RESUMO
A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94.
RESUMO
Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.
Assuntos
Proteínas de Choque Térmico HSP70 , Dobramento de Proteína , Humanos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Nucleotídeos , Trifosfato de Adenosina/metabolismoRESUMO
Hsp70 and Hsp90 chaperones provide protein quality control to the cytoplasm, endoplasmic reticulum (ER), and mitochondria. Hsp90 activity is often enhanced by cochaperones that drive conformational changes needed for ATP-dependent closure and capture of client proteins. Hsp90 activity is also enhanced when working with Hsp70, but, in this case, the underlying mechanistic explanation is poorly understood. Here we examine the ER-specific Hsp70/Hsp90 paralogs (BiP/Grp94) and discover that BiP itself acts as a cochaperone that accelerates Grp94 closure. The BiP nucleotide binding domain, which interacts with the Grp94 middle domain, is responsible for Grp94 closure acceleration. A client protein initiates a coordinated progression of steps for the BiP/Grp94 system, in which client binding to BiP causes a conformational change that enables BiP to bind to Grp94 and accelerate its ATP-dependent closure. Single-molecule fluorescence resonance energy transfer measurements show that BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that otherwise acts as an energetic barrier to closure. These findings provide an explanation for enhanced activity of BiP and Grp94 when working as a pair, and demonstrate the importance of a high-energy conformational state in controlling the timing of the Grp94 conformational cycle. Given the high conservation of the Hsp70/Hsp90 system, other Hsp70s may also serve dual roles as both chaperones and closure-accelerating cochaperones to their Hsp90 counterparts.
Assuntos
Chaperona BiP do Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Camundongos , Dobramento de ProteínaRESUMO
Glucose-regulated protein 94 (Grp94) is an isoform of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Inhibiting Grp94 has been implicated for many diseases. Co-crystal structures of two generations of Grp94 inhibitors revealed the importance of investigating the ester group, which is projected into the site 2 pocket unique to Grp94. Therefore, a series of KUNG65 benzamide analogs was designed and synthesized to evaluate their impact on the affinity and selectivity for Grp94. The data demonstrated that substituents with small and saturated ring systems that contain hydrogen bond acceptors exhibited increased affinity for Grp94, whereas larger saturated ring system manifested increased selectivity for Grp94 over Hsp90α.
Assuntos
Benzamidas , Benzamidas/química , Benzamidas/síntese química , Benzamidas/farmacologia , Relação Estrutura-Atividade , Humanos , Sítios de Ligação , Estrutura Molecular , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
GRP94 (glucose-regulated protein 94) is a well-studied chaperone with a lysine, aspartic acid, glutamic acid and leucine (KDEL) motif at its C-terminal, which is responsible for GRP94 localization in the endoplasmic reticulum (ER). GRP94 is upregulated during ER stress to help fold unfolded proteins or direct proteins to ER-associated degradation. In a previous study, engineered GRP94 without the KDEL motif stimulated a powerful immune response in vaccine cells. In this report, we show that endogenous GRP94 is naturally secreted into the medium in a truncated form that lacks the KDEL motif in Chinese hamster ovary cells. The secretion of the truncated form of GRP94 was stimulated by the induction of ER stress. These truncations prevent GRP94 recognition by KDEL receptors and retention inside the cell. This study sheds light on a potential trafficking phenomenon during the unfolded protein response that may help understand the functional role of GRP94 as a trafficking molecule.
Assuntos
Estresse do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70 , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de MembranaRESUMO
Receptor-mediated vesicular transport has been extensively developed to penetrate the blood-brain barrier (BBB) and has emerged as a class of powerful brain-targeting delivery technologies. However, commonly used BBB receptors such as transferrin receptor and low-density lipoprotein receptor-related protein 1, are also expressed in normal brain parenchymal cells and can cause drug distribution in normal brain tissues and subsequent neuroinflammation and cognitive impairment. Here, the endoplasmic reticulum residing protein GRP94 is found upregulated and relocated to the cell membrane of both BBB endothelial cells and brain metastatic breast cancer cells (BMBCCs) by preclinical and clinical investigations. Inspired by that Escherichia coli penetrates the BBB via the binding of its outer membrane proteins with GRP94, avirulent DH5α outer membrane protein-coated nanocapsules (Omp@NCs) are developed to cross the BBB, avert normal brain cells, and target BMBCCs via recognizing GRP94. Embelin (EMB)-loaded Omp@EMB specifically reduce neuroserpin in BMBCCs, which inhibits vascular cooption growth and induces apoptosis of BMBCCs by restoring plasmin. Omp@EMB plus anti-angiogenic therapy prolongs the survival of mice with brain metastases. This platform holds the translational potential to maximize therapeutic effects on GRP94-positive brain diseases.
Assuntos
Neoplasias Encefálicas , Nanocápsulas , Camundongos , Animais , Células Endoteliais/metabolismo , Biomimética , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Proteínas de Membrana/metabolismo , Barreira Hematoencefálica/metabolismoRESUMO
The 94 kDa molecular chaperone, glucose-regulated protein 94 (Grp94), has garnered interest during the last decade due to its direct association with endoplasmic reticulum (ER) stress and disease. Grp94 belongs to the Hsp90 family of molecular chaperones and is a master regulator of ER homeostasis due to its ability to fold and stabilize proteins/receptors, and to chaperone misfolded proteins for degradation. Multiple studies have demonstrated that Grp94 knockdown or inhibition leads to the degradation of client protein substrates, which leads to disruption of disease-dependent signaling pathways. As a result, small molecule inhibitors of Grp94 have become a promising therapeutic approach to target a variety of disease states. Specifically, Grp94 has proven to be a promising target for cancer, glaucoma, immune-mediated inflammation, and viral infection. Moreover, Grp94-peptide complexes have been utilized effectively as adjuvants for vaccines against a variety of disease states. This work highlights the significance of Grp94 biology and the development of therapeutics that target this molecular chaperone in multiple disease states.
Assuntos
Proteínas de Choque Térmico HSP70 , Glicoproteínas de Membrana , Biologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Chaperonas Moleculares/metabolismoRESUMO
Autosomal dominant polycystic kidney disease is a common inherited renal disorder that results from mutations in either PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Downregulation or overexpression of PKD1 or PKD2 in mouse models results in renal cyst formation, suggesting that the quantity of PC1 and PC2 needs to be maintained within a tight functional window to prevent cystogenesis. Here we show that enhanced PC2 expression is a common feature of PKD1 mutant tissues, in part due to an increase in Pkd2 mRNA. However, our data also suggest that more effective protein folding contributes to the augmented levels of PC2. We demonstrate that the unfolded protein response is activated in Pkd1 knockout kidneys and in Pkd1 mutant cells and that this is coupled with increased levels of GRP94, an endoplasmic reticulum protein that is a member of the HSP90 family of chaperones. GRP94 was found to physically interact with PC2 and depletion or chemical inhibition of GRP94 led to a decrease in PC2, suggesting that GRP94 serves as its chaperone. Moreover, GRP94 is acetylated and binds to histone deacetylase 6 (HDAC6), a known deacetylase and activator of HSP90 proteins. Inhibition of HDAC6 decreased PC2 suggesting that HDAC6 and GRP94 work together to regulate PC2 levels. Lastly, we showed that inhibition of GRP94 prevents cAMP-induced cyst formation in vitro. Taken together our data uncovered a novel HDAC6-GRP94-related axis that likely participates in maintaining elevated PC2 levels in Pkd1 mutant cells.
Assuntos
Cistos/patologia , Retículo Endoplasmático/metabolismo , Nefropatias/patologia , Glicoproteínas de Membrana/metabolismo , Fator de Transcrição PAX8/fisiologia , Canais de Cátion TRPP/fisiologia , Animais , Cálcio/metabolismo , Cistos/etiologia , Cistos/metabolismo , Nefropatias/etiologia , Nefropatias/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Resposta a Proteínas não DobradasRESUMO
Colorectal cancer (CRC) is ranked the third driving reason for cancer death in the world. Surgery and chemotherapy have long been the first choices for cancer patients. However, the prognosis of CRC has never been satisfying, necessitating new effective treatment strategies. In our previous study, we synthesized compound5othat showed high anticancer potential with a 6-acrylic phenethyl ester-2-pyranone backbone, but its mechanism of action (MOA) is not understood. To articulate the MOA of 5o against colon cancer, we evaluated the anti-cancer effect of compound5oon CRC cells by cell proliferation assays. The MOA of5owas explored through cell cycle assays and apoptosis assays. The target of 5o was identified by molecular dynamic assays, ATPase assays, and surface plasmon resonance (SPR) analysis. We discovered 5o, a compound capable of inhibiting CRC cell proliferation with 1/25 folds in IC50 values compared with NCM460 cells (normal human colonic epithelial cell line). 5o induces cell apoptosis in a dose-dependent manner through PI3K/Akt/FoxO1 and NF-κB signaling pathways. In addition, 5o arrests cell cycle at G2/M by regulating MAPKs (ERK1/2 and p38) pathway. We further confirmed that 5o inhibits ATPase activity of GRP94 (Glucose-regulated protein 94) with the IC50 1.45 ± 0.06 µM. Compound 5o inhibits GRP94 to trigger regulation of PI3K/Akt and MAPKs pathways. This study reveals that 5o is a promising therapeutic agent against CRC as a novel GRP94 inhibition.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Adenosina Trifosfatases , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteínas de Choque Térmico HSP70 , Humanos , Proteínas de Membrana , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , PironasRESUMO
MZB1 (pERp1) is a B-cell-specific and endoplasmic reticulum (ER)-localized protein implicated in antibody secretion and integrin-mediated cell adhesion. Here, we examine the role of MZB1 in vivo by conditional gene inactivation in the mouse germline and at different stages of B lymphopoiesis. Deletion of MZB1 impairs humoral immune responses and antibody secretion in plasma cells that naturally undergo ER stress. In addition, we found that experimental induction of ER stress by tunicamycin injections in mice results in a block of pro-B-cell to pre-B-cell differentiation specifically in Mzb1(-/-) mice. A similar developmental block was observed in Mzb1(fl/fl)mb1(Cre) mice, whereby a Cre recombinase-induced genotoxic stress unmasks a role for MZB1 in the surface expression of immunoglobulin µ heavy chains (µHCs). MZB1 associates directly with the substrate-specific chaperone GRP94 (also called HSP90B1 or gp96) in an ATP-sensitive manner and is required for the interaction of GRP94 with µHCs upon ER stress. Thus, MZB1 seems to act as a substrate-specific cochaperone of GRP94 that enables proper biosynthesis of µHCs under conditions of ER stress.
Assuntos
Estresse do Retículo Endoplasmático/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Chaperonas Moleculares/metabolismo , Animais , Linfócitos B/metabolismo , Técnicas de Inativação de Genes , Imunidade Humoral/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias mu de Imunoglobulina/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/genéticaRESUMO
Endoplasmic reticulum stress (ERS) is closely related to the occurrence and progression of metabolic liver disease. The treatment targeting glucose-regulated protein 94 (GRP94) for liver disease has gotten much attention, but the specific effect of GRP94 on hepatocyte apoptosis is still unclear. So far, all the studies on GRP94 have been conducted in mice or rats, and little study has been reported on pigs, which share more similarities with humans. In this study, we used low-dose (LD) and high-dose (HD) tunicamycin (TM) to establish ERS models on piglet livers and immortalized porcine hepatic stellate cells (HSCs). On the piglet ERS model we found that ERS could significantly (p < 0.01) stimulate the secretion and synthesis of insulin-like growth factor (IGF-1), IGF-1 receptor (IGF-1R), and IGF-binding protein (IGFBP)-1 and IGFBP-3; however, with the increase in ERS degree, the effect of promoting secretion and synthesis significantly (p < 0.01) decreased. In addition, the ubiquitin protein and ubiquitination-related gene were significantly increased (p < 0.05) in the LD group compared with the vehicle group. The protein level of Active-caspase 3 was significantly increased (p < 0.01) in the HD group, however, the TUNEL staining showed there was no significant apoptosis in the piglet liver ERS model. To explore the biofunction of ER chaperone GRP94, we used shRNA to knock down the expression of GRP94 in porcine HSCs. Interestingly, on porcine HSCs, the knockdown of GRP94 significantly (p < 0.05) decreased the secretion of IGF-1, IGFBP-1 and IGFBP-3 under ERS, but had no significant effect on these under normal condition, and knockdown GRP94 had a significant (p < 0.01) effect on the UBE2E gene and ubiquitin protein from the analysis of two-way ANOVA. On porcine HSCs apoptosis, the knockdown of GRP94 increased the cell apoptosis in TUNEL staining, and the two-way ANOVA analysis shows that knockdown GRP94 had a significant (p < 0.01) effect on the protein levels of Bcl-2 and Caspase-3. For CCK-8 assay, ERS had a significant inhibitory(p < 0.05) effect on cell proliferation when treated with ERS for 24 h, and both knockdown GRP94 and ERS had a significant inhibitory(p < 0.05) effect on cell proliferation when treated with ERS for 36 h and 48 h. We concluded that GRP94 can protect the cell from ERS-induced apoptosis by promoting the IGF-1 system and ubiquitin. These results provide valuable information on the adaptive mechanisms of the liver under ERS, and could help identify vital functional genes to be applied as possible diagnostic biomarkers and treatments for diseases induced by ERS in the future.
Assuntos
Estresse do Retículo Endoplasmático , Células Estreladas do Fígado , Humanos , Animais , Ratos , Camundongos , Suínos , Células Estreladas do Fígado/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ubiquitina/metabolismo , ApoptoseRESUMO
The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca2+ homeostasis, antioxidant cytoprotection and protein-protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca2+ storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca2+ homeostasis.
Assuntos
Neoplasias da Mama , Citoproteção , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Feminino , HumanosRESUMO
Autophagy of vascular endothelial cells (VECs) plays an important role in maintaining vascular homeostasis. Lipid droplets (LDs) are organelles that can be formed in response to various stimuli, including excessive lipid or various stresses. LDs sequester toxic lipids, thereby preventing lipotoxic cell damage and have a complex relationship with autophagy. In the previous study, we identified a novel Grp94 inhibitor HCP1 inhibited apoptosis in VECs. Here we found that HCP1 targeted LDs and promoted the accumulation of LDs in VECs. Our results showed that HCP1 upregulated the protein levels of autophagy-related proteins. We demonstrated that HCP1 upregulated the number of LDs and suppressed autophagy by inhibiting Grp94. Therefore, we provided HCP1 as a new VECs autophagy inhibitor targeting LDs, which might be a potential compound in the treatment of VECs autophagy related vascular diseases.
Assuntos
Autofagia/efeitos dos fármacos , Cumarínicos/farmacologia , Células Endoteliais/efeitos dos fármacos , Gotículas Lipídicas/efeitos dos fármacos , Pirazóis/farmacologia , Células Cultivadas , Cumarínicos/química , Humanos , Pirazóis/químicaRESUMO
Colorectal cancer (CRC) is a worldwide health problem. Glucose-regulated protein 94 (GRP94) is known as an important endoplasmic reticulum-stress response protein that shows correlation with aggressive cancer behavior. However, the role of GRP94 in CRC is still unclear. Our results showed that silencing GRP94 (GRP94-KD) reduced cell proliferation, invasion and migration of CRC cells and suppressed tumorigenesis in the xenograft mouse model. Rescue assay showed that ETV1 overexpression reversed the effect of GRP94 on cell proliferation and migration. In the molecular mechanism, we found that knockdown of GRP94 inhibited the level of MAPK pathway, including ERK/p-ERK, JNK/p-JNK, and p38/p-p38 signals. Cyclooxygenase-2 and epithelial-mesenchymal transformation biomarkers, such as N-cadherin, vimentin, and ß-catenin were suppressed in GRP94 knockdown cells. Treatment of specific inhibitors of MAPK pathway showed that ERK/p-ERK, and p38/p-p38 inhibitors significantly influenced ETV1 expression as compared to JNK/p-JNK inhibitor. Our results indicated that silencing GRP94 repressed the ability of EMT process, cancer cell proliferation, metastasis, and CRC tumorigenesis. Therefore, GRP94 may play an important role in CRC by regulating ETV1 and MAPK pathway.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Glicoproteínas de Membrana/genética , CamundongosRESUMO
Papillary thyroid cancer (PTC) usually has favorable prognosis; however, distant metastasis is a leading cause of death associated with PTC. MicroRNA-99a-3p (miR-99a-3p) is a member of the miR-99 family that is shown to be a tumor suppressor in various human cancers including the anaplastic thyroid cancer, another type of thyroid cancer. The Cancer Genome Atlas database and our previous study reported that miR-99a-3p is downregulated in human PTC tissues as well as human papillary thyroid carcinoma B-CPAP and TPC-1 cell lines. However, its pathological role in PTC remains unclear, especially its impact on PTC metastasis. In the present study, the role of miR-99a-3p in PTC metastasis was molecularly evaluated in in vitro and in vivo models. Our functional study revealed that overexpressing miR-99a-3p significantly suppresses epithelial-mesenchymal transition (EMT) and anoikis resistance as well as migration and invasion of B-CPAP and TPC-1 cells. The mechanical study indicated that glucose-regulated protein 94 (GRP94) is the direct target of miR-99a-3p. Moreover, GRP94 overexpression reverses the inhibitory effect of miR-99a-3p on PTC metastasis. In addition, the miR-99a-3p/GRP94 axis exerts its effect via inhibiting the expression and cytoplasmic relocation of integrin 2α (ITGA2). Furthermore, in vivo experiments confirmed that miR-99a-3p significantly inhibits tumor growth and lung metastasis in PTC xenograft mice. Overall, our findings suggested that the miR-99a-3p/GRP94/ITGA2 axis may be a novel therapeutic target for the prevention of PTC metastasis.
Assuntos
Integrina alfa2/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Animais , Anoikis/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Feminino , Xenoenxertos/metabolismo , Humanos , Camundongos Nus , Metástase Neoplásica/genética , Câncer Papilífero da Tireoide/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1-/-Prdm1+/gfp mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1-/- plasmablasts show a reduced activation of ß1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Integrina beta1/metabolismo , Chaperonas Moleculares/metabolismo , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Animais , Células da Medula Óssea/citologia , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Integrina beta1/genética , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Plasmócitos/citologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/genéticaRESUMO
Patients with advanced colorectal cancer (CRC) with distant metastases have a poor prognosis. We evaluated the clinicopathological relevance of GRP94 expression in these cases. The immunohistochemical expression of GRP94 was studied in 189 CRC patients with synchronous (SM; n = 123) and metachronous metastases (MM; n = 66), using tissue microarray; the association between GRP94 expression, outcome, and tumor-infiltrating lymphocytes (TILs) was also evaluated. GRP94 was expressed in 64.6% (122/189) patients with CRC; GRP94 positivity was found in 67.5% and 59.1% patients with SM and MM, respectively. In the SM group, high GRP94 expression was more common in patients with a higher density of CD4+ TILs (p = 0.002), unlike in the MM group. Survival analysis showed that patients with GRP94 positivity had significantly favorable survival (p = 0.030); after multivariate analysis, GRP94 only served as an independent prognostic factor (p = 0.034; hazard ratio, 0.581; 95% confidence interval, 0.351-0.961) in the SM group. GRP94 expression was detected in 49.4% of metastatic sites and showed significant heterogeneity between primary and metastatic lesions (p = 0.012). GRP94 is widely expressed in CRC with distant metastases; its expression was associated with favorable prognosis in the SM group, unlike in the MM group.
Assuntos
Neoplasias Colorretais/patologia , Glicoproteínas de Membrana/metabolismo , Neoplasias Primárias Múltiplas/patologia , Segunda Neoplasia Primária/patologia , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/metabolismo , Segunda Neoplasia Primária/metabolismo , Prognóstico , Análise de Sobrevida , Análise Serial de TecidosRESUMO
Curcumin administration attenuates muscle disuse atrophy, but its effectiveness against aging-induced, selective loss of mass or force (presarcopenia or asthenia/dynopenia), or combined loss (sarcopenia), remains controversial. A new systemic curcumin treatment was developed and tested in 18-month-old C57BL6J and C57BL10ScSn male mice. The effects on survival, liver toxicity, loss of muscle mass and force, and satellite cell responsivity and commitment were evaluated after 6-month treatment. Although only 24-month-old C57BL10ScSn mice displayed age-related muscle impairment, curcumin significantly increased survival of both strains (+20-35%), without signs of liver toxicity. Treatment prevented sarcopenia in soleus and presarcopenia in EDL of C57BL10ScSn mice, whereas it did not affect healthy-aged muscles of C57BL6J. Curcumin-treated old C57BL10ScSn soleus preserved type-1 myofiber size and increased type-2A one, whereas EDL maintained adult values of total myofiber number and fiber-type composition. Mechanistically, curcumin only partially prevented the age-related changes in protein level and subcellular distribution of major costamere components and regulators. Conversely, it affected satellite cells, by maintaining adult levels of myofiber maturation in old regenerating soleus and increasing percentage of isolated, MyoD-positive satellite cells from old hindlimb muscles. Therefore, curcumin treatment successfully prevents presarcopenia and sarcopenia development by improving satellite cell commitment and recruitment.
Assuntos
Envelhecimento , Curcumina/farmacologia , Músculo Esquelético , Sarcopenia , Envelhecimento/efeitos dos fármacos , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Masculino , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Sarcopenia/tratamento farmacológico , Sarcopenia/metabolismo , Sarcopenia/patologiaRESUMO
HSP90 is a vital chaperone protein conserved across all organisms. As a chaperone protein, it correctly folds client proteins. Structurally, this protein is a dimer with monomer subunits that consist of three main conserved domains known as the N-terminal domain, middle domain, and the C-terminal domain. Multiple isoforms of HSP90 exist, and these isoforms share high homology. These isoforms are present both within the cell and outside the cell. Isoforms HSP90α and HSP90ß are present in the cytoplasm; TRAP1 is present in the mitochondria; and GRP94 is present in the endoplasmic reticulum and is likely secreted due to post-translational modifications (PTM). HSP90 is also secreted into an extracellular environment via an exosome pathway that differs from the classic secretion pathway. Various co-chaperones are necessary for HSP90 to function. Elevated levels of HSP90 have been observed in patients with cancer. Despite this observation, the possible role of HSP90 in cancer was overlooked because the chaperone was also present in extreme amounts in normal cells and was vital to normal cell function, as observed when the drastic adverse effects resulting from gene knockout inhibited the production of this protein. Differences between normal HSP90 and HSP90 of the tumor phenotype have been better understood and have aided in making the chaperone protein a target for cancer drugs. One difference is in the conformation: HSP90 of the tumor phenotype is more susceptible to inhibitors. Since overexpression of HSP90 is a factor in tumorigenesis, HSP90 inhibitors have been studied to combat the adverse effects of HSP90 overexpression. Monotherapies using HSP90 inhibitors have shown some success; however, combination therapies have shown better results and are thus being studied for a more effective cancer treatment.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Neoplasias/metabolismo , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Humanos , Neoplasias/patologia , Isoformas de Proteínas/metabolismoRESUMO
The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90ß, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.