RESUMO
Heat shock proteins are constitutively expressed chaperones induced by cellular stress, such as changes in temperature, pH, and osmolarity. These proteins, present in all organisms, are highly conserved and are recruited for the assembly of protein complexes, transport, and compartmentalization of molecules. In fungi, these proteins are related to their adaptation to the environment, their evolutionary success in acquiring new hosts, and regulation of virulence and resistance factors. These characteristics are interesting for assessment of the host adaptability and ecological transitions, given the emergence of infections by these microorganisms. Based on phylogenetic inferences, we compared the sequences of HSP9, HSP12, HSP30, HSP40, HSP70, HSP90, and HSP110 to elucidate the evolutionary relationships of different fungal organisms to suggest evolutionary patterns employing the maximum likelihood method. By the different reconstructions, our inference supports the hypothesis that these classes of proteins are associated with pathogenic gains against endothermic hosts, as well as adaptations for phytopathogenic fungi.
Assuntos
Proteínas de Choque Térmico , Chaperonas Moleculares , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Filogenia , Sequência de Aminoácidos , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismoRESUMO
J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.
Assuntos
Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico , Humanos , Adenosina Trifosfatases/metabolismo , Ácido Edético , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico HSP70/química , Chaperonas Moleculares/químicaRESUMO
Molecular chaperones aid proteins to fold and assemble without modifying their final structure, requiring, in several folding processes, the interplay between members of the Hsp70 and Hsp40 families. Here, we report the NMR chemical shift assignments for 1 H, 15 N, and 13 C nuclei of the backbone and side chains of the J-domain of the class B Hsp40 from Saccharomyces cerevisiae, Sis1, complexed with the C-terminal EEVD motif of Hsp70. The data revealed information on the structure and backbone dynamics that add significantly to the understanding of the J-domain-Hsp70-EEVD mechanism of interaction.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Ligação Proteica , Ressonância Magnética Nuclear Biomolecular , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos/químicaRESUMO
BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.
Assuntos
Proteínas de Choque Térmico , Nucleotídeos , Humanos , Nucleotídeos/metabolismo , Temperatura , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70/química , Adenosina Trifosfatases/química , Ligação ProteicaRESUMO
New SARS-CoV-2 variants emerged in the United Kingdom and South Africa in December 2020 in concomitant with the Brazillian variant in February 2021 (B.1.1.248 lineage) and currently sparking worldwide during the last few months. The new strain 501.V2 in South Africa bears three mutations in the spike receptor-binding domain (RBD); K417 N, E484K, and N501Y, while the Brazilian B.1.1.248 lineage has 12 mutations. In the current study, we simulate the complex ACE2-SARS-CoV-2 spike RBD system in which the RBD is in the wild-type and mutated isoforms. Additionally, the cell-surface Glucose Regulated Protein 78 (CS-GRP78) associated with the ACE2-SARS-CoV-2 spike RBD complex (ACE2-S RBD) is modeled at the presence of these mutant variants of the viral spike. The results showed that E484K and N501Y are critical in viral spike recognition through either ACE2 or CS-GRP78. The mutated variants (the UK, South African, and Brazilian) of the spike RBD tightly bind to GRP78 more than in the case of the wild-type RBD. These results point to the potent role of GRP78 with ACE2 in the attachment of the new variants, which could be a key for the design of inhibitors to block SARS-CoV-2 attachment and entry to the host cell.
Assuntos
Simulação por Computador , Proteínas de Choque Térmico/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Brasil , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Receptores Virais/química , Receptores Virais/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , África do Sul , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Especificidade por Substrato , Reino Unido , Internalização do VírusRESUMO
The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (a) functions as a receptor for many ligands, and (b) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF), and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders.
Assuntos
Doenças Autoimunes do Sistema Nervoso/imunologia , COVID-19/transmissão , Proteínas de Choque Térmico/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Virais/fisiologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes do Sistema Nervoso/metabolismo , Sobrevivência Celular , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/fisiologia , Exossomos , Proteínas Ligadas por GPI/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/imunologia , Humanos , Ligantes , Invasividade Neoplásica , Proteínas de Neoplasias/imunologia , Proteínas do Tecido Nervoso/imunologia , Domínios Proteicos , Transporte Proteico , Transdução de Sinais , Microambiente Tumoral , Resposta a Proteínas não Dobradas/fisiologia , Internalização do VírusRESUMO
Recent reports have shown that small and big felines could be infected by SARS-CoV-2, while other animals, like swines and mice, are apparently not susceptible to this infection. These findings raise the question of the role of cell factors associated with early stages of the viral infection in host selectivity. The cellular receptor for SARS-CoV-2 is the Angiotensin Converting Enzyme (ACE2). Transmembrane protease serine 2 (TMPRSS2) has been shown to prime the viral spike for its interaction with its receptor. GRP78 has also been proposed as a possible co-receptor. In this study, we used several bioinformatics approaches to bring clues in the interaction of ACE2, TMPRSS2, and GRP78 with SARS-CoV-2. We selected several mammalian hosts that could play a key role in viral spread by acting as secondary hosts (cats, dogs, pigs, mice, and ferrets) and evaluated their predicted permissiveness by in silico analysis. Results showed that ionic pairs (salt bridges, N-O pair, and long-range interactions) produced between ACE2 and the viral spike has an essential function in the host interaction. On the other hand, TMPRSS2 and GRP78 are proteins with high homology in all the evaluated hosts. Thus, these proteins do not seem to play a role in host selectivity, suggesting that other factors may play a role in the non-permissivity in some of these hosts. These proteins represent however interesting cell targets that could be explored in order to control the virus replication in humans and in the intermediary hosts.
Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Proteínas de Choque Térmico/química , Mamíferos/metabolismo , Peptidil Dipeptidase A/química , Pneumonia Viral/virologia , Receptores Virais/química , Serina Endopeptidases/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Tropismo Viral , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Antivirais/farmacologia , Benzamidinas , COVID-19 , Gatos , Cães , Chaperona BiP do Retículo Endoplasmático , Furões , Guanidinas/farmacologia , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/metabolismo , Conformação Proteica , Receptores Virais/metabolismo , SARS-CoV-2 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo , Especificidade da Espécie , Suínos , Ligação Viral , Internalização do VírusRESUMO
HOP is a cochaperone belonging to the foldosome, a system formed by the cytoplasmic Hsp70 and Hsp90 chaperones. HOP acts as an adapter protein capable of transferring client proteins from the first to the second molecular chaperone. HOP is a modular protein that regulates the ATPase activity of Hsp70 and Hsp90 to perform its function. To obtain more detailed information on the structure and function of this protein, we produced the recombinant HOP of Plasmodium falciparum (PfHOP). The protein was obtained in a folded form, with a high content of α-helix secondary structure. Unfolding experiments showed that PfHOP unfolds through two transitions, suggesting the presence of at least two domains with different stabilities. In addition, PfHOP primarily behaved as an elongated dimer in equilibrium with the monomer. Small-angle X-ray scattering data corroborated this interpretation and led to the reconstruction of a PfHOP ab initio model as a dimer. Finally, the PfHOP protein was able to inhibit and to stimulate the ATPase activity of the recombinant Hsp90 and Hsp70-1, respectively, of P. falciparum. Our results deepened the knowledge of the structure and function of PfHOP and further clarified its participation in the P. falciparum foldosome.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Choque Térmico/química , Proteínas de Protozoários/química , Proteínas de Choque Térmico/genética , Modelos Moleculares , Plasmodium falciparum , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes/químicaRESUMO
Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor, a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sorghum/química , Produtos Agrícolas , Proteínas de Choque Térmico/química , Humanos , Conformação Molecular , Proteínas de Plantas/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Chaperones belonging to the small heat shock protein (sHSP) family are ubiquitous and exhibit elevated expression under stresses conditions to protect proteins against aggregation, thereby contributing to the stress tolerance of the organism. Tropical plants are constantly exposed to high temperatures, and the mechanisms by which these plants tolerate heat stress are of foremost importance to basic science as well as applied agrobiotechnology. Therefore, this study aims to characterize sHSPs from different organelles from sugarcane, an important crop that is associated with sugar and bioenergy production. An expression sequence tag database of sugarcane was searched, and sHsp genes of mitochondrial and chloroplast organelles were selected and cloned. The proteins were expressed in Escherichia coli and isolated and purified by two chromatographic steps with high purity as single species. Circular dichroism and fluorescence spectroscopy showed that both proteins were purified in their folded states with a predominant ß-sheet secondary structure. Determination of the molecular weight, diffusion coefficient and Stokes radius parameters showed that both chaperones form large spherical-like oligomers in solution. The two sHSPs had different oligomeric states and substrate specificities. The mitochondrial sHSP was a 20-mer with ability to protect model substrates that differ from that of the 16-meric sHSP from chloroplasts. These results indicate that both sHSPs are key agents to protect against stress confirming the importance of the great diversity of sHSP chaperones in plants for homeostasis maintenance. Moreover, to our knowledge, this is the first report about small HSPs from sugarcane organelles.