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1.
Molecules ; 26(20)2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34684701

RESUMO

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid ß (Aß) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aß (Aß40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aß40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aß40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt ß-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aß40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.


Assuntos
Proteínas 14-3-3/metabolismo , Peptídeos beta-Amiloides/metabolismo , alfa-Sinucleína/metabolismo , Proteínas 14-3-3/fisiologia , Amiloide/metabolismo , Amiloide/fisiologia , Peptídeos beta-Amiloides/fisiologia , Humanos , Chaperonas Moleculares/fisiologia , Agregados Proteicos , Ligação Proteica/fisiologia , Conformação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas/fisiologia , Desdobramento de Proteína , alfa-Sinucleína/fisiologia
2.
Nucleic Acids Res ; 49(11): 6267-6280, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34096575

RESUMO

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.


Assuntos
Chaperonas Moleculares/fisiologia , Splicing de RNA , RNA Mensageiro/metabolismo , Transcrição Genética , Linhagem Celular , Humanos , Íntrons , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/fisiologia , Transcriptoma
3.
Biochim Biophys Acta Proteins Proteom ; 1869(9): 140680, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34051341

RESUMO

Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Choque Térmico/metabolismo , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/fisiologia , Citocinas/metabolismo , Diabetes Mellitus/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/fisiologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Camundongos , Mitocôndrias/fisiologia , Chaperonas Moleculares/fisiologia , Proteômica/métodos , Tapsigargina/farmacologia
5.
J Clin Invest ; 131(6)2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33529159

RESUMO

In inherited neurodevelopmental diseases, pathogenic processes unique to critical periods during early brain development may preclude the effectiveness of gene modification therapies applied later in life. We explored this question in a mouse model of DYT1 dystonia, a neurodevelopmental disease caused by a loss-of-function mutation in the TOR1A gene encoding torsinA. To define the temporal requirements for torsinA in normal motor function and gene replacement therapy, we developed a mouse line enabling spatiotemporal control of the endogenous torsinA allele. Suppressing torsinA during embryogenesis caused dystonia-mimicking behavioral and neuropathological phenotypes. Suppressing torsinA during adulthood, however, elicited no discernible abnormalities, establishing an essential requirement for torsinA during a developmental critical period. The developing CNS exhibited a parallel "therapeutic critical period" for torsinA repletion. Although restoring torsinA in juvenile DYT1 mice rescued motor phenotypes, there was no benefit from adult torsinA repletion. These data establish a unique requirement for torsinA in the developing nervous system and demonstrate that the critical period genetic insult provokes permanent pathophysiology mechanistically delinked from torsinA function. These findings imply that to be effective, torsinA-based therapeutic strategies must be employed early in the course of DYT1 dystonia.


Assuntos
Distonia Muscular Deformante/terapia , Terapia Genética/métodos , Chaperonas Moleculares/genética , Fatores Etários , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Modelos Animais de Doenças , Distonia Muscular Deformante/genética , Distonia Muscular Deformante/fisiopatologia , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/fisiologia , Mutação , Fenótipo , Análise Espaço-Temporal , Fatores de Tempo
6.
J Biol Chem ; 296: 100441, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33610544

RESUMO

The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMPs) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, is the entry point for clients to the guided entry of tail-anchored protein (GET) pathway, which targets endoplasmic reticulum-bound TA IMPs. Consisting of three structurally independent domains, the C terminus of Sgt2 binds to the hydrophobic transmembrane domain (TMD) of clients. However, the exact binding interface within Sgt2 and molecular details that underlie its binding mechanism and client preference are not known. Here, we reveal the mechanism of Sgt2 binding to hydrophobic clients, including TA IMPs. Through sequence analysis, biophysical characterization, and a series of capture assays, we establish that the Sgt2 C-terminal domain is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove approximately 15 Å long that is consistent with our observed higher affinity for client TMDs with a hydrophobic face and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing cochaperone proteins, demonstrating structural and sequence-based similarities to the DP domains in the yeast Hsp90 and Hsp70 coordinating protein, Sti1.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana/fisiologia , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitina/metabolismo
7.
Biochim Biophys Acta Proteins Proteom ; 1869(5): 140615, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33561579

RESUMO

CsaA is known to function as a protein secretion chaperone in bacteria. Homologs of CsaA are also found in archaea while they are absent in eukaryotes. This paper presents the biophysical, biochemical analysis and crystallographic structure determination of CsaA from a thermoacidophilic archaeon Picrophilus torridus (PtCsaA). The PtCsaA appears to prevent the aggregation of heat denatured Bovine Carbonic Anhydrase II (BCAII). Differential denaturation of PtCsaA by guanidine hydrochloride (Gdn-HCl) and urea indicates the stabilization of the protein via salt bridges. Denaturant mediated decrease in 8-Anilinonaphthalene-1-sulfonic acid (ANS) binding and shift in wavelength signifies the partial unfolding of the protein molecule and exposure of hydrophobic patches to solvent on denaturation. The crystal structure of PtCsaA was solved to a resolution of 1.7 Å. The structure of PtCsaA appears to be similar to bacterial CsaA in architecture. Docking of a six amino acid peptide in the substrate binding pocket of PtCsaA suggests conservation in the substrate binding cavity. Residues involved in the formation of the binding cavity and hydrogen bonds responsible for the dimerization of PtCsaA were compared with those observed in the structure of Bacillus subtilis CsaA. The similarities and differences in electrostatic surface potential of the substrate binding cavities in bacterial CsaA and PtCsaA are discussed.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/fisiologia , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiologia , Sequência de Aminoácidos , Bacillus subtilis/química , Proteínas de Bactérias/química , Sítios de Ligação , Dicroísmo Circular/métodos , Cristalografia por Raios X , Dimerização , Temperatura Alta , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta/métodos , Relação Estrutura-Atividade
8.
Acta Neuropathol Commun ; 9(1): 23, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33557929

RESUMO

Mutations in the DNAJB6 gene have been identified as rare causes of myofibrillar myopathies. However, the underlying pathophysiologica mechanisms remain elusive. DNAJB6 has two known isoforms, including the nuclear isoform DNAJB6a and the cytoplasmic isoform DNAJB6b, which was thought to be the pathogenic isoform. Here, we report a novel recessive mutation c.695_699del (p. Val 232 Gly fs*7) in the DNAJB6 gene, associated with an apparently recessively inherited late onset distal myofibrillar myopathy in a Chinese family. Notably, the novel mutation localizes to exon 9 and uniquely encodes DNAJB6a. We further identified that this mutation decreases the mRNA and protein levels of DNAJB6a and results in an age-dependent recessive toxic effect on skeletal muscle in knock-in mice. Moreover, the mutant DNAJB6a showed a dose-dependent anti-aggregation effect on polyglutamine-containing proteins in vitro. Taking together, these findings reveal the pathogenic role of DNAJB6a insufficiency in myofibrillar myopathies and expand upon the molecular spectrum of DNAJB6 mutations.


Assuntos
Miopatias Distais/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Mutação , Miopatias Congênitas Estruturais/genética , Proteínas do Tecido Nervoso/genética , Idoso , Animais , Grupo com Ancestrais do Continente Asiático , Miopatias Distais/diagnóstico por imagem , Miopatias Distais/patologia , Miopatias Distais/fisiopatologia , Técnicas de Introdução de Genes , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Miopatias Congênitas Estruturais/diagnóstico por imagem , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Fenótipo
9.
Sci Rep ; 11(1): 856, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441685

RESUMO

Adequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.


Assuntos
Proteínas Correpressoras/metabolismo , Chaperonas Moleculares/metabolismo , Terapia Viral Oncolítica/métodos , Replicação Viral/fisiologia , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/fisiologia , Proteínas E2 de Adenovirus/metabolismo , Proteínas E2 de Adenovirus/fisiologia , Linhagem Celular Tumoral , Proteínas Correpressoras/fisiologia , Humanos , Chaperonas Moleculares/fisiologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/metabolismo , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Replicação Viral/genética
10.
J Orthop Surg Res ; 16(1): 72, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478532

RESUMO

BACKGROUND: Heat shock protein 22 (HSP22) belongs to class I of the small HSP family that displays ubiquitous expression in osteoblasts. We previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling factor, induces the synthesis of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether HSP22 is implicated in the PGF2α-induced synthesis of IL-6 and VEGF and the mechanism of MC3T3-E1 cells. METHODS: MC3T3-E1 cells were transfected with HSP22-siRNA. IL-6 and VEGF release was assessed by ELISA. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was detected by Western blotting. RESULTS: The PGF2α-induced release of IL-6 in HSP22 knockdown cells was significantly suppressed compared with that in the control cells. HSP22 knockdown also reduced the VEGF release by PGF2α. Phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was attenuated by HSP22 downregulation. CONCLUSIONS: Our results strongly suggest that HSP22 acts as a positive regulator in the PGF2α-induced synthesis of IL-6 and VEGF in osteoblasts.


Assuntos
Dinoprosta/farmacologia , Proteínas de Choque Térmico/fisiologia , Interleucina-6/metabolismo , Chaperonas Moleculares/fisiologia , Osteoblastos/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/farmacologia , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Gene ; 774: 145420, 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33434627

RESUMO

ClpXP in Escherichia coli is a proteasome degrading protein substrates. It consists of one hexamer of ATPase (ClpX) and two heptamers of peptidase (ClpP). The ClpX binds ATP and translocates the substrate protein into the ClpP chamber by binding and hydrolysis of ATP. At single molecular level, ClpX harnesses cycles of power stroke (dwell and burst) to unfold the substrates, then releases the ADP and Pi. Based on the construction and function of ClpXP, especially the recent progress on how ClpX unfold protein substrates, in this mini-review, a currently proposed single ClpX molecular model system detected by optical tweezers, and its prospective for the elucidation of the mechanism of force generation of ClpX in its power stroke and the subunit interaction with each other, were discussed in detail.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/fisiologia , Endopeptidase Clp/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Chaperonas Moleculares/fisiologia , Imagem Individual de Molécula , ATPases Associadas a Diversas Atividades Celulares/química , Pesquisa Biomédica , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Redes e Vias Metabólicas , Mitocôndrias/fisiologia , Modelos Moleculares , Chaperonas Moleculares/química , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Estrutura Molecular , Relação Estrutura-Atividade
12.
Hum Cell ; 34(1): 177-186, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32936424

RESUMO

The aim of this study was to investigate the genes associated with ferroptosis and the progression of hepatocellular carcinoma (HCC). The RNA sequencing data of erastin-induced ferroptosis in HCC cells were downloaded from the Sequence Read Archive database with accession number SRP119173. The microarray dataset GSE89377 of HCC progression was downloaded from the Gene Expression Omnibus database. The ferroptosis-related genes were screened by differential analysis and HCC progression-related genes were screened by cluster analysis using Mfuzz. Then, the genes associated with ferroptosis and HCC progression were screened by Venn analysis, followed by functional enrichment, protein-protein interaction (PPI) analysis, and transcription factor (TF) prediction. Finally, survival analysis was performed using data from the Cancer Genome Atlas database. A total of 33 upregulated and 52 downregulated genes associated with HCC progression and ferroptosis were obtained, and these genes were significantly involved in the negative regulation of ERK1 and ERK2 cascades; the NAD biosynthetic process; alanine, aspartate, and glutamate metabolism; and other pathways. The PPI network contained 52 genes and 78 interactions, of which, cell division cycle 20 (CDC20) and heat shock protein family B (small) member 1 (HSPB1) were hub genes found in higher degrees. Among the 85 genes associated with HCC progression and ferroptosis, two TFs (activating TF 3 (ATF3) and HLF) were predicted, with HSPB1 targeted by ATF3. In addition, 26 genes that were found to be significantly correlated with the overall survival of HCC patients were screened, including CDC20 and thyroid hormone receptor interactor 13. Several genes associated with HCC progression and ferroptosis were screened based on a comprehensive bioinformatics analysis. These genes played roles in HCC progression and ferroptosis via the negative regulation of the ERK1 and ERK2 cascades; the NAD biosynthetic process; and alanine, aspartate, and glutamate metabolism. ATF3 and HSPB1 played important roles in HCC progression and ferroptosis, with HSPB1 possibly regulated by ATF3.


Assuntos
Fator 3 Ativador da Transcrição/fisiologia , Carcinoma Hepatocelular/genética , Ferroptose/genética , Regulação Neoplásica da Expressão Gênica/genética , Estudos de Associação Genética , Proteínas de Choque Térmico/fisiologia , Neoplasias Hepáticas/genética , Chaperonas Moleculares/fisiologia , Alanina/metabolismo , Progressão da Doença , Ácido Glutâmico/metabolismo , Humanos , Neoplasias Hepáticas/mortalidade , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , NAD/biossíntese , Taxa de Sobrevida
13.
Mol Microbiol ; 115(3): 383-394, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33217073

RESUMO

The bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighboring bacterial or eukaryotic cells. Antibacterial effectors are mostly toxins that inhibit the growth of other species and help to dominate the niche. A broad variety of these toxins cause cell lysis of the prey cell by disrupting the cell envelope. Other effectors are delivered into the cytoplasm where they affect DNA integrity, cell division or exhaust energy resources. The modular nature of T6SS machinery allows different means of recruitment of toxic effectors to secreted inner tube and spike components that act as carriers. Toxic effectors can be translationally fused to the secreted components or interact with them through specialized structural domains. These interactions can also be assisted by dedicated chaperone proteins. Moreover, conserved sequence motifs in effector-associated domains are subject to genetic rearrangements and therefore engage in the diversification of the arsenal of toxic effectors. This review discusses the diversity of T6SS secreted toxins and presents current knowledge about their loading on the T6SS machinery.


Assuntos
Proteínas de Bactérias/fisiologia , Chaperonas Moleculares/fisiologia , Sistemas de Secreção Tipo VI/fisiologia , Antibacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Sequência Conservada , Citoplasma/efeitos dos fármacos , Interações Microbianas , Periplasma/efeitos dos fármacos , Domínios Proteicos
14.
Annu Rev Phys Chem ; 72: 143-163, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33321054

RESUMO

α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged ß- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state nuclear magnetic resonance (NMR) and solution NMR, as well as cryo-electron microscopy.


Assuntos
Cristalino/química , Chaperonas Moleculares/química , alfa-Cristalinas/química , Animais , Cristalografia por Raios X , Peixes , Humanos , Cristalino/fisiologia , Chaperonas Moleculares/fisiologia , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Solubilidade , alfa-Cristalinas/fisiologia
15.
Nucleic Acids Res ; 49(1): 206-220, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330942

RESUMO

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.


Assuntos
Chaperonas Moleculares/fisiologia , Complexos Multiproteicos/fisiologia , Elongação Traducional da Cadeia Peptídica/fisiologia , Dobramento de Proteína , Proteostase/fisiologia , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Alelos , Mutação com Perda de Função , Chaperonas Moleculares/genética , Mutação de Sentido Incorreto , Peptidil Transferases/fisiologia , Mutação Puntual , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/fisiologia , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
Am J Hum Genet ; 107(6): 1078-1095, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33217308

RESUMO

The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Doenças Musculares/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Alelos , Animais , Caenorhabditis elegans , Feminino , Variação Genética , Humanos , Mutação com Perda de Função , Masculino , Músculo Esquelético/patologia , Miofibrilas , Miosinas , Sarcômeros/metabolismo , Análise de Sequência de RNA , Transgenes , Sequenciamento Completo do Exoma , Adulto Jovem
17.
Fungal Biol ; 124(12): 1024-1031, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33213782

RESUMO

Oomycetes are fungal-like eukaryotes and many of them are pathogens that threaten natural ecosystems and cause huge financial losses for the aqua- and agriculture industry. Amongst them, Aphanomyces invadans causes Epizootic Ulcerative Syndrome (EUS) in fish which can be responsible for up to 100% mortality in aquaculture. As other eukaryotic pathogens, in order to establish and promote an infection, A. invadans secretes proteins, which are predicted to overcome host defence mechanisms and interfere with other processes inside the host. We investigated the role of Lhs1 which is part of an ER-resident complex that generally promotes the translocation of proteins from the cytoplasm into the ER for further processing and secretion. Interestingly, proteomic studies reveal that only a subset of virulence factors are affected by the silencing of AiLhs1 in A. invadans indicating various secretion pathways for different proteins. Importantly, changes in the secretome upon silencing of AiLhs1 significantly reduces the virulence of A. invadans in the infection model Galleriamellonella. Furthermore, we show that AiLhs1 is important for the production of zoospores and their cluster formation. This renders proteins required for protein ER translocation as interesting targets for the potential development of alternative disease control strategies in agri- and aquaculture.


Assuntos
Aphanomyces , Doenças dos Peixes , Chaperonas Moleculares/fisiologia , Virulência , Animais , Aphanomyces/patogenicidade , Doenças dos Peixes/microbiologia , Proteômica
18.
Life Sci Alliance ; 3(11)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32913112

RESUMO

Histone acetylation is one of many posttranslational modifications that affect nucleosome accessibility. Vps75 is a histone chaperone that stimulates Rtt109 acetyltransferase activity toward histones H3-H4 in yeast. In this study, we use sedimentation velocity and light scattering to characterize various Vps75-Rtt109 complexes, both with and without H3-H4. These complexes were previously ill-defined because of protein multivalency and oligomerization. We determine both relative and absolute stoichiometry and define the most pertinent and homogeneous complexes. We show that the Vps75 dimer contains two unequal binding sites for Rtt109, with the weaker binding site being dispensable for H3-H4 acetylation. We further show that the Vps75-Rtt109-(H3-H4) complex is in equilibrium between a 2:1:1 species and a 4:2:2 species. Using a dimerization mutant of H3, we show that this equilibrium is mediated by the four-helix bundle between the two copies of H3. We optimize the purity, yield, and homogeneity of Vps75-Rtt109 complexes and determine optimal conditions for solubility when H3-H4 is added. Our comprehensive biochemical and biophysical approach ultimately defines the large-scale preparation of Vps75-Rtt109-(H3-H4) complexes with precise stoichiometry. This is an essential prerequisite for ongoing high-resolution structural and functional analysis of this important multi-subunit complex.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/química , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X/métodos , Dimerização , Histona Acetiltransferases/genética , Histona Acetiltransferases/fisiologia , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia
19.
Biochem Biophys Res Commun ; 533(3): 613-619, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-32981679

RESUMO

c-Myc modulator 1 (MM1), also known as PFDN5, is the fifth subunit of prefoldin. It was previously reported that MM1-based prefoldin promotes folding of actin during assembly of cytoskeleton, which plays key roles in cell migration. However, no evidence supports that MM1 affects cell migration. In the present study, we found that MM1 promotes cell migration in multiple cell lines. Further study revealed that MM1 promotes polymerization of ß-actin into filamentous form and increases both density and length of filopodia. Effects of MM1 on filopodia formation and cell migration depend on its prefoldin activity. Though c-Myc is repressed by MM1, simultaneous knock-down of c-Myc fails to rescue migration inhibition induced by MM1 ablation. Taken together, we here, for the first time, report that prefoldin subunit MM1 is involved in cell migration; this involvement of MM1 in cell migration is due to its prefoldin activity to boost polymerization of ß-actin during filopodia formation. Our findings may be helpful to elucidate the mechanism of cell migration and cancer metastasis.


Assuntos
Movimento Celular , Chaperonas Moleculares/fisiologia , Pseudópodes/metabolismo , Actinas/metabolismo , Linhagem Celular , Humanos , Chaperonas Moleculares/metabolismo , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia
20.
FASEB J ; 34(10): 13561-13572, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32844451

RESUMO

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/fisiologia , Córion/fisiologia , Proteínas de Insetos/fisiologia , Folículo Ovariano/fisiologia , Rhodnius/fisiologia , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Doença de Chagas , Retículo Endoplasmático/fisiologia , Feminino , Proteínas de Insetos/deficiência , Chaperonas Moleculares/fisiologia
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