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1.
Environ Toxicol ; 35(1): 55-65, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31497924

RESUMO

Overexposure to manganese (Mn) is widely known to induce alpha-synuclein (α-Syn) oligomerization, which has been attributed to the oxidative damage of α-Syn protein. Trehalose has been shown to induce autophagy and serve as a chemical chaperone, but little information has been reported about its effect on Mn-induced α-Syn oligomerization. In this study, we investigate whether trehalose can effectively interfere with Mn-induced α-Syn oligomerization, using different concentrations of trehalose (2% and 4% (g/vol [mL])) in a mouse model of manganism. After 6 weeks of exposure to Mn, both oxidative stress and autophagy were activated and resulted in α-Syn oligomerization and neuronal cell damage in the mouse brain tissue. Our results also revealed that pretreatment with trehalose significantly reduced the oxidative damage to α-Syn protein and increased autophagy activation. These findings clearly demonstrated that trehalose can relieve Mn-induced α-Syn oligomerization and neuronal cell damage through its anti-oxidative and autophagy-inducing effects.


Assuntos
Autofagia/efeitos dos fármacos , Manganês/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Trealose/farmacologia , alfa-Sinucleína/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Multimerização Proteica
2.
Adv Exp Med Biol ; 1206: 435-452, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31776997

RESUMO

Protein homeostasis is essential for maintaining cell survival. Protein synthesis and degradation coordinately regulate protein homeostasis. Chaperone-mediated autophagy (CMA) was the first lysosomal process to be discovered by which intracellular components are selectively degraded. This process involves the recognition of the substrate, the unfolding and translocation of the substrate, and the degradation of the substrate. By degrading specific target proteins in a timely manner, CMA is involved in a variety of cellular activities. In the past few years, we have acquired a better understanding of how CMA is regulated. It has been reported that peroxide accumulation, aging and/or other pathological signals interfere with CMA function, which in turn induces neurodegenerative diseases, cancer, and other diseases. Combining results from the current research, we summarize the basic processes, regulatory mechanisms, and physiological functions of CMA and discuss its critical role in the development of diseases.


Assuntos
Autofagia , Chaperonas Moleculares , Humanos , Lisossomos/metabolismo , Chaperonas Moleculares/metabolismo , Neoplasias/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia
3.
J Biochem ; 166(6): 455-462, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31755921

RESUMO

A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.


Assuntos
Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Anticorpos de Cadeia Única/biossíntese , Citoplasma/química , Fluorometria , Chaperonas Moleculares/química , Redobramento de Proteína , Anticorpos de Cadeia Única/química , Ressonância de Plasmônio de Superfície
4.
Nat Commun ; 10(1): 4781, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31636255

RESUMO

Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Cristalização , Técnicas In Vitro , Insetos , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Terciária de Proteína
5.
Postepy Biochem ; 65(3): 173-182, 2019 10 01.
Artigo em Polonês | MEDLINE | ID: mdl-31643164

RESUMO

Endoribonuclease III Dicer plays a crucial role in the biogenesis of small regulatory RNAs, such as microRNAs (miRNAs) and small inter­fering RNAs (siRNAs). However, this is not the only role that Dicer plays in cells. For example, it has been shown that Dicer is involved in processing of diverse classes of RNA, including tRNA and snoRNA, cleavage of repeat-element-derived RNAs, and maintenance of genome integrity. Dicer has also been found to participate in the chromosome fragmentation during apoptosis or in the inflammatory processes. More­over, a recent discovery of Dicer-binding passive sites in mRNAs and long non-coding RNAs, and its putative nucleic acid chaperone activity, has pointed out a novel regulatory role of the enzyme. Here we focus on human Dicer and review its structure and function including recent findings on miRNA-independent roles and their impact on cell biology.


Assuntos
Ribonuclease III/química , Ribonuclease III/metabolismo , Fragmentação do DNA , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/metabolismo
6.
F1000Res ; 82019.
Artigo em Inglês | MEDLINE | ID: mdl-31583082

RESUMO

Hypochlorous acid (HOCl; bleach) is a powerful weapon used by our immune system to eliminate invading bacteria. Yet the way HOCl actually kills bacteria and how they defend themselves from its oxidative action have only started to be uncovered. As this molecule induces both protein oxidation and aggregation, bacteria need concerted efforts of chaperones and antioxidants to maintain proteostasis during stress. Recent advances in the field identified several stress-activated chaperones, like Hsp33, RidA, and CnoX, which display unique structural features and play a central role in protecting the bacterial proteome during HOCl stress.


Assuntos
Bactérias/metabolismo , Ácido Hipocloroso/química , Chaperonas Moleculares/metabolismo , Oxidantes/química , Estresse Fisiológico , Infecções Bacterianas/imunologia , Proteínas de Bactérias/metabolismo , Humanos , Oxirredução , Proteólise
7.
PLoS Pathog ; 15(9): e1008065, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31557263

RESUMO

Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27°C and are unable to survive prolonged exposure to 37°C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.


Assuntos
Proteínas de Protozoários/metabolismo , Tiorredoxinas/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Técnicas de Silenciamento de Genes , Genes de Protozoários , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Oxirredução , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/patogenicidade
8.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514384

RESUMO

Alpha-synuclein (α-Syn) can misfold and aggregate, causing the degeneration of dopaminergic neurons, as seen in Parkinson's disease (PD). We recently demonstrated that DNAJB6, a co-chaperone found in Lewy bodies (LB), suppresses the aggregation of α-Syn in cells and in vitro. In this study, we compared the capacities of DNAJB1 and DNAJB6 to suppress the seeded α-Syn aggregation in HEK293 cells expressing α-Syn tagged with cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). The aggregation of α-Syn was seeded by the transfection of the cells with recombinant α-Syn pre-formed fibrils (PFFs), following the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated knockout (KO) of these two genes, respectively. We quantified the α-Syn aggregation by fluorescence microscopy and fluorescence resonance energy transfer (FRET) analysis. We detected significantly more aggregates in the DNAJB6 KO cells compared with the parental cells, whereas the DNAJB1 KO had no effect on the α-Syn aggregation. This is the first evidence that DNAJB6 can suppress α-Syn aggregation, induced by exogenous α-Syn seeds, in cells. Next, we explored whether this mechanism could be dependent on protein degradation pathways. We observed that the increase in the α-Syn PFF-induced aggregation in the DNAJB6 KO cells compared with the parental cells was strongly diminished upon the incubation of the cells with the proteasomal inhibitor MG132. These results consolidate that DNAJB6 is a suppressor of α-Syn aggregation, and suggest that DNAJB6 may target misfolded and/or aggregated α-Syn for proteasomal degradation.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Agregados Proteicos , alfa-Sinucleína/metabolismo , Células HEK293 , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Inibidores de Proteassoma/farmacologia
9.
Elife ; 82019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31518229

RESUMO

Loss of proteostasis and cellular senescence are key hallmarks of aging, but direct cause-effect relationships are not well understood. We show that most yeast cells arrest in G1 before death with low nuclear levels of Cln3, a key G1 cyclin extremely sensitive to chaperone status. Chaperone availability is seriously compromised in aged cells, and the G1 arrest coincides with massive aggregation of a metastable chaperone-activity reporter. Moreover, G1-cyclin overexpression increases lifespan in a chaperone-dependent manner. As a key prediction of a model integrating autocatalytic protein aggregation and a minimal Start network, enforced protein aggregation causes a severe reduction in lifespan, an effect that is greatly alleviated by increased expression of specific chaperones or cyclin Cln3. Overall, our data show that proteostasis breakdown, by compromising chaperone activity and G1-cyclin function, causes an irreversible arrest in G1, configuring a molecular pathway postulating proteostasis decay as a key contributing effector of cell senescence.


Assuntos
Pontos de Checagem do Ciclo Celular , Senescência Celular , Chaperonas Moleculares/metabolismo , Proteostase , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ciclinas/metabolismo
10.
Nat Commun ; 10(1): 4121, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511508

RESUMO

The functionality of most secreted proteins depends on their assembly into a defined quaternary structure. Despite this, it remains unclear how cells discriminate unassembled proteins en route to the native state from misfolded ones that need to be degraded. Here we show how chaperones can regulate and control assembly of heterodimeric proteins, using interleukin 23 (IL-23) as a model. We find that the IL-23 α-subunit remains partially unstructured until assembly with its ß-subunit occurs and identify a major site of incomplete folding. Incomplete folding is recognized by different chaperones along the secretory pathway, realizing reliable assembly control by sequential checkpoints. Structural optimization of the chaperone recognition site allows it to bypass quality control checkpoints and provides a secretion-competent IL-23α subunit, which can still form functional heterodimeric IL-23. Thus, locally-restricted incomplete folding within single-domain proteins can be used to regulate and control their assembly.


Assuntos
Interleucina-23/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Células COS , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Meia-Vida , Humanos , Interleucina-23/química , Modelos Biológicos , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína
11.
Nat Commun ; 10(1): 4046, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492860

RESUMO

Nucleosomes containing the histone H3 variant CENP-A are the epigenetic mark of centromeres, the kinetochore assembly sites required for chromosome segregation. HJURP is the CENP-A chaperone, which associates with Mis18α, Mis18ß, and M18BP1 to target centromeres and deposit new CENP-A. How these proteins interact to promote CENP-A deposition remains poorly understood. Here we show that two repeats in human HJURP proposed to be functionally distinct are in fact interchangeable and bind concomitantly to the 4:2:2 Mis18α:Mis18ß:M18BP1 complex without dissociating it. HJURP binds CENP-A:H4 dimers, and therefore assembly of CENP-A:H4 tetramers must be performed by two Mis18αß:M18BP1:HJURP complexes, or by the same complex in consecutive rounds. The Mis18α N-terminal tails blockade two identical HJURP-repeat binding sites near the Mis18αß C-terminal helices. These were identified by photo-cross-linking experiments and mutated to separate Mis18 from HJURP centromere recruitment. Our results identify molecular underpinnings of eukaryotic chromosome inheritance and shed light on how centromeres license CENP-A deposition.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteína Centromérica A/química , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Histonas/química , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Ligação Proteica , Interferência de RNA , Homologia de Sequência de Aminoácidos
12.
PLoS Genet ; 15(8): e1008328, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31404065

RESUMO

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5' untranslated regions (5'UTRs) of 20-60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5'UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5'UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.


Assuntos
Proteínas Arqueais/metabolismo , Mathanococcus/fisiologia , Chaperonas Moleculares/metabolismo , RNA Arqueal/metabolismo , Regiões 5' não Traduzidas/genética , Proteínas Arqueais/genética , Genoma Arqueal/genética , Chaperonas Moleculares/genética , Transcrição Genética , Transcriptoma/genética
13.
Nat Commun ; 10(1): 3626, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399574

RESUMO

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.


Assuntos
Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Isoformas de Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Resposta ao Choque Térmico/fisiologia , Ligantes , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
14.
Int J Mol Sci ; 20(17)2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-31450862

RESUMO

Proteins must fold into their native structure and maintain it during their lifespan to display the desired activity. To ensure proper folding and stability, and avoid generation of misfolded conformations that can be potentially cytotoxic, cells synthesize a wide variety of molecular chaperones that assist folding of other proteins and avoid their aggregation, which unfortunately is unavoidable under acute stress conditions. A protein machinery in metazoa, composed of representatives of the Hsp70, Hsp40, and Hsp110 chaperone families, can reactivate protein aggregates. We revised herein the phosphorylation sites found so far in members of these chaperone families and the functional consequences associated with some of them. We also discuss how phosphorylation might regulate the chaperone activity and the interaction of human Hsp70 with its accessory and client proteins. Finally, we present the information that would be necessary to decrypt the effect that post-translational modifications, and especially phosphorylation, could have on the biological activity of the Hsp70 system, known as the "chaperone code".


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Proteínas de Choque Térmico HSP70/química , Humanos , Chaperonas Moleculares/química , Fosforilação , Agregados Proteicos , Ligação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade
15.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140267, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31470132

RESUMO

Spectrin, the major protein component of the erythrocyte membrane skeleton has chaperone like activity and is known to bind membrane phospholipids and hemoglobin. We have probed the chaperone activity of spectrin in presence of hemoglobin and phospholipid SUVs of different compositions to elucidate the effect of phospholipid/hemoglobin binding on chaperone function. It is seen that spectrin displays a preference for hemoglobin over other substrates leading to a decrease in chaperone activity in presence of hemoglobin. A competition is seen to exist between phospholipid binding and chaperone function of spectrin, in a dose dependent manner with the greatest extent of decrease being seen in case of phospholipid vesicles containing aminophospholipids e.g. PS and PE which may have implications in diseases like hereditary spherocytosis where mutation in spectrin is implicated in its detachment from cell membrane. To gain a clearer understanding of the chaperone like activity of spectrin under in-vivo like conditions we have investigated the effect of macromolecular crowders as well as phosphorylation and glycation states on chaperone activity. It is seen that the presence of non-specific, protein and non-protein macromolecular crowders do not appreciably affect chaperone function. Phosphorylation also does not affect the chaperone function unlike glycation which progressively diminishes chaperone activity. We propose a model where chaperone clients adsorb onto spectrin's surface and processes that bind to and occlude these surfaces decrease chaperone activity.


Assuntos
Membrana Eritrocítica/química , Hemoglobinas/química , Chaperonas Moleculares/química , Espectrina/química , Animais , Bovinos , Membrana Eritrocítica/metabolismo , Hemoglobinas/metabolismo , Chaperonas Moleculares/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ovinos , Espectrina/metabolismo
16.
Nat Commun ; 10(1): 3435, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387991

RESUMO

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.


Assuntos
Histona Acetiltransferases/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Acetilação , Domínio Catalítico , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferases/isolamento & purificação , Chaperonas de Histonas/isolamento & purificação , Histonas/isolamento & purificação , Lisina/metabolismo , Chaperonas Moleculares/isolamento & purificação , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo
17.
Chemosphere ; 233: 786-795, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31340409

RESUMO

Microbial volatile organic compounds (mVCs) are formed in the metabolism of microorganisms and widely distributed in nature and pose threats to human health. However, the air pollution by microorganisms is a situation which is poorly understood. In this study, the cytotoxicity of E. aerogenes VCs was evaluated in the model organism Saccharomyces cerevisiae. E. aerogenes VCs inhibited the survival of yeast and triggered the formation of intracellular reactive oxygen species (ROS). The hypersensitive of MAP kinase mpk1/slt2 and 19S regulatory assembly chaperone adc17 mutants to the E. aerogenes VCs indicated cell wall integrity (CWI) pathway together with stress-inducible proteasome assembly regulation are essentially involved in mVCs tolerance mechanism. Furthermore, exposure to the mVCs resulted in the transcriptional upregulation of the CWI pathway, the regulatory particle assembly chaperones, and genes involved in proteasome regulations. Our research suggested that the ROS/MAPK signaling and proteasome regulatory pathway play pivotal roles in the integration and fine-tuning of the mVCs stress response. This study provides a molecular framework for future study of the effects of mVCs on more complex organisms, such as humans.


Assuntos
Enterobacter aerogenes/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Compostos Orgânicos Voláteis/farmacologia , Poluentes Atmosféricos/análise , Poluição do Ar/análise , Parede Celular/metabolismo , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ativação Transcricional
18.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1556-1566, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326538

RESUMO

Chaperone-assisted selective autophagy (CASA) initiated by the cochaperone Bcl2-associated athanogene 3 (BAG3) represents an important mechanism for the disposal of misfolded and damaged proteins in mammalian cells. Under mechanical stress, the cochaperone cooperates with the small heat shock protein HSPB8 and the cytoskeleton-associated protein SYNPO2 to degrade force-unfolded forms of the actin-crosslinking protein filamin. This is essential for muscle maintenance in flies, fish, mice and men. Here, we identify the serine/threonine protein kinase 38 (STK38), which is part of the Hippo signaling network, as a novel interactor of BAG3. STK38 was previously shown to facilitate cytoskeleton assembly and to promote mitophagy as well as starvation and detachment induced autophagy. Significantly, our study reveals that STK38 exerts an inhibitory activity on BAG3-mediated autophagy. Inhibition relies on a disruption of the functional interplay of BAG3 with HSPB8 and SYNPO2 upon binding of STK38 to the cochaperone. Of note, STK38 attenuates CASA independently of its kinase activity, whereas previously established regulatory functions of STK38 involve target phosphorylation. The ability to exert different modes of regulation on central protein homeostasis (proteostasis) machineries apparently allows STK38 to coordinate the execution of diverse macroautophagy pathways and to balance cytoskeleton assembly and degradation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteostase/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Citoesqueleto/metabolismo , Filaminas/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas dos Microfilamentos , Chaperonas Moleculares/metabolismo , Ligação Proteica , Proteômica , Transdução de Sinais , Estresse Mecânico
19.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1544-1555, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326539

RESUMO

Plasma membrane transporter SLC6A14 transports all neutral and basic amino acids in a Na/Cl - dependent way and it is up-regulated in many types of cancer. Mass spectrometry analysis of overexpressed SLC6A14-associated proteins identified, among others, the presence of cytosolic heat shock proteins (HSPs) and co-chaperones. We detected co-localization of overexpressed and native SLC6A14 with HSP90-beta and HSP70 (HSPA14). Proximity ligation assay confirmed a direct interaction of overexpressed SLC6A14 with both HSPs. Treatment with radicicol and VER155008, specific inhibitors of HSP90 and HSP70, respectively, attenuated these interactions and strongly reduced transporter presence at the cell surface, what resulted from the diminished level of the total transporter protein. Distortion of SLC6A14 proper folding by both HSPs inhibitors directed the transporter towards endoplasmic reticulum-associated degradation pathway, a process reversed by the proteasome inhibitor - bortezomib. As demonstrated in an in vitro ATPase assay of recombinant purified HSP90-beta, the peptides corresponding to C-terminal amino acid sequence following the last transmembrane domain of SLC6A14 affected the HSP90-beta activity. These results indicate that a plasma membrane protein folding can be controlled not only by chaperones in the endoplasmic reticulum, but also those localized in the cytosol.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Transporte Proteico/fisiologia , Adenosina Trifosfatases/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Biotinilação , Bortezomib/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Células MCF-7 , Macrolídeos/farmacologia , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Dobramento de Proteína , Transporte Proteico/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia
20.
Nat Commun ; 10(1): 3262, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332180

RESUMO

TorsinA is an ER-resident AAA + ATPase, whose deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is an unusual AAA + ATPase that needs an external activator. Also, it likely does not thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of its still enigmatic function. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 Å structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner channel of ~ 4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA + ATPases. We discuss the implications of these observations for TorsinA function.


Assuntos
Adenosina Trifosfatases/química , Modelos Moleculares , Chaperonas Moleculares/química , Polímeros/química , Conformação Proteica , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Polimerização , Polímeros/metabolismo
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