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1.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638699

RESUMO

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein-protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α-Sec61ß, TRAPα-TRAPß) or protein complexes in juxtaposition (inter-complex interactions: Sec61α-TRAPα, Sec61α-Sec63, and Sec61ß-Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein-protein interactions in response to changing cellular conditions in living cells.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Canais de Translocação SEC/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismo , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/genética , Chaperonas Moleculares/genética , Transporte Proteico , Proteínas de Ligação a RNA/genética , Canais de Translocação SEC/genética , Fosfatase Ácida Resistente a Tartarato/genética
2.
Int J Mol Sci ; 22(19)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34638658

RESUMO

HSP90 is a vital chaperone protein conserved across all organisms. As a chaperone protein, it correctly folds client proteins. Structurally, this protein is a dimer with monomer subunits that consist of three main conserved domains known as the N-terminal domain, middle domain, and the C-terminal domain. Multiple isoforms of HSP90 exist, and these isoforms share high homology. These isoforms are present both within the cell and outside the cell. Isoforms HSP90α and HSP90ß are present in the cytoplasm; TRAP1 is present in the mitochondria; and GRP94 is present in the endoplasmic reticulum and is likely secreted due to post-translational modifications (PTM). HSP90 is also secreted into an extracellular environment via an exosome pathway that differs from the classic secretion pathway. Various co-chaperones are necessary for HSP90 to function. Elevated levels of HSP90 have been observed in patients with cancer. Despite this observation, the possible role of HSP90 in cancer was overlooked because the chaperone was also present in extreme amounts in normal cells and was vital to normal cell function, as observed when the drastic adverse effects resulting from gene knockout inhibited the production of this protein. Differences between normal HSP90 and HSP90 of the tumor phenotype have been better understood and have aided in making the chaperone protein a target for cancer drugs. One difference is in the conformation: HSP90 of the tumor phenotype is more susceptible to inhibitors. Since overexpression of HSP90 is a factor in tumorigenesis, HSP90 inhibitors have been studied to combat the adverse effects of HSP90 overexpression. Monotherapies using HSP90 inhibitors have shown some success; however, combination therapies have shown better results and are thus being studied for a more effective cancer treatment.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Neoplasias/metabolismo , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Humanos , Neoplasias/patologia , Isoformas de Proteínas/metabolismo
3.
Molecules ; 26(20)2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34684720

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in or on various cell types and have diverse functions. In immune cells nAChRs regulate proliferation, differentiation and cytokine release. Specifically, activation of the α7 nAChR reduces inflammation as part of the cholinergic anti-inflammatory pathway. Here we review numerous effects of α7 nAChR activation on immune cell function and differentiation. Further, we also describe evidence implicating this receptor and its chaperone RIC-3 in diseases of the central nervous system and in neuroinflammation, focusing on multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Deregulated neuroinflammation due to dysfunction of α7 nAChR provides one explanation for involvement of this receptor and of RIC-3 in neurodegenerative diseases. In this review, we also provide evidence implicating α7 nAChRs and RIC-3 in neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) involving neuroinflammation. Besides, we will describe the therapeutic implications of activating the cholinergic anti-inflammatory pathway for diseases involving neuroinflammation.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças do Sistema Nervoso/metabolismo , Neuroimunomodulação/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Humanos , Inflamação/metabolismo , Chaperonas Moleculares/metabolismo , Neuroimunomodulação/imunologia , Transdução de Sinais
4.
Nat Commun ; 12(1): 5999, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34650037

RESUMO

Molecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson's disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Chaperonas Moleculares/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Escherichia coli , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Cinética , Doença de Parkinson/metabolismo
5.
Nat Commun ; 12(1): 5996, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34650047

RESUMO

Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1-mediated H3K9me3 and DNA methylation. However, the protein network which regulates the deposition of these chromatin modifications is still incompletely understood. Here, we perform a genome-wide single guide RNA (sgRNA) screen for genes involved in ERV silencing and identify the GHKL ATPase protein Morc3 as a top-scoring hit. Morc3 knock-out (ko) cells display de-repression, reduced H3K9me3, and increased chromatin accessibility of distinct ERV families. We find that the Morc3 ATPase cycle and Morc3 SUMOylation are important for ERV chromatin regulation. Proteomic analyses reveal that Morc3 mutant proteins fail to interact with the histone H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation and Daxx SUMO binding. Notably, in Morc3 ko cells, we observe strongly reduced histone H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as a critical regulator of Daxx-mediated histone H3.3 incorporation to ERV regions.


Assuntos
Adenosina Trifosfatases/genética , Proteínas Correpressoras/genética , Proteínas de Ligação a DNA/genética , Retrovirus Endógenos/genética , Inativação Gênica , Chaperonas Moleculares/genética , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Cromatina , Proteínas Correpressoras/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/metabolismo , Técnicas de Inativação de Genes , Histona-Lisina N-Metiltransferase , Histonas/genética , Histonas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Ligação Proteica , Proteômica , Sumoilação
6.
Int J Mol Sci ; 22(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34639110

RESUMO

Previously, we showed that the removal of the 54-61 residues from αB-crystallin (αBΔ54-61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone-substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54-61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54-61 protein.


Assuntos
Apoptose , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/química , Epitélio Pigmentado da Retina/patologia , Cadeia B de alfa-Cristalina/química , Células Cultivadas , Humanos , Estrutura Secundária de Proteína , Epitélio Pigmentado da Retina/metabolismo , Deleção de Sequência , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo
7.
Invest Ophthalmol Vis Sci ; 62(12): 4, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34499705

RESUMO

Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown. Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11-transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking. Results: SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11-transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11-transfected fibroblasts. Conclusions: HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.


Assuntos
Amônia/farmacologia , Proteínas de Transporte de Ânions/genética , Distrofias Hereditárias da Córnea/genética , Endotélio Corneano/metabolismo , Mitocôndrias/patologia , Proteômica/métodos , Simportadores/genética , Animais , Proteínas de Transporte de Ânions/metabolismo , Células Cultivadas , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transporte Proteico , Simportadores/metabolismo
8.
Nat Commun ; 12(1): 5207, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471127

RESUMO

Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Escherichia coli Uropatogênica/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Escherichia coli Uropatogênica/genética
9.
Nat Commun ; 12(1): 5666, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34580293

RESUMO

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/isolamento & purificação , Hidrólise , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Simulação de Acoplamento Molecular , Domínios Proteicos/genética , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Espectrometria de Massas em Tandem
10.
BMC Cancer ; 21(1): 1003, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34493236

RESUMO

BACKGROUND: Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in head and neck. Platinum-based chemotherapy is an important treatment for NPC. However, the molecular mechanism of resistance to platinum drug remains unknown. Endoplasmic reticulum resident protein 44(ERp44), an unfolded protein response (UPR)-induced endoplasmic reticulum(ER) protein, is induced during ER stress. This research explored the mechanism of ERp44 in strengthening cisplatin resistance in NPC. METHODS: Western blot and immunohistochemistry were used to investigate the expression of ERp44 and Glucose-Regulated Protein 78(GRP78) in NPC. We took CCK8 to detect the role of ERp44 on cell chemosensitivity. Flow cytometric analysis and western blot were taken to analyze cell apoptosis. We performed differential centrifugation to isolate exosomes from serum or conditioned media of cells and analyzed the impact of exosomal ERp44 on cells cisplatin sensitivity. Finally, the results were confirmed in vivo. RESULTS: We found the increased expression of ERp44 and GRP78 in NPC and ERp44 was highly expressed in ER-stressed tissues. Cell proliferation was inhibited after cisplatin treatment when ERp44 was knocked down and ERp44 strengthened cisplatin resistance by influencing cell apoptosis and pyroptosis. Then we also collected exosomes and cell viability was increased after the addition of NPC-derived-exosomes with cisplatin treatment. More importantly, our results showed under ERS, NPC cells secreted exosomes containing ERp44 and could transfer them to adjacent cells to strengthen chemoresistance. CONCLUSION: Our data suggested that exosomal ERp44 derived from ER-stressed NPC cells took an inevitable role in NPC chemoresistance and might act as a treatment target.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático , Exossomos/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Carcinoma Nasofaríngeo/tratamento farmacológico , Resposta a Proteínas não Dobradas , Animais , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Exossomos/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Chaperonas Moleculares/genética , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Int J Mol Sci ; 22(18)2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34576306

RESUMO

Streptomycetes are important biotechnological bacteria that produce several clinically bioactive compounds. They have a complex development, including hyphae differentiation and sporulation. Cytosolic copper is a well-known modulator of differentiation and secondary metabolism. The interruption of the Streptomyces coelicolor SCO2730 (copper chaperone, SCO2730::Tn5062 mutant) blocks SCO2730 and reduces SCO2731 (P-type ATPase copper export) expressions, decreasing copper export and increasing cytosolic copper. This mutation triggers the expression of 13 secondary metabolite clusters, including cryptic pathways, during the whole developmental cycle, skipping the vegetative, non-productive stage. As a proof of concept, here, we tested whether the knockdown of the SCO2730/31 orthologue expression can enhance secondary metabolism in streptomycetes. We created a SCO2730/31 consensus antisense mRNA from the sequences of seven key streptomycetes, which helped to increase the cytosolic copper in S. coelicolor, albeit to a lower level than in the SCO2730::Tn5062 mutant. This antisense mRNA affected the production of at least six secondary metabolites (CDA, 2-methylisoborneol, undecylprodigiosin, tetrahydroxynaphtalene, α-actinorhodin, ε-actinorhodin) in the S. coelicolor, and five (phenanthroviridin, alkylresorcinol, chloramphenicol, pikromycin, jadomycin G) in the S. venezuelae; it also helped to alter the S. albus metabolome. The SCO2730/31 consensus antisense mRNA designed here constitutes a tool for the knockdown of SCO2730/31 expression and for the enhancement of Streptomyces' secondary metabolism.


Assuntos
Proteínas de Bactérias/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Metabolismo Secundário , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Cobre/metabolismo , ATPases Transportadoras de Cobre/genética , Chaperonas Moleculares/genética , Streptomyces coelicolor/genética
12.
Nat Commun ; 12(1): 5338, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504072

RESUMO

Molecular chaperones, including Hsp70/J-domain protein (JDP) families, play central roles in binding substrates to prevent their aggregation. How JDPs select different conformations of substrates remains poorly understood. Here, we report an interaction between the JDP DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a ß-turn structural element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau, but not mutant, ß-turn structural elements can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. Our work identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.


Assuntos
Amiloide/química , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Agregados Proteicos/genética , Tauopatias/genética , Proteínas tau/química , Amiloide/antagonistas & inibidores , Amiloide/genética , Amiloide/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Encéfalo/patologia , Clonagem Molecular , Modelos Animais de Doenças , Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tauopatias/metabolismo , Tauopatias/patologia , Termodinâmica , Proteínas tau/genética , Proteínas tau/metabolismo
13.
Elife ; 102021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34487489

RESUMO

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif of FUS as a key driver of condensate aging. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.


Assuntos
Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , RNA/metabolismo , Células HeLa , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Relação Estrutura-Atividade , Fatores de Tempo
14.
G3 (Bethesda) ; 11(9)2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34544131

RESUMO

Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate 3D structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here, we build upon our septin studies to develop a new approach for identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Hsp90 inhibition liberates mutant p53 to enter the nucleus. These findings provide new insights into the effects of missense mutations.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
15.
Biochem J ; 478(18): 3395-3421, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34554214

RESUMO

The ubiquitin-proteasome pathway (UPP) and autophagy play integral roles in cellular homeostasis. As part of their normal life cycle, most proteins undergo ubiquitination for some form of redistribution, localization and/or functional modulation. However, ubiquitination is also important to the UPP and several autophagic processes. The UPP is initiated after specific lysine residues of short-lived, damaged or misfolded proteins are conjugated to ubiquitin, which targets these proteins to proteasomes. Autophagy is the endosomal/lysosomal-dependent degradation of organelles, invading microbes, zymogen granules and macromolecules such as protein, carbohydrates and lipids. Autophagy can be broadly separated into three distinct subtypes termed microautophagy, chaperone-mediated autophagy and macroautophagy. Although autophagy was once thought of as non-selective bulk degradation, advancements in the field have led to the discovery of several selective forms of autophagy. Here, we focus on the mechanisms of primary and selective mammalian autophagy pathways and highlight the current knowledge gaps in these molecular pathways.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Endossomos/metabolismo , Lisossomos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Homeostase/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteólise , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
16.
Nat Commun ; 12(1): 5438, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521831

RESUMO

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.


Assuntos
Proteínas de Arabidopsis/metabolismo , Técnicas Biossensoriais , Proteínas Intrinsicamente Desordenadas/metabolismo , Chaperonas Moleculares/metabolismo , Pressão Osmótica , Água/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Linhagem Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Concentração Osmolar , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Termodinâmica
17.
Cells ; 10(9)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34571827

RESUMO

During type 1 diabetes mellitus (T1DM) development, beta-cells undergo intense endoplasmic reticulum (ER) stress that could result in apoptosis through the failure of adaptation to the unfolded protein response (UPR). Islet transplantation is considered an attractive alternative among beta-cell replacement therapies for T1DM. To avoid the loss of beta-cells that will jeopardize the transplant's outcome, several strategies are being studied. We have previously shown that prolactin induces protection against proinflammatory cytokines and redox imbalance-induced beta-cell death by increasing heat-shock protein B1 (HSPB1) levels. Since the role of HSPB1 in beta cells has not been deeply studied, we investigated the mechanisms involved in unbalanced protein homeostasis caused by intense ER stress and overload of the proteasomal protein degradation pathway. We tested whether HSPB1-mediated cytoprotective effects involved UPR modulation and improvement of protein degradation via the ubiquitin-proteasome system. We demonstrated that increased levels of HSPB1 attenuated levels of pro-apoptotic proteins such as CHOP and BIM, as well as increased protein ubiquitination and the speed of proteasomal protein degradation. Our data showed that HSPB1 induced resistance to proteotoxic stress and, thus, enhanced cell survival via an increase in beta-cell proteolytic capacity. These results could contribute to generate strategies aimed at the optimization of beta-cell replacement therapies.


Assuntos
Proteínas de Choque Térmico/metabolismo , Células Secretoras de Insulina/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/fisiologia , Proteólise , Resposta a Proteínas não Dobradas/fisiologia
18.
Am J Physiol Heart Circ Physiol ; 321(5): H963-H975, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34477462

RESUMO

Small heat shock proteins (sHsps) protect the heart from chemotherapeutics-induced heart failure by inhibiting p53-dependent apoptosis. However, mechanism of such protection has not been elucidated yet. Here we test a hypothesis that serine phosphorylation of sHsps is essential to inhibit the doxorubicin-induced and p53-dependent apoptotic pathway. Three transgenic mice (TG) lines with cardiomyocyte-specific overexpression of human heat shock protein 27 (hHsp27), namely, wild-type [myosin heavy chain (MHC)-hHsp27], S82A single mutant [MHC-mut-hHsp27(S82A)], and trimutant [MHC-mut-hHsp27(S15A/S78A/S82A)] were generated. TG mice were treated with Dox (6 mg/kg body wt; once in a week; 4 wk) along with age-matched nontransgenic (non-TG) controls. The Dox-treated MHC-hHsp27 mice showed improved survival and cardiac function (both MRI and echocardiography) in terms of contractility [ejection fraction (%EF)] and left ventricular inner diameter (LVID) compared with the Dox-treated non-TG mice. However, both MHC-mut-hHsp27(S82A) and MHC-mut-hHsp27(S15A/S78A/S82A) mutants overexpressing TG mice did not show such a cardioprotection. Furthermore, transactivation of p53 was found to be attenuated only in Dox-treated MHC-hHsp27 mice-derived cardiomyocytes in vitro, as low p53 was detected in the nuclei, not in mutant hHsp27 overexpressing cardiomyocytes. Similarly, only in MHC-hHsp27 overexpressing cardiomyocytes, low Bax, higher mechanistic target of rapamycin (mTOR) phosphorylation, and low apoptotic poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (89 kDa fragment) were detected. Pharmacological inhibition of p53 was more effective in mutant TG mice compared with MHC-hHsp27 mice. We conclude that phosphorylation of overexpressed Hsp27 at S82 and its association with p53 are essential for the cardioprotective effect of overexpressed Hsp27 against Dox-induced dilated cardiomyopathy. Only phosphorylated Hsp27 protects the heart by inhibiting p53 transactivation.NEW & NOTEWORTHY Requirement of serine phosphorylation in Hsp27 for cardioprotective effect against Dox is tested in various mutants overexpressing mice. Cardioprotective effect was found to be compromised in Hsp27 serine mutants overexpressed mice compared with wild-type overexpressing mice. These results indicate that cancer patients, who carry these mutations, may have higher risk of aggravated cardiomyopathy on treated with cardiotoxic chemotherapeutics such as doxorubicin.


Assuntos
Apoptose , Cardiomiopatia Dilatada/metabolismo , Doxorrubicina , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Mutação , Miocárdio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Cardiomiopatia Dilatada/induzido quimicamente , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiotoxicidade , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Fosforilação , Serina , Transdução de Sinais
19.
Science ; 373(6556)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385370

RESUMO

The neurotransmitter acetylcholine (ACh) acts in part through a family of nicotinic ACh receptors (nAChRs), which mediate diverse physiological processes including muscle contraction, neurotransmission, and sensory transduction. Pharmacologically, nAChRs are responsible for tobacco addiction and are targeted by medicines for hypertension and dementia. Nicotinic AChRs were the first ion channels to be isolated. Recent studies have identified molecules that control nAChR biogenesis, trafficking, and function. These nAChR accessories include protein and chemical chaperones as well as auxiliary subunits. Whereas some factors act on many nAChRs, others are receptor specific. Discovery of these regulatory mechanisms is transforming nAChR research in cells and tissues ranging from central neurons to spinal ganglia to cochlear hair cells. Nicotinic AChR-specific accessories also enable drug discovery on high-confidence targets for psychiatric, neurological, and auditory disorders.


Assuntos
Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Membrana Celular/metabolismo , Descoberta de Drogas , Retículo Endoplasmático/metabolismo , Humanos , Ligantes , Músculo Esquelético/metabolismo , Neurofarmacologia , Nicotina/metabolismo , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/química
20.
Nat Commun ; 12(1): 4810, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376666

RESUMO

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation.


Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mucosa Intestinal/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Humanos , Mucosa Intestinal/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Ligação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo
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