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1.
Cells ; 10(12)2021 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-34944015

RESUMO

The inflammatory response of macrophages is an orderly and complex process under strict regulation accompanied by drastic changes in morphology and functions. It is predicted that proteins will undergo structural changes during these finely regulated processes. However, changes in structural proteome in macrophages during the inflammatory response remain poorly characterized. In the present study, we applied limited proteolysis coupled mass spectrometry (LiP-MS) to identify proteome-wide structural changes in lipopolysaccharide (LPS)-activated macrophages. We identified 386 structure-specific proteolytic fingerprints from 230 proteins. Using the Gene Ontology (GO) biological process enrichment, we discovered that proteins with altered structures were enriched into protein folding-related terms, in which HSP60 was ranked as the most changed protein. We verified the structural changes in HSP60 by using cellular thermal shift assay (CETSA) and native CETSA. Our results showed that the thermal stability of HSP60 was enhanced in activated macrophages and formed an HSP10-less complex. In conclusion, we demonstrate that in situ structural systems biology is an effective method to characterize proteomic structural changes and reveal that the structures of chaperone proteins vary significantly during macrophage activation.


Assuntos
Proteínas de Choque Térmico/química , Ativação de Macrófagos , Macrófagos/metabolismo , Animais , Chaperonina 60/química , Chaperonina 60/metabolismo , Ontologia Genética , Proteínas de Choque Térmico/metabolismo , Espectrometria de Massas , Camundongos , Análise de Componente Principal , Proteólise , Proteoma/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo
2.
Int J Mol Sci ; 22(19)2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34639171

RESUMO

Diabetes is a major risk factor for cardiovascular diseases, especially cardiomyopathy, a condition in which the smooth muscles of the heart become thick and rigid, affecting the functioning of cardiomyocytes, the contractile cells of the heart. Uncontrolled elevated glucose levels over time can result in oxidative stress, which could lead to inflammation and altered epigenetic mechanisms. In the current study, we investigated whether hyperglycemia can modify cardiac function by directly affecting these changes in cardiomyocytes. To evaluate the adverse effect of high glucose, we measured the levels of gap junction protein, connexin 43, which is responsible for modulating cardiac electric activities and Troponin I, a part of the troponin complex in the heart muscles, commonly used as cardiac markers of ischemic heart disease. AC16 human cardiomyocyte cells were used in this study. Under hyperglycemic conditions, these cells demonstrated altered levels of connexin 43 and Troponin-I after 24 h of exposure. We also examined hyperglycemia induced changes in epigenetic markers: H3K9me1, Sirtuin-1 (SIRT1), and histone deacetylase (HDAC)-2 as well as in inflammatory and stress-related mediators, such as heat shock protein (HSP)-60, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1 and CXC chemokine receptor (CXCR)-4. Cardiomyocytes exposed to 25mM glucose resulted in the downregulation of HSP60 and SIRT1 after 48 h. We further examined that hyperglycemia mediated the decrease in the gap junction protein CX43, as well as CXC chemokine receptor CXCR4 which may affect the physiological functions of the cardiomyocytes when exposed to high glucose for 24 and 48 h. Upregulated expression of DNA-binding nuclear protein HMGB1, along with changes in histone methylation marker H3K9me1 have demonstrated hyperglycemia-induced damage to cardiomyocyte at 24 h of exposure. Our study established that 24 to 48 h of hyperglycemic exposure could stimulate stress-mediated inflammatory mediators in cardiomyocytes in vitro. These stress-related changes in hyperglycemia-induced cardiomyocytes may further initiate an increase in injury markers which eventually could alter the epigenetic processes. Therefore, epigenetic and inflammatory mechanisms in conjunction with alterations in a downstream signaling pathway could have a direct effect on the functionality of the cardiomyocytes exposed to high glucose during short and long-term exposures.


Assuntos
Biomarcadores/metabolismo , Epigênese Genética , Hiperglicemia/fisiopatologia , Mediadores da Inflamação/metabolismo , Miócitos Cardíacos/patologia , Estresse Fisiológico , Chaperonina 60/genética , Chaperonina 60/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo
3.
Sci Rep ; 11(1): 14809, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285302

RESUMO

Human mitochondrial chaperonin mHsp60 is essential for mitochondrial function by assisting folding of mitochondrial proteins. Unlike the double-ring bacterial GroEL, mHsp60 exists as a heptameric ring that is unstable and dissociates to subunits. The structural dynamics has been implicated for a unique mechanism of mHsp60. We purified active heptameric mHsp60, and determined a cryo-EM structure of mHsp60 heptamer at 3.4 Å. Of the three domains, the equatorial domains contribute most to the inter-subunit interactions, which include a four-stranded ß sheet. Our structural comparison with GroEL shows that mHsp60 contains several unique sequences that directly decrease the sidechain interactions around the ß sheet and indirectly shorten ß strands by disengaging the backbones of the flanking residues from hydrogen bonding in the ß strand conformation. The decreased inter-subunit interactions result in a small inter-subunit interface in mHsp60 compared to GroEL, providing a structural basis for the dynamics of mHsp60 subunit association. Importantly, the unique sequences are conserved among higher eukaryotic mitochondrial chaperonins, suggesting the importance of structural dynamics for eukaryotic chaperonins. Our structural comparison with the single-ring mHsp60-mHsp10 shows that upon mHsp10 binding the shortened inter-subunit ß sheet is restored and the overall inter-subunit interface of mHsp60 increases drastically. Our structural basis for the mHsp10 induced stabilization of mHsp60 subunit interaction is consistent with the literature that mHsp10 stabilizes mHsp60 quaternary structure. Together, our studies provide structural bases for structural dynamics of the mHsp60 heptamer and for the stabilizing effect of mHsp10 on mHsp60 subunit association.


Assuntos
Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas da Gravidez/química , Proteínas da Gravidez/metabolismo , Fatores Supressores Imunológicos/química , Fatores Supressores Imunológicos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína
4.
Front Immunol ; 12: 647987, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248935

RESUMO

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.


Assuntos
Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/metabolismo , Chaperonina 60/administração & dosagem , Chaperonina 60/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Lactococcus lactis/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Mycobacterium leprae/enzimologia , Administração Oral , Animais , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Feminino , Inflamação/tratamento farmacológico , Inflamação/imunologia , Lactococcus lactis/genética , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia
5.
Biochem Biophys Res Commun ; 569: 17-22, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34216993

RESUMO

The pathogenesis of Parkinson's disease (PD) remains elusive, but mitochondrial dysfunction is believed to be one crucial step in its pathogenesis. The mitochondrial unfolded protein response (UPRmt) is an important mitochondrial quality control strategy that maintains mitochondrial function in response to disturbances of mitochondrial protein homeostasis. Activation of the UPRmt and the beneficial effect of rescuing mitochondrial proteostasis have been reported in several genetic models of PD. However, the pathogenic relevance of the UPRmt in idiopathic PD is unknown. The present study examined the link between the UPRmt and mitochondrial dysfunction in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells. Treatment with MPP + induced activation of the UPRmt, reflected by an increase in the expression of UPRmt-related chaperones, proteases, and transcription mediators. UPRmt activation that was induced by overexpressing mutant ornithine transcarbamylase significantly reduced the production of mitochondrial reactive oxygen species (ROS) and improved cell survival in SH-SY5Y cells following MPP+ treatment. Moreover, the overexpression of activating transcription factor 5 (mammalian UPRmt transcription factor) conferred protection against MPP+-induced ROS production and against cell death in SH-SY5Y cells. Overall, our results demonstrate the beneficial effect of UPRmt activation in MPP + -treated cells, shedding new light on the mechanism of mitochondrial dysfunction in the pathogenesis of PD.


Assuntos
1-Metil-4-fenilpiridínio/farmacologia , Mitocôndrias/metabolismo , Modelos Biológicos , Doença de Parkinson/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fatores Ativadores da Transcrição/genética , Fatores Ativadores da Transcrição/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Chaperonina 60/genética , Chaperonina 60/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/genética , Substâncias Protetoras/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Resposta a Proteínas não Dobradas/genética
6.
J Phys Chem Lett ; 12(24): 5723-5730, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34129341

RESUMO

The GroE molecular chaperone system is a critical protein machine that assists the folding of substrate proteins in its cavity. Water in the cavity is suspected to play a role in substrate protein folding, but the mechanism is currently unknown. Herein, we report measurements of water dynamics in the equatorial and apical domains of the GroEL cavity in the apo and football states, using site-specific tryptophanyl mutagenesis as an intrinsic optical probe with femtosecond resolution combined with molecular dynamics simulations. We observed clearly different water dynamics in the two domains with a slowdown of the cavity water from the apical to equatorial region in the football state. The results suggest that the GroEL cavity provides a unique water environment that may facilitate substrate protein folding.


Assuntos
Chaperonina 60/química , Chaperonina 60/metabolismo , Água/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Cinética , Modelos Moleculares , Domínios Proteicos
7.
J Biol Chem ; 296: 100744, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33957121

RESUMO

This review contains a personal account of the role played by the PDB in the development of the field of molecular chaperones and protein homeostasis, from the viewpoint of someone who experienced the concurrent advances in the structural biology, electron microscopy, and chaperone fields. The emphasis is on some key structures, including those of Hsp70, GroEL, Hsp90, and small heat shock proteins, that were determined as the molecular chaperone concept and systems for protein quality control were emerging. These structures were pivotal in demonstrating how seemingly nonspecific chaperones could assist the specific folding pathways of a variety of substrates. Moreover, they have provided mechanistic insights into the ATPase machinery of complexes such as GroEL/GroES that promote unfolding and folding and the disaggregases that extract polypeptides from large aggregates and disassemble amyloid fibers. The PDB has provided a framework for the current success in curating, evaluating, and distributing structural biology data, through both the PDB and the EMDB.


Assuntos
Chaperonina 10 , Chaperonina 60 , Bases de Dados de Proteínas , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico HSP90 , Proteólise , Animais , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos
8.
Bioorg Med Chem ; 40: 116129, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33971488

RESUMO

Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.


Assuntos
Antineoplásicos/farmacologia , Benzoxazóis/farmacologia , Chaperonina 10/antagonistas & inibidores , Chaperonina 60/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Salicilanilidas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Benzoxazóis/síntese química , Benzoxazóis/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Salicilanilidas/síntese química , Salicilanilidas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
9.
PLoS One ; 16(4): e0249358, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857160

RESUMO

Chlamydia trachomatis infects squamous and columnar epithelia at the mucosal surface. Research on gene expression patterns of C. trachomatis has predominantly focused on non-native host cells, with limited data on growth kinetics and gene expression of chlamydia in keratinocytes. Here, we investigated whether early, mid, and late chlamydial genes observed in HeLa cell line studies were co-ordinately regulated at the transcriptional level even in the keratinized cell line model and whether the expression was stage-specific during the developmental cycle. HaCaT cell lines were infected with chlamydia clinical isolates (US151and serovar E) and reference strain (L2 434). Expression of groEL-1, incB, pyk-F, tal, hctA, and omcB genes was conducted with comparative real-time PCR and transcriptional events during the chlamydial developmental cycle using transmission electron microscopy. The relative expression level of each gene and fold difference were calculated using the 2-ΔΔCT method. The expression of groEL-1 and pyk-F genes was highest at 2 hours post-infection (hpi) in the L2 434 and serovar E. The expression of incB gene increased at 2 hpi in L2 434 and serovar E but peaked at 12 hpi in serovar E. L2 434 and US151 had similar tal expression profiles. Increased expression of hctA and omcB genes were found at 2 and 36 hpi in L2 434. Both clinical isolates and reference strains presented the normal chlamydial replication cycle comprising elementary bodies and reticulate bodies within 36 hpi. We show different gene expression patterns between clinical isolates and reference strain during in vitro infection of keratinocytes, with reference strain-inducing consistent expression of genes. These findings confirm that keratinocytes are appropriate cell lines to interrogate cell differentiation, growth kinetics, and gene expression of C. trachomatis infection. Furthermore, more studies with different clinical isolates and genes are needed to better understand the Chlamydial pathogenesis in keratinocytes.


Assuntos
Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Regulação Bacteriana da Expressão Gênica , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Chaperonina 60/genética , Chaperonina 60/metabolismo , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Fatores de Tempo , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo
10.
Int J Mol Sci ; 22(8)2021 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-33919591

RESUMO

Thyroid cancers are the most common of the endocrine system malignancies and progress must be made in the areas of differential diagnosis and treatment to improve patient management. Advances in the understanding of carcinogenic mechanisms have occurred in various fronts, including studies of the chaperone system (CS). Components of the CS are found to be quantitatively increased or decreased, and some correlations have been established between the quantitative changes and tumor type, prognosis, and response to treatment. These correlations provide the basis for identifying distinctive patterns useful in differential diagnosis and for planning experiments aiming at elucidating the role of the CS in tumorigenesis. Here, we discuss studies of the CS components in various thyroid cancers (TC). The chaperones belonging to the families of the small heat-shock proteins Hsp70 and Hsp90 and the chaperonin of Group I, Hsp60, have been quantified mostly by immunohistochemistry and Western blot in tumor and normal control tissues and in extracellular vesicles. Distinctive differences were revealed between the various thyroid tumor types. The most frequent finding was an increase in the chaperones, which can be attributed to the augmented need for chaperones the tumor cells have because of their accelerated metabolism, growth, and division rate. Thus, chaperones help the tumor cell rather than protect the patient, exemplifying chaperonopathies by mistake or collaborationism. This highlights the need for research on chaperonotherapy, namely the development of means to eliminate/inhibit pathogenic chaperones.


Assuntos
Chaperonas Moleculares/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Animais , Chaperonina 60/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos
11.
FASEB J ; 35(2): e21328, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33433932

RESUMO

To date, there are limited and incomplete data on possible sex-based differences in fiber-types of skeletal muscle and their response to physical exercise. Adult healthy male and female mice completed a single bout of endurance exercise to examine the sex-based differences of the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), heat shock protein 60 (Hsp60), interleukin 6 (IL-6) expression, as well as the Myosin Heavy Chain (MHC) fiber-type distribution in soleus and extensor digitorum longus (EDL) muscles. Our results showed for the first time that in male soleus, a muscle rich of type IIa fibers, endurance exercise activates specifically genes involved in mitochondrial biogenesis such as PGC1 α1 isoform, Hsp60 and IL-6, whereas the expression of PGC1 α2 and α3 was significantly upregulated in EDL muscle, a fast-twitch skeletal muscle, independently from the gender. Moreover, we found that the acute response of different PGC1α isoforms was muscle and gender dependent. These findings add a new piece to the huge puzzle of muscle response to physical exercise. Given the importance of these genes in the physiological response of the muscle to exercise, we strongly believe that our data could support future research studies to personalize a specific and sex-based exercise training protocol.


Assuntos
Atividade Motora , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Chaperonina 60/genética , Chaperonina 60/metabolismo , Feminino , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores Sexuais
12.
mBio ; 12(1)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436430

RESUMO

As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coli groESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coli groESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.


Assuntos
Chaperonina 10/genética , Chaperonina 60/genética , Escherichia coli/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Enterobacter/efeitos dos fármacos , Enterobacter/genética , Enterobacter/metabolismo , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Cinética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
13.
Biochemistry ; 60(6): 460-464, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33464880

RESUMO

The Escherichia coli ATP-consuming chaperonin machinery, a complex between GroEL and GroES, has evolved to facilitate folding of substrate proteins (SPs) that cannot do so spontaneously. A series of kinetic experiments show that the SPs are encapsulated in the GroEL/ES nanocage for a short duration. If confinement of the SPs is the mechanism by which GroEL/ES facilitates folding, it follows that the assisted folding rate, relative to the bulk value, should always be enhanced. Here, we show that this is not the case for the folding of rhodanese in the presence of the full machinery of GroEL/ES and ATP. The assisted folding rate of rhodanese decreases. On the basis of our finding and those reported in other studies, we suggest that the ATP-consuming chaperonin machinery has evolved to optimize the product of the folding rate and the yield of the folded SPs on the biological time scale. Neither the rate nor the yield is separately maximized.


Assuntos
Chaperonina 10/química , Chaperonina 60/química , Dobramento de Proteína , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Conformação Proteica
14.
Microscopy (Oxf) ; 70(3): 289-296, 2021 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-33173948

RESUMO

Escherichia coli chaperonin GroEL, which is a large cylindrical protein complex comprising two heptameric rings with cavities of 4.5 nm each in the center, assists in intracellular protein folding with the aid of GroES and adenosine triphosphate (ATP). Here, we investigated the possibility that GroEL can also encapsulate metal nanoparticles (NPs) up to ∼5 nm in diameter into the cavities with the aid of GroES and ATP. The slow ATP-hydrolyzing GroELD52A/D398A mutant, which forms extremely stable complexes with GroES (half-time of ∼6 days), made it possible to analyze GroEL/GroES complexes containing metal NPs. Scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy analysis proved distinctly that FePt NPs and Au NPs were encapsulated in the GroEL/GroES complexes. Dynamic light scattering measurements showed that the NPs in the GroEL/GroES complex were able to maintain their dispersibility in solution. We previously described that the incubation of GroEL and GroES in the presence of ATP·BeFx and adenosine diphosphate·BeFx resulted in the formation of symmetric football-shaped and asymmetric bullet-shaped complexes, respectively. Based on this knowledge, we successfully constructed the football-shaped complex in which two compartments were occupied by Pt or Au NPs (first compartment) and FePt NPs (second compartment). This study showed that metal NPs were sequentially encapsulated according to the GroEL reaction in a step-by-step manner. In light of these results, chaperonin can be used as a tool for handling nanomaterials.


Assuntos
Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Nanopartículas Metálicas/química , Trifosfato de Adenosina/metabolismo , Chaperonina 60/genética , Chaperoninas/química , Chaperoninas/genética , Chaperoninas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ouro/química , Ouro/metabolismo , Ferro/química , Ferro/metabolismo , Microscopia Eletrônica de Transmissão e Varredura , Microscopia Eletrônica de Transmissão , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Platina/química , Platina/metabolismo , Ligação Proteica , Dobramento de Proteína
15.
Biochim Biophys Acta Gen Subj ; 1865(1): 129758, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33031906

RESUMO

BACKGROUND: Bacterial surface proteins act as potential adhesins or invasins. The GroEL is a signal peptide-free surface expressed protein that aids adhesion in Escherichia coli by binding to LOX-1 receptor of the host cells. Mycobacterium tuberculosis (Mtb) expresses GroEL2 protein, having high level sequence identity with E. coli GroEL. This study investigates the interaction mechanism of GroEL2 protein of Mtb with LOX-1 of macrophages using integrated computational and experimental approach. METHODS: Mtb GroEL2 protein was purified as histidine tagged protein using Ni-NTA chromatography. Confocal and scanning electron microscopies were used to study the uptake of GroEL2 coated fluorescent latex beads through the LOX-1 receptor in RAW264.7 macrophage cell line. Docking studies were performed to understand the interaction between the GroEL2 and LOX-1 proteins. Polyinosinic acid (PIA) was used as a LOX-1 inhibitor in both in silico and in vitro experiments. RESULTS: GroEL2 protein coating enhances uptake of latex beads into macrophages through LOX-1 receptor. LOX-1 inhibitor PIA decreased the uptake of GroEL2 coated latex beads. GroEL2 interacts with the key ligand binding regions of the LOX-1 receptor, such as the basic spine and the saddle hydrophobic patch. PIA molecule destabilized the LOX-1-GroEL2 docked complex. CONCLUSION: Surface associated GroEL2 protein of Mtb is a potential ligand for macrophage LOX-1 receptor. Interaction between GroEL2 and LOX-1 receptor may be utilized by Mtb to gain its intracellular access. GENERAL SIGNIFICANCE: Surface associated GroEL2 of Mtb may bind to the macrophage LOX-1 receptor, enabling the internalization of the bacteria and progression of the infection.


Assuntos
Chaperonina 60/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Mycobacterium tuberculosis/fisiologia , Receptores Depuradores Classe E/metabolismo , Tuberculose/metabolismo , Animais , Macrófagos/microbiologia , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , Células RAW 264.7 , Tuberculose/microbiologia
16.
Cancer Biomark ; 30(1): 85-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32986659

RESUMO

BACKGROUND: HSP60 and its partner HSP10 are members of heat shock proteins (HSPs) family, which help mitochondrial protein to fold correctly. Mcl-1, a member of the Bcl-2 family, plays a crucial role in regulation of cell apoptosis. Aberrant expression of HSP10, HSP60 and Mcl-1 is involved in the development of many tumors. OBJECTIVE: To examine the association between expression of HSP10, HSP60 and Mcl-1 and clinicopathological features of non-small cell lung cancer (NSCLC). METHODS: Tissue microarrays including 53 non-cancerous lung tissues (Non-CLT) and 354 surgically resected NSCLC were stained with anti-HSP10, anti-HSP60 and anti-Mcl-1 antibodies respectively by immunohistochemistry. RESULTS: Higher expression of HSP10, HSP60 and Mcl-1 was found in NSCLC compared with Non-CLT. Both individual and combined HSP10 and HSP60 expression in patients with clinical stage III was higher than that in stage I ∼ II. Expression of HSP10 showed a positive correlation with HSP60 and Mcl-1. Overall survival time of NSCLC patients was remarkably shorter with elevated expression of HSP10, HSP60 and Mcl-1 alone and in combination. Moreover overexpression of HSP10 and Mcl-1 was poor independent prognostic factor for lung adenocarcinoma patients. CONCLUSIONS: High expression of HSP10, HSP60 and Mcl-1 might act as novel biomarker of poor prognosis for NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Chaperonina 10/biossíntese , Chaperonina 60/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Chaperonina 10/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise de Sobrevida
17.
Arch Oral Biol ; 122: 104991, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33307322

RESUMO

OBJECTIVE: The aim of this study was to demonstrate the influence of the virulence factor GroEL on osteoblast behavior by characterizing the changes of secreted gelatinases. DESIGN: ELISA was performed to detect GroEL from samples from patients with or without apical periodontitis. An apical periodontitis model was established in rats and the expression of MMP-2, MMP-9 and NF-κB was evaluated by immunofluorescence staining. The primary osteoblasts and osteoblast-like MC3T3 cells were stimulated with recombinant GroEL, and gelatin zymography was used to determine the activity and expression of MMP-2 and MMP-9. Western blot was used to screen signaling pathways, and immunofluorescence staining was performed to confirm the activated signaling. RESULTS: First, we found expression of GroEL to be higher in oral saliva, gingival crevicular fluid and periradicular granulation tissue of patients with apical periodontitis than it was in healthy control patients. We next found that recombinant GroEL could increase the activity of the gelatinases, MMP-2 and MMP-9, which were secreted by both primary osteoblasts and MC3T3 cells. In a rat apical periodontitis model, strong expression of gelatinases was confirmed. Then, we found that GroEL-enhanced gelatinase activity was mediated through activation of NF-κB signaling. Acetylated NF-κB accumulated in the cell nucleus and bound to the promoter of MMP-2 and MMP-9 genes, thus initiating their high expression. CONCLUSION: This study reveals a direct interaction between oral bacteria and adult cells by demonstrating that gelatinase secretion is induced by GroEL, which partially explains bone resorption through gelatinase activation.


Assuntos
Chaperonina 60/metabolismo , Gelatinases/metabolismo , Osteoblastos/enzimologia , Periodontite/enzimologia , Animais , Bactérias/patogenicidade , Reabsorção Óssea , Linhagem Celular , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Camundongos , NF-kappa B , Ratos , Fatores de Virulência/metabolismo
18.
J Ethnopharmacol ; 267: 113361, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32891819

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Overweight/obesity was mentioned by many countries as an obstacle to good health and long life, which increases risk of diseases and disorders. Previous studies suggested that the chronic low-grade inflammation present in the body was considered as the essential pathogenesis for obesity. Chrysin is extracted from traditional Chinese medicine Oroxylum indicum (Linn.) Kurz and plays a superior anti-obesity role. Chrysin could reduce the lipid depot by inhibiting the obesity-related inflammation in adipose tissue. However, the target protein for chrysin to exert its anti-obesity role are not verified. AIM OF STUDY: The present study aimed to screen and validate the target protein for chrysin to reduce the lipid depot in palmitic acid-induced 3T3-L1 adipocytes. MATERIALS AND METHODS: Obesity model was established employing 0.5 mmol/L palmitic acid-induced 3T3-L1 adipocytes through "Cocktails" method. Two-dimensional gel electrophoresis (2-DE) combined with liquid chromatography-mass spectrometry (LC-MS) was applied to analyze the differentially expressed proteins for chrysin intervention by lipid formation in adipocytes. Gene silencing was utilized to decrease gene expression of the candidate proteins, then production of triglyceride in 3T3-L1 was detected by triglycerides assay to determine the target proteins. Ultraviolet (UV) absorption together with fluorescence spectra validated the direct target proteins of chrysin. They also computed the correlation constants of combination between chrysin and the target proteins. Molecular docking was further employed to identify the main binding amino acids between chrysin and the target protein. RESULTS: 2-DE combined with LC-MS screened four candidate proteins which were related to metabolism and inflammation. The production of triglycerides in 3T3-L1 was reduced after decreasing gene expression of Annexin A2 (ANXA2), 60 kDa heat shock protein (HSP-60) and succinyl-CoA:3-ketoacid coenzyme A transferase 1 (SCOT-S), respectively. UV spectrum showed that the absorbance spectra of ANXA2 from 260 to 300 nm shifted upwards along with the increase in chrysin concentration, meanwhile the absorbance spectra of HSP-60 from 200 to 220 nm and from 265 to 280 nm shifted slightly upwards along with the increase in chrysin concentrations. The results indicated the conjugated structures between chrysin and ANXA2 or HSP-60. Fluorescence quenching further suggested a spontaneous interaction between chrysin and ANXA2 or HSP-60. Finally, molecular docking identified the main binding amino acids between ANXA2 and chrysin were Ser22, Tyr24, Pro267, Val298, Asp299, and Lys302. CONCLUSIONS: Chrysin can reduce the amount of triglycerides by directly downregulating the inflammation-related target proteins ANXA2 and HSP-60, exerting an anti-obesity role.


Assuntos
Adipócitos/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Flavonoides/farmacologia , Hipolipemiantes/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteômica , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Anexina A2/genética , Anexina A2/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Inativação Gênica , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Transdução de Sinais , Triglicerídeos/metabolismo
19.
J Biochem ; 169(3): 351-361, 2021 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-32997746

RESUMO

In contrast to Escherichia coli, cyanobacteria have multiple GroELs, the bacterial homologues of chaperonin/Hsp60. We have shown that cyanobacterial GroELs are mutually distinct and different from E. coli GroEL with which the paradigm for chaperonin structure/function has been established. However, little is known about regulation of cyanobacterial GroELs. This study investigated effect of pH (varied from 7.0 to 8.5) on chaperone activity of GroEL1 and GroEL2 from the cyanobacterium Synechococcus elongatus PCC7942 and E. coli GroEL. GroEL1 and GroEL2 showed pH dependency in suppression of aggregation of heat-denatured malate dehydrogenase, lactate dehydrogenase and citrate synthase. They exhibited higher anti-aggregation activity at more alkaline pHs. Escherichia coli GroEL showed a similar pH-dependence in suppressing aggregation of heat-denatured lactate dehydrogenase. No pH dependence was observed in all the GroELs when urea-denatured lactate dehydrogenase was used for anti-aggregation assay, suggesting that the pH-dependence is related to some denatured structures. There was no significant influence of pH on the chaperone activity of all the GroELs to promote refolding of heat-denatured malate dehydrogenase. It is known that pH in cyanobacterial cytoplasm increases by one pH unit following a shift from darkness to light, suggesting that the pH-change modulates chaperone activity of cyanobacterial GroEL1 and GroEL2.


Assuntos
Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Escherichia coli/metabolismo , Synechococcus/metabolismo , Citrato (si)-Sintase/metabolismo , Cianobactérias/metabolismo , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Fotossíntese , Agregação Patológica de Proteínas , Desnaturação Proteica , Dobramento de Proteína
20.
Sci Rep ; 10(1): 21024, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33273609

RESUMO

The system is developed for efficient biosynthetic production of difficult-to-express polypeptides. A target polypeptide is produced fused into T. thermophilus GroEL chaperonin polypeptide chain in such a way that it is presented inside the GroEL cavity near the substrate binding surface. Such presentation allows alleviating potential problems of instability, toxicity or hydrophobicity of the fused peptide. Thermostability of thermophilic GroEL can be used for its one-step separation from the host cell proteins by heating. The target polypeptide may be released by any of amino acid-specific chemical treatments. In this study, GroEL was adapted for methionine-specific cleavage with cyanogen bromide by total replacement of methionine residues to facilitate further purification of the target polypeptide. The procedure is simple, robust and easy to scale-up. The capacity of this system to produce difficult-to-express polypeptides is demonstrated by production in bacterial system of one of the most potent antibacterial peptides polyphemusin I.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Microbiologia Industrial/métodos , Thermus thermophilus/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo
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