental AAV biology as well as engineering studies to enhance delivery vehicles. Driven by the high clinical potential, a new generation of synthetic-biologically engineered AAV vectors is on the rise. Concepts from synthetic biology enable the control and fine-tuning of vector function at different stages of cellular transduction and gene expression. It is anticipated that the emerging field of synthetic-biologically engineered AAV vectors can shape future gene therapeutic approaches and thus the design of tomorrow's gene delivery vectors. This review describes and discusses the recent trends in capsid and vector genome engineering, with particular emphasis on synthetic-biological approaches.
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Affiliation(s)
- Hanna J. Wagner
- Department of Biosystems Science and EngineeringETH ZurichMattenstrasse 26Basel4058Switzerland
- Faculty of BiologyUniversity of FreiburgSchänzlestraße 1Freiburg79104Germany
- Signalling Research Centres BIOSS and CIBSSUniversity of FreiburgSchänzlestraße 18Freiburg79104Germany
| | - Wilfried Weber
- Faculty of BiologyUniversity of FreiburgSchänzlestraße 1Freiburg79104Germany
- Signalling Research Centres BIOSS and CIBSSUniversity of FreiburgSchänzlestraße 18Freiburg79104Germany
| | - Martin Fussenegger
- Department of Biosystems Science and EngineeringETH ZurichMattenstrasse 26Basel4058Switzerland
- Faculty of ScienceUniversity of BaselKlingelbergstrasse 50Basel4056Switzerland
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38
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Rodriques SG, Chen LM, Liu S, Zhong ED, Scherrer JR, Boyden ES, Chen F. RNA timestamps identify the age of single molecules in RNA sequencing. Nat Biotechnol 2021; 39:320-325. [PMID: 33077959 PMCID: PMC7956158 DOI: 10.1038/s41587-020-0704-z] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 08/27/2020] [Accepted: 09/07/2020] [Indexed: 12/28/2022]
Abstract
Current approaches to single-cell RNA sequencing (RNA-seq) provide only limited information about the dynamics of gene expression. Here we present RNA timestamps, a method for inferring the age of individual RNAs in RNA-seq data by exploiting RNA editing. To introduce timestamps, we tag RNA with a reporter motif consisting of multiple MS2 binding sites that recruit the adenosine deaminase ADAR2 fused to an MS2 capsid protein. ADAR2 binding to tagged RNA causes A-to-I edits to accumulate over time, allowing the age of the RNA to be inferred with hour-scale accuracy. By combining observations of multiple timestamped RNAs driven by the same promoter, we can determine when the promoter was active. We demonstrate that the system can infer the presence and timing of multiple past transcriptional events. Finally, we apply the method to cluster single cells according to the timing of past transcriptional activity. RNA timestamps will allow the incorporation of temporal information into RNA-seq workflows.
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Affiliation(s)
- Samuel G Rodriques
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of Harvard and MIT, Cambridge, MA, USA
| | - Linlin M Chen
- Broad Institute of Harvard and MIT, Cambridge, MA, USA
| | - Sophia Liu
- Biophysics Program, Harvard University, Boston, MA, USA
- Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Ellen D Zhong
- Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Joseph R Scherrer
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Edward S Boyden
- Department of Media Arts and Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
- MIT McGovern Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Koch Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Howard Hughes Medical Institute, Cambridge, MA, USA.
| | - Fei Chen
- Broad Institute of Harvard and MIT, Cambridge, MA, USA.
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.
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39
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Braga L, Ali H, Secco I, Giacca M. Non-coding RNA therapeutics for cardiac regeneration. Cardiovasc Res 2021; 117:674-693. [PMID: 32215566 PMCID: PMC7898953 DOI: 10.1093/cvr/cvaa071] [Citation(s) in RCA: 62] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/27/2019] [Revised: 03/02/2020] [Accepted: 03/20/2020] [Indexed: 12/19/2022] Open
Abstract
A growing body of evidence indicates that cardiac regeneration after myocardial infarction can be achieved by stimulating the endogenous capacity of cardiomyocytes (CMs) to replicate. This process is controlled, both positively and negatively, by a large set of non-coding RNAs (ncRNAs). Some of the microRNAs (miRNAs) that can stimulate CM proliferation is expressed in embryonic stem cells and is required to maintain pluripotency (e.g. the miR-302∼367 cluster). Others also govern the proliferation of different cell types, including cancer cells (e.g. the miR-17∼92 cluster). Additional miRNAs were discovered through systematic screenings (e.g. miR-199a-3p and miR-590-3p). Several miRNAs instead suppress CM proliferation and are involved in the withdrawal of CMs from the cell cycle after birth (e.g. the let-7 and miR-15 families). Similar regulatory roles on CM proliferation are also exerted by a few long ncRNAs. This body of information has obvious therapeutic implications, as miRNAs with activator function or short antisense oligonucleotides against inhibitory miRNAs or lncRNAs can be administered to stimulate cardiac regeneration. Expression of miRNAs can be achieved by gene therapy using adeno-associated vectors, which transduce CMs with high efficiency. More effective and safer for therapeutic purposes, small nucleic acid therapeutics can be obtained as chemically modified, synthetic molecules, which can be administered through lipofection or inclusion in lipid or polymer nanoparticles for efficient cardiac delivery. The notion that it is possible to reprogramme CMs into a regenerative state and that this property can be enhanced by ncRNA therapeutics remains exciting, however extensive experimentation in large mammals and rigorous assessment of safety are required to advance towards clinical application.
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Affiliation(s)
- Luca Braga
- British Heart Foundation Centre of Research Excellence, School of Cardiovascular Medicine & Sciences, King’s College London, The James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK
| | - Hashim Ali
- British Heart Foundation Centre of Research Excellence, School of Cardiovascular Medicine & Sciences, King’s College London, The James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK
| | - Ilaria Secco
- British Heart Foundation Centre of Research Excellence, School of Cardiovascular Medicine & Sciences, King’s College London, The James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK
| | - Mauro Giacca
- British Heart Foundation Centre of Research Excellence, School of Cardiovascular Medicine & Sciences, King’s College London, The James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK
- Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
- Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy
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40
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Sgro A, Blancafort P. Epigenome engineering: new technologies for precision medicine. Nucleic Acids Res 2021; 48:12453-12482. [PMID: 33196851 PMCID: PMC7736826 DOI: 10.1093/nar/gkaa1000] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Revised: 10/10/2020] [Accepted: 10/16/2020] [Indexed: 02/07/2023] Open
Abstract
Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.
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Affiliation(s)
- Agustin Sgro
- Cancer Epigenetics Laboratory, The Harry Perkins Institute of Medical Research, Nedlands, Western Australia 6009, Australia.,School of Human Sciences, The University of Western Australia, Crawley, Perth, Western Australia 6009, Australia
| | - Pilar Blancafort
- Cancer Epigenetics Laboratory, The Harry Perkins Institute of Medical Research, Nedlands, Western Australia 6009, Australia.,School of Human Sciences, The University of Western Australia, Crawley, Perth, Western Australia 6009, Australia.,The Greehey Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, TX 78229, USA
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Tristán-Manzano M, Justicia-Lirio P, Maldonado-Pérez N, Cortijo-Gutiérrez M, Benabdellah K, Martin F. Externally-Controlled Systems for Immunotherapy: From Bench to Bedside. Front Immunol 2020; 11:2044. [PMID: 33013864 PMCID: PMC7498544 DOI: 10.3389/fimmu.2020.02044] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 07/28/2020] [Indexed: 12/27/2022] Open
Abstract
Immunotherapy is a very promising therapeutic approach against cancer that is particularly effective when combined with gene therapy. Immuno-gene therapy approaches have led to the approval of four advanced therapy medicinal products (ATMPs) for the treatment of p53-deficient tumors (Gendicine and Imlygic), refractory acute lymphoblastic leukemia (Kymriah) and large B-cell lymphomas (Yescarta). In spite of these remarkable successes, immunotherapy is still associated with severe side effects for CD19+ malignancies and is inefficient for solid tumors. Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of immunotherapy. The aim is to develop smart immunogene therapy-based-ATMPs, which can be controlled by the addition of innocuous drugs or agents, allowing the clinicians to manage the intensity and durability of the therapy. In the present manuscript, we will review the different inducible, versatile and externally controlled gene delivery systems that have been developed and their applications to the field of immunotherapy. We will highlight the advantages and disadvantages of each system and their potential applications in clinics.
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Affiliation(s)
- María Tristán-Manzano
- Gene and Cell Therapy Unit, Genomic Medicine Department, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain
| | - Pedro Justicia-Lirio
- Gene and Cell Therapy Unit, Genomic Medicine Department, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain.,LentiStem Biotech, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain
| | - Noelia Maldonado-Pérez
- Gene and Cell Therapy Unit, Genomic Medicine Department, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain
| | - Marina Cortijo-Gutiérrez
- Gene and Cell Therapy Unit, Genomic Medicine Department, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain
| | - Karim Benabdellah
- Gene and Cell Therapy Unit, Genomic Medicine Department, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain
| | - Francisco Martin
- Gene and Cell Therapy Unit, Genomic Medicine Department, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain
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Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly. Nature 2020; 587:285-290. [PMID: 32879492 PMCID: PMC7666080 DOI: 10.1038/s41586-020-2672-3] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2019] [Accepted: 06/08/2020] [Indexed: 12/31/2022]
Abstract
Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation1. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores2. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix3. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores2. These pores have a size limit of 39 nanometres4-7, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis8, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.
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Design and Applications of Bifunctional Small Molecules in Biology. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2020; 1869:140534. [PMID: 32871274 DOI: 10.1016/j.bbapap.2020.140534] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Revised: 08/17/2020] [Accepted: 08/27/2020] [Indexed: 12/12/2022]
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Wong M, Gilmour D. Getting back on track: exploiting canalization to uncover the mechanisms of developmental robustness. Curr Opin Genet Dev 2020; 63:53-60. [DOI: 10.1016/j.gde.2020.04.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2020] [Accepted: 04/09/2020] [Indexed: 02/08/2023]
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45
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Lee YT, He L, Zhou Y. Expanding the Chemogenetic Toolbox by Circular Permutation. J Mol Biol 2020; 432:3127-3136. [PMID: 32277990 DOI: 10.1016/j.jmb.2020.03.033] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Revised: 03/25/2020] [Accepted: 03/31/2020] [Indexed: 12/18/2022]
Abstract
To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12-rapamycin binding domain (cpFRB). By permuting the topology of the four helices within FRB, we have created cpFRB-FKBP pairs that respond to ligand with varying activation kinetics and dynamics. The cpRAPID system enables chemical-controllable subcellular redistribution of proteins, as well as inducible transcriptional activation when coupled with the CRISPR activation (CRISPRa) technology to induce a GFP reporter and endogenous gene expression. We have further demonstrated the use of cpRAPID to generate chemically switchable split nanobody (designated Chessbody) for ligand-gated antigen recognition in living cells. Collectively, the circular permutation approach offers a powerful means for diversifying the chemogenetics toolbox to benefit the burgeoning synthetic biology field.
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Affiliation(s)
- Yi-Tsang Lee
- Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA
| | - Lian He
- Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA.
| | - Yubin Zhou
- Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, USA; Department of Medical Physiology, College of Medicine, Texas A&M University, Bryan, TX 77807, USA; Department of Translational Medical Sciences, College of Medicine, Texas A&M University, Houston, TX 77030, USA.
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46
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Donahue PS, Draut JW, Muldoon JJ, Edelstein HI, Bagheri N, Leonard JN. The COMET toolkit for composing customizable genetic programs in mammalian cells. Nat Commun 2020; 11:779. [PMID: 32034124 PMCID: PMC7005830 DOI: 10.1038/s41467-019-14147-5] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Accepted: 12/16/2019] [Indexed: 12/11/2022] Open
Abstract
Engineering mammalian cells to carry out sophisticated and customizable genetic programs requires a toolkit of multiple orthogonal and well-characterized transcription factors (TFs). To address this need, we develop the COmposable Mammalian Elements of Transcription (COMET)-an ensemble of TFs and promoters that enable the design and tuning of gene expression to an extent not, to the best of our knowledge, previously possible. COMET currently comprises 44 activating and 12 inhibitory zinc-finger TFs and 83 cognate promoters, combined in a framework that readily accommodates new parts. This system can tune gene expression over three orders of magnitude, provides chemically inducible control of TF activity, and enables single-layer Boolean logic. We also develop a mathematical model that provides mechanistic insights into COMET performance characteristics. Altogether, COMET enables the design and construction of customizable genetic programs in mammalian cells.
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Affiliation(s)
- Patrick S Donahue
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA
- Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, 60208, USA
- Medical Scientist Training Program, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA
| | - Joseph W Draut
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA
| | - Joseph J Muldoon
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA
- Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, 60208, USA
| | - Hailey I Edelstein
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA
| | - Neda Bagheri
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA
- Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, 60208, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA
- Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA
- Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208, USA
- Biology and Chemical Engineering, University of Washington, Seattle, WA, 98195, USA
| | - Joshua N Leonard
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, 60208, USA.
- Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, 60208, USA.
- Center for Synthetic Biology, Northwestern University, Evanston, IL, 60208, USA.
- Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA.
- Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208, USA.
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Barrett CM, McCracken R, Elmer J, Haynes KA. Components from the Human c-myb Transcriptional Regulation System Reactivate Epigenetically Repressed Transgenes. Int J Mol Sci 2020; 21:E530. [PMID: 31947658 PMCID: PMC7014047 DOI: 10.3390/ijms21020530] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2019] [Revised: 01/07/2020] [Accepted: 01/07/2020] [Indexed: 11/16/2022] Open
Abstract
A persistent challenge for mammalian cell engineering is the undesirable epigenetic silencing of transgenes. Foreign DNA can be incorporated into closed chromatin before and after it has been integrated into a host cell's genome. To identify elements that mitigate epigenetic silencing, we tested components from the c-myb and NF-kB transcriptional regulation systems in transiently transfected DNA and at chromosomally integrated transgenes in PC-3 and HEK 293 cells. DNA binding sites for MYB (c-myb) placed upstream of a minimal promoter enhanced expression from transiently transfected plasmid DNA. We targeted p65 and MYB fusion proteins to a chromosomal transgene, UAS-Tk-luciferase, that was silenced by ectopic Polycomb chromatin complexes. Transient expression of Gal4-MYB induced an activated state that resisted complete re-silencing. We used custom guide RNAs and dCas9-MYB to target MYB to different positions relative to the promoter and observed that transgene activation within ectopic Polycomb chromatin required proximity of dCas9-MYB to the transcriptional start site. Our report demonstrates the use of MYB in the context of the CRISPR-activation system, showing that DNA elements and fusion proteins derived from c-myb can mitigate epigenetic silencing to improve transgene expression in engineered cell lines.
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Affiliation(s)
- Cassandra M. Barrett
- School of Biological and Health Systems Engineering, Arizona State University, 501 East Tyler Mall, Tempe, AZ 85287, USA;
| | - Reilly McCracken
- Department of Chemical Engineering, Villanova University, 217 White Hall, 800 East Lancaster Avenue, Villanova, PA 19085, USA; (R.M.); (J.E.)
| | - Jacob Elmer
- Department of Chemical Engineering, Villanova University, 217 White Hall, 800 East Lancaster Avenue, Villanova, PA 19085, USA; (R.M.); (J.E.)
| | - Karmella A. Haynes
- School of Biological and Health Systems Engineering, Arizona State University, 501 East Tyler Mall, Tempe, AZ 85287, USA;
- Wallace H. Coulter Department of Biomedical Engineering, Emory University, Atlanta, GA 30322, USA
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Optogenetic Control of Gene Expression Using Cryptochrome 2 and a Light-Activated Degron. Methods Mol Biol 2020; 2173:151-158. [PMID: 32651916 DOI: 10.1007/978-1-0716-0755-8_10] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Optogenetic tools allow for use of light as an external input to control cellular processes. When applied to regulate the function of transcription factors, optogenetic approaches provide a tunable, reversible, and bidirectional method to control gene expression. Herein, we present a detailed method to induce gene expression in mammalian cells using the light dependent dimerization of cryptochrome 2 (CRY2) and CIB1 to complement a split transcription factor. We also describe a protocol to disrupt gene expression with light by fusing a dimeric transcription factor to CRY2. When combined with a light-induced degron attached to the gene product, this method allows for rapid modulation of target protein abundance.
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49
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He Y, Wu Z, Qiu C, Wang X, Xiang Y, Lu T, He Y, Shang T, Zhu Q, Wang X, Zeng Q, Zhang H, Li D. Long non-coding RNA GAPLINC promotes angiogenesis by regulating miR-211 under hypoxia in human umbilical vein endothelial cells. J Cell Mol Med 2019; 23:8090-8100. [PMID: 31589383 PMCID: PMC6850972 DOI: 10.1111/jcmm.14678] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Revised: 08/19/2019] [Accepted: 08/29/2019] [Indexed: 12/18/2022] Open
Abstract
In this study, we investigated the role of a long non-coding RNA GAPLINC in angiogenesis using human umbilical vein endothelial cells (HUVEC). We found that hypoxia and hypoxia-inducible factor 1α (HIF-1α) increased the expression of GAPLINC in HUVEC cells. Moreover, GAPLINC overexpression down-regulated miR-211 and up-regulated Bcl2 protein expression. Further rescue experiments confirmed that hypoxia directly increased GAPLINC expression. GAPLINC overexpression also increased cell migration and vessel formation which promoted angiogenesis, and these changes were attributed to the increased expression of vascular endothelial growth factor receptors (VEGFR) and delta-like canonical notch ligand 4 (DLL4) receptors. Finally, we demonstrated that GAPLINC promotes vessel formation and migration by regulating MAPK and NF-kB signalling pathways. Taken together, these findings comprehensively demonstrate that overexpression of GAPLINC increases HUVEC cells angiogenesis under hypoxia condition suggesting that GAPLINC can be a potential target for critical limb ischaemia (CLI) treatment.
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Affiliation(s)
- Yangyan He
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Ziheng Wu
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Chenyang Qiu
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Xiaohui Wang
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Yilang Xiang
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Tian Lu
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Yunjun He
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Tao Shang
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Qianqian Zhu
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Xun Wang
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Qinglong Zeng
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Hongkun Zhang
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
| | - Donglin Li
- Department of Vascular SurgeryThe First Affiliated Hospital, College of Medicine, Zhejiang UniversityHang ZhouChina
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50
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Surana S, Villarroel‐Campos D, Lazo OM, Moretto E, Tosolini AP, Rhymes ER, Richter S, Sleigh JN, Schiavo G. The evolution of the axonal transport toolkit. Traffic 2019; 21:13-33. [DOI: 10.1111/tra.12710] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Revised: 10/16/2019] [Accepted: 10/17/2019] [Indexed: 12/11/2022]
Affiliation(s)
- Sunaina Surana
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
| | - David Villarroel‐Campos
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
| | - Oscar M. Lazo
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
- UK Dementia Research InstituteUniversity College London London UK
| | - Edoardo Moretto
- UK Dementia Research InstituteUniversity College London London UK
| | - Andrew P. Tosolini
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
| | - Elena R. Rhymes
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
| | - Sandy Richter
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
| | - James N. Sleigh
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
- UK Dementia Research InstituteUniversity College London London UK
| | - Giampietro Schiavo
- Department of Neuromuscular Diseases, UCL Queen Square Institute of NeurologyUniversity College London London UK
- UK Dementia Research InstituteUniversity College London London UK
- Discoveries Centre for Regenerative and Precision MedicineUniversity College London London UK
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