RESUMO
A rise in temperature triggers a structural change in the human Type I 40 kDa heat shock protein (Hsp40/DnaJ), known as DNAJA1. This change leads to a less compact structure, characterized by an increased presence of solvent-exposed hydrophobic patches and ß-sheet-rich regions. This transformation is validated by circular dichroism, thioflavin T binding, and Bis-ANS assays. The formation of this ß-sheet-rich conformation, which is amplified in the absence of zinc, leads to protein aggregation. This aggregation is induced not only by high temperatures but also by low ionic strength and high protein concentration. The aggregated conformation exhibits characteristics of an amyloidogenic structure, including a distinctive X-ray diffraction pattern, seeding competence (which stimulates the formation of amyloid-like aggregates), cytotoxicity, resistance to SDS, and fibril formation. Interestingly, the yeast Type I Ydj1 also tends to adopt a similar ß-sheet-rich structure under comparable conditions, whereas Type II Hsp40s, whether human or from yeast, do not. Moreover, Ydj1 aggregates were found to be cytotoxic. Studies using DNAJA1- and Ydj1-deleted mutants suggest that the zinc-finger region plays a crucial role in amyloid formation. Our discovery of amyloid aggregation in a C-terminal deletion mutant of DNAJA1, which resembles a spliced homolog expressed in the testis, implies that Type I Hsp40 co-chaperones may generate amyloidogenic species in vivo.
RESUMO
DnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain. However, the interaction is transient, creating a challenge for detailed analysis. We investigated whether it would be possible to gain further understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale is to increase the proximity between the interacting partners to promote their natural interaction and facilitate the characterization of the interaction. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the full-length Hsp70 or in the SBD alone, for instance a higher protective effect against aggregation and being a monomer. Substrate binding also appear to exceed that of SBD alone as revealed by a decreased binding to bis-ANS, a probe for hydrophobic patches. This hypothesis is supported by the structural model created by small angle X-ray scattering, suggesting that the lid subdomain (SBDα) is partially opened in the J-SBD. Collectively, our results suggest a model in which J-domain binding may shift the Hsp70 equilibrium towards the monomer state, exposing hydrophobic sites prone to substrate accommodation.
Assuntos
Proteínas de Choque Térmico HSP70/química , Peptídeos/química , Domínios Proteicos , Sítios de Ligação , Proteínas de Choque Térmico HSP70/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Peptídeos/genética , Ligação Proteica , Espalhamento a Baixo ÂnguloRESUMO
Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the ß4-ß8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.
Assuntos
Calpaína/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Enzimológica da Expressão Gênica , Miócitos Cardíacos/citologia , Cadeia B de alfa-Cristalina/química , Animais , Aorta/metabolismo , Apoptose , Sobrevivência Celular , Transferência Ressonante de Energia de Fluorescência , Inativação Gênica , Homeostase , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Moleculares , Miocárdio/metabolismo , Fosforilação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Estresse Mecânico , Quinases da Família src/metabolismoRESUMO
Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce. This work describes the first cloning of a cDNA predicted to code for a cytosolic Saccharum spp. (sugarcane) Hsp70, named SsHsp70 here, the purification of the recombinant protein and the characterization of its structural conformation in solution by chemical cross-linking coupled to mass spectrometry. The in vivo expression of SsHsp70 in sugarcane extracts was confirmed by Western blot. Recombinant SsHsp70 was monomeric, both ADP and ATP binding increased its stability and it was efficient in cooperating with co-chaperones: ATPase activity was stimulated by Hsp40s, and it aided the refolding of an unfolded polypeptide delivered by a member of the small Hsp family. The structural conformation results favor a model in which nucleotide-free SsHsp70 is highly dynamic and may fluctuate among different conformations that may resemble those in which nucleotide is bound. BIOLOGICAL SIGNIFICANCE: Validation of a sugarcane EST as a true mRNA that encodes a cytosolic Hsp70 (SsHsp70) as confirmed by in vivo expression and characterization of the structure and function of the recombinant protein. SsHsp70 was monomeric, both ADP and ATP binding increased its stability and was efficient in interacting and cooperating with co-chaperones to enhance ATPase activity and refold unfolded proteins. The conformation of nucleotide-free SsHsp70 in solution was much more dynamic than suggested by crystal structures of other Hsp70s. This article is part of a Special Issue entitled: Environmental and structural proteomics.
Assuntos
Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/ultraestrutura , Espectrometria de Massas/métodos , Modelos Químicos , Modelos Moleculares , Saccharum/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Ativação Enzimática , Dados de Sequência Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Cells require a protein quality control (PQC) system to obtain a correct balance between folding and the degradation of incorrectly folded or misfolded proteins. This system maintains protein homeostasis and is essential for life. Key components of the PQC are molecular chaperones, which compose a ubiquitous class of proteins that mediate protein quality control by aiding in both the correct folding of proteins and the elimination of proteins that are misfolded due to cellular stress or mutation. Recent studies showed that protein homeostasis has an important role in nutrition and aging, increasing the relevance of the heat shock response to human health. This review summarizes our current knowledge of the molecular chaperone system and its role in protein homeostasis.
Assuntos
Chaperonas Moleculares/fisiologia , Animais , Homeostase , Humanos , Chaperonas Moleculares/química , Dobramento de Proteína , Deficiências na Proteostase/etiologia , Deficiências na Proteostase/metabolismoRESUMO
Small heat shock proteins (sHsp) constitute an important chaperone family linked to conformational diseases. In plants, sHsps prevent protein aggregation by acting as thermosensors and to enhance cell stress tolerance. SsHsp17.2 and SsHsp17.9 are the most highly expressed class I sHsps in sugarcane. They exist as dodecamers at 20 degrees C and have distinct substrate specificities. Therefore, they are useful models to study how class I SHsps work. Here we present data on the effects of heat on the oligomerization and chaperone activity of SsHsp17.2 and SsHsp17.9. Using several biophysical and biochemical probes, we show that the effects of heat are completely reversible, an important property for proteins that act at heat shock temperatures. SsHsp17.2 and SsHsp17.9 dodecamers dissociated to dimers at temperatures ranging from 40 to 45 degrees C and this dissociation was followed by enhanced chaperone activity. We conclude that high temperature affects the oligomeric state of these chaperones, resulting in enhanced chaperone activity.