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1.
Cell ; 186(5): 1039-1049.e17, 2023 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-36764293

RESUMO

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.


Assuntos
Chaperonas Moleculares , Dobramento de Proteína , Microscopia Crioeletrônica , Chaperonas Moleculares/metabolismo , Oxirredução , Chaperoninas/química , Chaperoninas/metabolismo , Chaperonina 60/química , Chaperonina 10/metabolismo
2.
Cell ; 186(16): 3443-3459.e24, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37480851

RESUMO

Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.


Assuntos
Chaperonina com TCP-1 , Ubiquitina-Proteína Ligases , Chaperonina com TCP-1/química , Ubiquitina-Proteína Ligases/genética , Humanos
3.
Cell ; 185(25): 4770-4787.e20, 2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36493755

RESUMO

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of ß-tubulin using human prefoldin and TRiC. We find unstructured ß-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded ß-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.


Assuntos
Chaperonina com TCP-1 , Tubulina (Proteína) , Humanos , Chaperonina com TCP-1/química , Tubulina (Proteína)/metabolismo , Dobramento de Proteína , Proteostase , Trifosfato de Adenosina/metabolismo
4.
Cell ; 185(8): 1325-1345.e22, 2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35366418

RESUMO

Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.


Assuntos
Chaperonina com TCP-1 , Macroautofagia , Agregados Proteicos , Animais , Camundongos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Chaperonina com TCP-1/metabolismo , Proteína Sequestossoma-1/metabolismo
5.
Cell ; 177(3): 751-765.e15, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30955883

RESUMO

Maintaining proteostasis in eukaryotic protein folding involves cooperation of distinct chaperone systems. To understand how the essential ring-shaped chaperonin TRiC/CCT cooperates with the chaperone prefoldin/GIMc (PFD), we integrate cryoelectron microscopy (cryo-EM), crosslinking-mass-spectrometry and biochemical and cellular approaches to elucidate the structural and functional interplay between TRiC/CCT and PFD. We find these hetero-oligomeric chaperones associate in a defined architecture, through a conserved interface of electrostatic contacts that serves as a pivot point for a TRiC-PFD conformational cycle. PFD alternates between an open "latched" conformation and a closed "engaged" conformation that aligns the PFD-TRiC substrate binding chambers. PFD can act after TRiC bound its substrates to enhance the rate and yield of the folding reaction, suppressing non-productive reaction cycles. Disrupting the TRiC-PFD interaction in vivo is strongly deleterious, leading to accumulation of amyloid aggregates. The supra-chaperone assembly formed by PFD and TRiC is essential to prevent toxic conformations and ensure effective cellular proteostasis.


Assuntos
Chaperonina com TCP-1/metabolismo , Chaperonas Moleculares/metabolismo , Proteostase/fisiologia , Actinas/química , Actinas/metabolismo , Chaperonina com TCP-1/química , Chaperonina com TCP-1/genética , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Eletricidade Estática
6.
Cell ; 174(6): 1507-1521.e16, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30100183

RESUMO

The hetero-oligomeric chaperonin of eukarya, TRiC, is required to fold the cytoskeletal protein actin. The simpler bacterial chaperonin system, GroEL/GroES, is unable to mediate actin folding. Here, we use spectroscopic and structural techniques to determine how TRiC promotes the conformational progression of actin to the native state. We find that actin fails to fold spontaneously even in the absence of aggregation but populates a kinetically trapped, conformationally dynamic state. Binding of this frustrated intermediate to TRiC specifies an extended topology of actin with native-like secondary structure. In contrast, GroEL stabilizes bound actin in an unfolded state. ATP binding to TRiC effects an asymmetric conformational change in the chaperonin ring. This step induces the partial release of actin, priming it for folding upon complete release into the chaperonin cavity, mediated by ATP hydrolysis. Our results reveal how the unique features of TRiC direct the folding pathway of an obligate eukaryotic substrate.


Assuntos
Actinas/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Actinas/química , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Chaperonina 10/química , Chaperonina 60/química , Microscopia Crioeletrônica , Desoxirribonuclease I/química , Desoxirribonuclease I/metabolismo , Medição da Troca de Deutério , Humanos , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína
7.
Cell ; 172(3): 605-617.e11, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29336887

RESUMO

The bacterial chaperonin GroEL and its cofactor, GroES, form a nano-cage for a single molecule of substrate protein (SP) to fold in isolation. GroEL and GroES undergo an ATP-regulated interaction cycle to close and open the folding cage. GroEL consists of two heptameric rings stacked back to back. Here, we show that GroEL undergoes transient ring separation, resulting in ring exchange between complexes. Ring separation occurs upon ATP-binding to the trans ring of the asymmetric GroEL:7ADP:GroES complex in the presence or absence of SP and is a consequence of inter-ring negative allostery. We find that a GroEL mutant unable to perform ring separation is folding active but populates symmetric GroEL:GroES2 complexes, where both GroEL rings function simultaneously rather than sequentially. As a consequence, SP binding and release from the folding chamber is inefficient, and E. coli growth is impaired. We suggest that transient ring separation is an integral part of the chaperonin mechanism.


Assuntos
Chaperonina 60/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Mutação , Ligação Proteica
8.
Mol Cell ; 83(17): 3123-3139.e8, 2023 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-37625406

RESUMO

How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes.


Assuntos
Chaperonina com TCP-1 , Actinas , Chaperonina com TCP-1/química , Chaperonina com TCP-1/metabolismo , Humanos , Animais
9.
Mol Cell ; 82(17): 3193-3208.e8, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35853451

RESUMO

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.


Assuntos
Chaperonas Moleculares , Dobramento de Proteína , Chaperonas Moleculares/metabolismo
10.
Mol Cell ; 74(1): 88-100.e9, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30876804

RESUMO

Eukaryotic elongation factor 2 (eEF2) is an abundant and essential component of the translation machinery. The biogenesis of this 93 kDa multi-domain protein is assisted by the chaperonin TRiC/CCT. Here, we show in yeast cells that the highly conserved protein Hgh1 (FAM203 in humans) is a chaperone that cooperates with TRiC in eEF2 folding. In the absence of Hgh1, a substantial fraction of newly synthesized eEF2 is degraded or aggregates. We solved the crystal structure of Hgh1 and analyzed the interaction of wild-type and mutant Hgh1 with eEF2. These experiments revealed that Hgh1 is an armadillo repeat protein that binds to the dynamic central domain III of eEF2 via a bipartite interface. Hgh1 binding recruits TRiC to the C-terminal eEF2 module and prevents unproductive interactions of domain III, allowing efficient folding of the N-terminal GTPase module. eEF2 folding is completed upon dissociation of TRiC and Hgh1.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Chaperonas Moleculares/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/genética , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade
11.
Semin Cell Dev Biol ; 155(Pt A): 37-47, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-37085353

RESUMO

Rubisco catalyses the entry of almost all CO2 into the biosphere and is often the rate-limiting step in plant photosynthesis and growth. Its notoriety as the most abundant protein on Earth stems from the slow and error-prone catalytic properties that require plants, cyanobacteria, algae and photosynthetic bacteria to produce it in high amounts. Efforts to improve the CO2-fixing properties of plant Rubisco has been spurred on by the discovery of more effective isoforms in some algae with the potential to significantly improve crop productivity. Incompatibilities between the protein folding machinery of leaf and algae chloroplasts have, so far, prevented efforts to transplant these more effective Rubisco variants into plants. There is therefore increasing interest in improving Rubisco catalysis by directed (laboratory) evolution. Here we review the advances being made in, and the ongoing challenges with, improving the solubility and/or carboxylation activity of differing non-plant Rubisco lineages. We provide perspectives on new opportunities for the directed evolution of crop Rubiscos and the existing plant transformation capabilities available to evaluate the extent to which Rubisco activity improvements can benefit agricultural productivity.


Assuntos
Dióxido de Carbono , Ribulose-Bifosfato Carboxilase , Ribulose-Bifosfato Carboxilase/genética , Folhas de Planta , Dobramento de Proteína
12.
Proc Natl Acad Sci U S A ; 119(34): e2200106119, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35969751

RESUMO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) has long been studied from many perspectives. As a multisubunit (large subunits [LSUs] and small subunits[SSUs]) protein encoded by genes residing in the chloroplast (rbcL) and nuclear (rbcS) genomes, RuBisCo also is a model for cytonuclear coevolution following allopolyploid speciation in plants. Here, we studied the genomic and transcriptional cytonuclear coordination of auxiliary chaperonin and chaperones that facilitate RuBisCo biogenesis across multiple natural and artificially synthesized plant allopolyploids. We found similar genomic and transcriptional cytonuclear responses, including respective paternal-to-maternal conversions and maternal homeologous biased expression, in chaperonin/chaperon-assisted folding and assembly of RuBisCo in different allopolyploids. One observation is about the temporally attenuated genomic and transcriptional cytonuclear evolutionary responses during early folding and later assembly process of RuBisCo biogenesis, which were established by long-term evolution and immediate onset of allopolyploidy, respectively. Our study not only points to the potential widespread and hitherto unrecognized features of cytonuclear evolution but also bears implications for the structural interaction interface between LSU and Cpn60 chaperonin and the functioning stage of the Raf2 chaperone.


Assuntos
Chaperoninas/metabolismo , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase , Núcleo Celular/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo
13.
Infect Immun ; 92(10): e0023424, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39248465

RESUMO

Chaperonins/Heat shock protein 60 are ubiquitous multimeric protein complexes that assist in the folding of partially and/or misfolded proteins using metabolic energy into their native stage. The eukaryotic group II chaperonin, also referred as T-complex protein-1 ring complex (TRiC)/T-complex protein-1 (TCP1)/chaperonin containing T-complex protein (CCT), contains 8-9 paralogous subunits, arranged in each of the two rings of hetero-oligomeric complex. In Leishmania, till date, only one subunit, LdTCP1γ, has been well studied. Here, we report the molecular, structural, and functional characterization of TCP1δ subunit of Leishmania donovani (LdTCP1δ), the causative agent of Indian kala-azar. LdTCP1δ gene exhibited only 27.9% identity with LdTCP1γ and clustered in a separate branch in the phylogenic tree of LdTCP1 subunits. The purified recombinant protein formed a high molecular weight complex (0.75 MDa), arranged into 16-mer assembly, and performed in vitro chaperonin activity as assayed by ATP-dependent luciferase folding. LdTCP1δ exhibits 1.8-fold upregulated expression in metabolically active, rapidly dividing log phase promastigotes. Over-expression of LdTCP1δ in promastigotes results in increased infectivity and rate of multiplication of intracellular amastigotes. The study thus establishes the existence of an individual functionally active homo-oligomeric complex of LdTCP1δ chaperonin with its role in parasite infectivity and multiplication.


Assuntos
Leishmania donovani , Leishmania donovani/genética , Leishmania donovani/metabolismo , Chaperonina com TCP-1/metabolismo , Chaperonina com TCP-1/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Animais , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Filogenia , Camundongos , Sequência de Aminoácidos
14.
Mol Microbiol ; 120(2): 210-223, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37350285

RESUMO

Mycobacterium tuberculosis encodes two chaperonin proteins, MtbCpn60.1 and MtbCpn60.2, that share substantial sequence similarity with the Escherichia coli chaperonin, GroEL. However, unlike GroEL, MtbCpn60.1 and MtbCpn60.2 purify as lower-order oligomers. Previous studies have shown that MtbCpn60.2 can functionally replace GroEL in E. coli, while the function of MtbCpn60.1 remained an enigma. Here, we demonstrate the molecular chaperone function of MtbCpn60.1 and MtbCpn60.2, by probing their ability to assist the folding of obligate chaperonin clients, DapA, FtsE and MetK, in an E. coli strain depleted of endogenous GroEL. We show that both MtbCpn60.1 and MtbCpn60.2 support cell survival and cell division by assisting the folding of DapA and FtsE, but only MtbCpn60.2 completely rescues GroEL-depleted E. coli cells. We also show that, unlike MtbCpn60.2, MtbCpn60.1 has limited ability to support cell growth and proliferation and assist the folding of MetK. Our findings suggest that the client pools of GroEL and MtbCpn60.2 overlap substantially, while MtbCpn60.1 folds only a small subset of GroEL clients. We conclude that the differences between MtbCpn60.1 and MtbCpn60.2 may be a consequence of their intrinsic sequence features, which affect their thermostability, efficiency, clientomes and modes of action.


Assuntos
Proteínas de Escherichia coli , Mycobacterium tuberculosis , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteostase , Chaperoninas/genética , Chaperoninas/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Escherichia coli/metabolismo
15.
Mol Genet Metab ; 142(3): 108495, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38772223

RESUMO

PURPOSE: To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene. METHODS: Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing. RESULTS: In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink. CONCLUSION: Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease.


Assuntos
Fibroblastos , Glutaratos , Fenilbutiratos , Humanos , Fenilbutiratos/farmacologia , Fenilbutiratos/uso terapêutico , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Glutaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/genética , Metabolômica , Sequenciamento do Exoma , Citrato (si)-Sintase/metabolismo , Citrato (si)-Sintase/genética , Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Encefalopatias Metabólicas/tratamento farmacológico , Encefalopatias Metabólicas/genética , Encefalopatias Metabólicas/metabolismo , Encefalopatias Metabólicas/patologia , Multiômica , Proteínas Mitocondriais , Transportadores de Ânions Orgânicos
16.
FASEB J ; 37(2): e22757, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36607310

RESUMO

Vesicle trafficking is a fundamental cellular process that controls the transport of various proteins and cargos between cellular compartments in eukaryotes. Using a combination of genome-wide CRISPR screening in mammalian cells and RNAi screening in Caenorhabditis elegans, we identify chaperonin containing TCP-1 subunit 4 (CCT4) as a critical regulator of protein secretion and vesicle trafficking. In C. elegans, deficiency of cct-4 as well as other CCT subunits impairs the trafficking of endocytic markers in intestinal cells, and this defect resembles that of dyn-1 RNAi worms. Consistent with these findings, the silencing of CCT4 in human cells leads to defective endosomal trafficking, and this defect can be rescued by the dynamin activator Ryngo 1-23. These results suggest that the cytosolic chaperonin CCT may regulate vesicle trafficking by promoting the folding of dynamin in addition to its known substrate tubulin. Our findings establish an essential role for the CCT chaperonin in regulating vesicle trafficking, and provide new insights into the regulation of vesicle trafficking and the cellular function of the cytosolic chaperonin.


Assuntos
Caenorhabditis elegans , Chaperonina com TCP-1 , Animais , Humanos , Chaperonina com TCP-1/genética , Chaperonina com TCP-1/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Chaperoninas/genética , Chaperoninas/metabolismo , Tubulina (Proteína)/metabolismo , Citosol/metabolismo , Dobramento de Proteína , Mamíferos/metabolismo
17.
J Biomed Sci ; 31(1): 65, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38943128

RESUMO

BACKGROUND: Enterovirus 71 (EV-A71) causes Hand, Foot and Mouth Disease (HFMD) in children and has been associated with neurological complications. The molecular mechanisms involved in EV-A71 pathogenesis have remained elusive. METHODS: A siRNA screen in EV-A71 infected-motor neurons was performed targeting 112 genes involved in intracellular membrane trafficking, followed by validation of the top four hits using deconvoluted siRNA. Downstream approaches including viral entry by-pass, intracellular viral genome quantification by qPCR, Western blot analyses, and Luciferase reporter assays allowed determine the stage of the infection cycle the top candidate, RAB11A was involved in. Proximity ligation assay, co-immunoprecipitation and multiplex confocal imaging were employed to study interactions between viral components and RAB11A. Dominant negative and constitutively active RAB11A constructs were used to determine the importance of the protein's GTPase activity during EV-A71 infection. Mass spectrometry and protein interaction analyses were employed for the identification of RAB11A's host interacting partners during infection. RESULTS: Small GTPase RAB11A was identified as a novel pro-viral host factor during EV-A71 infection. RAB11A and RAB11B isoforms were interchangeably exploited by strains from major EV-A71 genogroups and by Coxsackievirus A16, another major causative agent of HFMD. We showed that RAB11A was not involved in viral entry, IRES-mediated protein translation, viral genome replication, and virus exit. RAB11A co-localized with replication organelles where it interacted with structural and non-structural viral components. Over-expression of dominant negative (S25N; GDP-bound) and constitutively active (Q70L; GTP-bound) RAB11A mutants had no effect on EV-A71 infection outcome, ruling out RAB11A's involvement in intracellular trafficking of viral or host components. Instead, decreased ratio of intracellular mature viral particles to viral RNA copies and increased VP0:VP2 ratio in siRAB11-treated cells supported a role in provirion maturation hallmarked by VP0 cleavage into VP2 and VP4. Finally, chaperones, not trafficking and transporter proteins, were found to be RAB11A's top interacting partners during EV-A71 infection. Among which, CCT8 subunit from the chaperone complex TRiC/CCT was further validated and shown to interact with viral structural proteins specifically, representing yet another novel pro-viral host factor during EV-A71 infection. CONCLUSIONS: This study describes a novel, unconventional role for RAB11A during viral infection where it participates in the complex process of virus morphogenesis by recruiting essential chaperone proteins.


Assuntos
Enterovirus Humano A , Proteínas rab de Ligação ao GTP , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Enterovirus Humano A/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Replicação Viral
18.
Arch Microbiol ; 206(7): 299, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38861015

RESUMO

Chaperonins from psychrophilic bacteria have been shown to exist as single-ring complexes. This deviation from the standard double-ring structure has been thought to be a beneficial adaptation to the cold environment. Here we show that Cpn60 from the psychrophile Pseudoalteromonas haloplanktis (Ph) maintains its double-ring structure also in the cold. A strongly reduced ATPase activity keeps the chaperonin in an energy-saving dormant state, until binding of client protein activates it. Ph Cpn60 in complex with co-chaperonin Ph Cpn10 efficiently assists in protein folding up to 55 °C. Moreover, we show that recombinant expression of Ph Cpn60 can provide its host Escherichia coli with improved viability under low temperature growth conditions. These properties of the Ph chaperonin may make it a valuable tool in the folding and stabilization of psychrophilic proteins.


Assuntos
Proteínas de Bactérias , Temperatura Baixa , Escherichia coli , Dobramento de Proteína , Pseudoalteromonas , Pseudoalteromonas/genética , Pseudoalteromonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Chaperonina 60/metabolismo , Chaperonina 60/genética , Chaperonina 60/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Chaperoninas/metabolismo , Chaperoninas/genética , Chaperoninas/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética
19.
Mol Biol Rep ; 51(1): 54, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38165547

RESUMO

BACKGROUND: Epithelial ovarian cancer (EOC) is featured by rapid progression and dismal outcomes clinically. Chaperonin Containing TCP1 Subunit 2 (CCT2) was identified as a crucial regulator for tumor progression, however, its exact role in EOC remained largely unknown. METHODS: CCT2 expression and prognostic value in EOC samples were assessed according to TCGA dataset. Proliferation and mobility potentials were assessed by CCK8, colony-formation, wound healing, and Transwell assays. Cancer stem cell (CSC) traits were evaluated by RT-PCR, WB assays, sphere-forming assay and chemoresistance analysis. Bioinformatic analysis, co-IP assays and ubiquitin assays were performed to explore the mechanisms of CCT2 on EOC cells. RESULTS: CCT2 highly expressed in EOC tissues and predicted poor prognosis of EOC patients by TCGA analysis. Silencing CCT2 significantly restrained cell proliferation, migration, and invasion. Moreover, CCT2 could effectively trigger epithelial-mesenchymal transition to confer extensive invasion potentials to EOC cells, Importantly, CCT2 positively correlated with CSC markers in EOC, and CCT2 knockdown impaired CSC traits and sensitize EOC cells to conventional chemotherapy regimens. Contrarily, overexpressing CCT2 achieved opposite results. Mechanistically, CCT2 exerted its pro-oncogene function by triggering Wnt/ß-catenin signaling. Specifically, CCT2 could recruit HSP105-PP2A complex, a well-established dephosphorylation complex, to ß-catenin via direct physical interaction to prevent phosphorylation-induced proteasomal degradation of ß-catenin, resulting in intracellular accumulation of active ß-catenin and increased signaling activity. CONCLUSIONS: CCT2 was a novel promotor for EOC progression and a crucial sustainer for CSC traits mainly by preventing ß-catenin degradation. Targeting CCT2 may represent a promising therapeutic strategy for EOC.


Assuntos
Neoplasias Ovarianas , Humanos , Feminino , Carcinoma Epitelial do Ovário/metabolismo , Neoplasias Ovarianas/patologia , beta Catenina/genética , beta Catenina/metabolismo , Via de Sinalização Wnt , Proliferação de Células , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Transição Epitelial-Mesenquimal/genética , Movimento Celular , Chaperonina com TCP-1/metabolismo
20.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649236

RESUMO

Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the m6A methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in Drosophila and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing m6A levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with m6A RNA methylation and demonstrates their roles in suppressing autophagy.


Assuntos
Autofagia , Proteínas de Drosophila/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metiltransferases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Metilação , Metiltransferases/genética , Receptores Nucleares Órfãos , Estabilidade de RNA , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética
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