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Background: Aggressive mature T-cell lymphoma (TCL) is a disease that carries a poor prognosis. Methods: We analyzed the expression of 22 tumor cell functional proteins in 16 randomly selected patients with TCL. Immunohistochemistry was performed in paraffin-embedded tumor tissue sections to determine the protein expression statuses in tumor cells. Results: Glucose-regulated protein 94 (GRP94), a protein that serves as a pro-survival component under endoplasmic reticulum (ER) stress in the tumor microenvironment, was significantly associated with a shortened survival. Furthermore, significant differences were observed when GRP94 was combined with six other factors. The six factors were (1) programmed cell death-ligand 1 (PD-L1); (2) programmed cell death 1 (PD-1); (3) aldo-keto reductase family 1 member C3 (AKR1C3); (4) P53, a tumor suppressor; (5) glucose-regulated protein 78 (GRP78), an ER stress protein; and (6) thymidine phosphorylase (TP). Based on the combination of GRP94 and the six other factors expressed in the tumors, we propose a new prognostic classification system for TCL (TCL Urayasu classification). Group 1 (relatively good prognosis): GRP94-negative (n = 6; median OS, 88 months; p < 0.01); Group 2 (poor prognosis): GRP94-positive, plus expression of two of the six factors mentioned above (n = 5; median OS, 25 months; p > 0.05); and Group 3 (very poor prognosis): GRP94-positive, plus expression of at least three of the six factors mentioned above (n = 5; median OS, 10 months; p < 0.01). Conclusions: Thus, the TCL Urayasu prognostic classification may be a simple, useful, and innovative classification that also explains the mechanism of resistance to treatment for each functional protein. If validated in a larger number of patients, the TCL Urayasu classification will enable a targeted treatment using selected inhibitors acting on the abnormal protein found in each patient.
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Glycosylation, a prevalent post-translational modification, plays a pivotal role in regulating intricate cellular processes by covalently attaching glycans to macromolecules. Dysregulated glycosylation is linked to a spectrum of diseases, encompassing cancer, neurodegenerative disorders, congenital disorders, infections, and inflammation. This review delves into the intricate interplay between glycosylation and protein conformation, with a specific focus on the profound impact of N-glycans on the selection of distinct protein conformations characterized by distinct interactomes-namely, protein assemblies-under normal and pathological conditions across various diseases. We begin by examining the spike protein of the SARS virus, illustrating how N-glycans regulate the infectivity of pathogenic agents. Subsequently, we utilize the prion protein and the chaperone glucose-regulated protein 94 as examples, exploring instances where N-glycosylation transforms physiological protein structures into disease-associated forms. Unraveling these connections provides valuable insights into potential therapeutic avenues and a deeper comprehension of the molecular intricacies that underlie disease conditions. This exploration of glycosylation's influence on protein conformation effectively bridges the gap between the glycome and disease, offering a comprehensive perspective on the therapeutic implications of targeting conformational mutants and their pathologic assemblies in various diseases. The goal is to unravel the nuances of these post-translational modifications, shedding light on how they contribute to the intricate interplay between protein conformation, assembly, and disease.
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Príons , Processamento de Proteína Pós-Traducional , Glicosilação , Polissacarídeos/química , Conformação Proteica , Príons/metabolismoRESUMO
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.
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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
The pursuit of single drugs targeting multiple targets has become a prominent trend in modern cancer therapeutics. Natural products, known for their multi-targeting capabilities, accessibility, and cost-effectiveness, hold great potential for the development of multi-target drugs. However, their therapeutic efficacy is often hindered by complex structural modifications and limited anti-tumor activity. In this study, we present a novel approach using celastrol (CST)-based Proteolysis Targeting Chimeras (PROTACs) for breast cancer therapy. Through rational design, we have successfully developed compound 6a, a potent multiple protein degrader capable of selectively degrading GRP94 and CDK1/4 in tumor cells via the endogenous ubiquitin-proteasome system. Furthermore, compound 6a has demonstrated remarkable inhibitory effects on cell proliferation and migration, and induction of apoptosis in 4T1 cells through cell cycle arrest and activation of the Bcl-2/Bax/cleaved Caspase-3 apoptotic pathway. In vivo administration of compound 6a has effectively suppressed tumor growth with an acceptable safety profile. Our findings suggest that the CST-based PROTACs described herein can be readily extended to other natural products, offering a potential avenue for the development of natural product-based PROTACs for cancer treatment.
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Produtos Biológicos , Neoplasias de Mama Triplo Negativas , Humanos , Proteólise , Quimera de Direcionamento de Proteólise , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
The present study was designed to investigate potential biomarkers of depression and targets of antidepressants from the perspective of hippocampal endoplasmic reticulum stress (ERS) based on cerebrospinal fluid (CSF) proteomics. Firstly, a six-week depression model was established and treated with fluoxetine (FLX). We found antidepressant-FLX could ameliorate depression-like behaviors and cognition in depressed rats caused by chronic unpredictable mild stress (CUMS). FLX significantly increased neuronal numbers in dentate gyrus (DG) and CA3 regions of hippocampus. CSF proteome data revealed thirty-seven differentially expressed proteins (DEPs) co-regulated by CUMS and FLX, including GRP94 and EIF2α. Results of Gene Oncology (GO) annotation and KEGG pathway enrichment for DEPs mainly included PERK-mediated unfolded protein response, endoplasmic reticulum, and translational initiation. The expression levels of GRP94, p-PERK, p-EIF2α, CHOP and Caspase-12 were increased in hippocampus of CUMS rats, and FLX worked the opposite way. FLX had strong affinity and binding activity with GRP94 protein, and four key proteins on the PERK pathway (PERK, EIF2α, p-EIF2α, CHOP). We proposed that FLX may exert antidepressant effects and neuroprotective action by alleviating excessive activation of the hippocampal PERK pathway and reducing neuronal deficits in depressed rats. PERK, EIF2α, p-EIF2α, and CHOP may be potential targets for antidepressant-FLX. GRP94 in CSF may be a potential biomarker of depression and the therapeutic effects of antidepressants.
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Depressão , Proteínas de Membrana , Proteômica , Animais , Ratos , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Depressão/etiologia , Depressão/metabolismo , Estresse do Retículo Endoplasmático/genética , Fluoxetina/farmacologia , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismoRESUMO
We conducted a retrospective analysis of GRP94 immunohistochemical (IHC) staining, an ER stress protein, on large B-cell lymphoma (LBCL) cells, intracellular p53, and 15 factors involved in the metabolism of the CHOP regimen: AKR1C3 (HO metabolism), CYP3A4 (CHOP metabolism), and HO efflux pumps (MDR1 and MRP1). The study subjects were 42 patients with LBCL at our hospital. The IHC staining used antibodies against the 17 factors. The odds ratios by logistic regression analysis used a dichotomous variable of CR and non-CR/relapse were statistically significant for MDR1, MRP1, and AKR1C3. The overall survival (OS) after R-CHOP was compared by the log-rank test. The four groups showed that Very good (5-year OS, 100%) consisted of four patients who showed negative IHC staining for both GRP94 and CYP3A4. Very poor (1-year OS, 0%) consisted of three patients who showed positive results in IHC for both GRP94 and CYP3A4. The remaining 35 patients comprised two subgroups: Good (5-year OS 60-80%): 15 patients who showed negative staining for both MDR1 and AKR1C3 and Poor (5-year OS, 10-20%): 20 patients who showed positive staining for either MDR, AKR1C3, MRP1, or p53. The Histological Prognostic Index (HPI) (the four groups: Very poor, Poor, Good, and Very good) is a breakthrough method for stratifying patients based on the factors involved in the development of treatment resistance.
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Glucose regulatory protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family, that plays an important role in secreted protein folding. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the main pathogens in sericulture, causing serious economic losses every year. Previous studies showed that HSP90 members promote BmNPV replication in silkworm, but the function of BmGRP94 in BmNPV infection and proliferation is still not understood. In this study, we investigated the interplay between BmGRP94 and BmNPV infection in silkworm. We first identified a single gene of BmGRP94 in the Bombyx mori genome, which encodes a polypeptide with 810 amino acids in length. Spatio-temporal expression profiles showed that BmGRP94 was highly expressed in hemocytes and midgut, and was significantly induced by BmNPV infection. Furthermore, overexpression of BmGRP94 facilitates viral proliferation, while BmGRP94 inhibition evidently decreased BmNPV proliferation in BmN cells and in silkworm midgut. Mechanistically, BmGRP94 inhibition triggers ER stress, as judged by increased expression of PERK/ATF4/ERO1, H2O2 production, and ER calcium efflux, which promotes cell apoptosis to restrict BmNPV replication in silkworm. These results suggest that BmGRP94 plays an important role in facilitating BmNPV proliferation, and provides a potential molecular target for BmNPV prevention.
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Bombyx , Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/fisiologia , Peróxido de Hidrogênio/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Bombyx/metabolismo , Apoptose/genética , Proliferação de Células , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismoRESUMO
[This corrects the article DOI: 10.3389/fonc.2022.829520.].
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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.
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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.
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Proteínas de Choque Térmico HSP70 , Glicoproteínas de Membrana , Biologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Chaperonas Moleculares/metabolismoRESUMO
Colorectal cancer (CRC) is one of the life-threatening malignancies worldwide. Thus, novel potential therapeutic targets and therapeutics for the treatment of CRC need to be identified to improve the clinical outcomes of patients with CRC. In this study, we found that glucose-regulated protein 94 (GRP94) is overexpressed in CRC tissues, and its high expression is correlated with increased microvessel density. Next, through phage display technology and consecutive in vitro functional isolations, we generated a novel human monoclonal antibody that specifically targets cell surface GRP94 and shows superior internalizing activity comparable to trastuzumab. We found that this antibody specifically inhibits endothelial cell tube formation and simultaneously promotes the downregulation of GRP94 expression on the endothelial cell surface. Finally, we demonstrated that this antibody effectively suppresses tumor growth and angiogenesis of HCT116 human CRC cells without causing severe toxicity in vivo. Collectively, these findings suggest that cell surface GRP94 is a novel potential anti-angiogenic target in CRC and that antibody targeting of GRP94 on the endothelial cell surface is an effective strategy to suppress CRC tumor angiogenesis.
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Neoplasias Colorretais , Glicoproteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Proteínas de Choque Térmico HSP70 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas de Membrana/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismoRESUMO
BACKGROUND: Nasopharyngeal carcinoma (NPC) is highly prevalent in southern China. The remote metastasis of advanced NPC requires chemotherapeutic treatments to reduce the mortality. Our previous work revealed that saucerneol (SN) showed cytotoxicity against several nasopharyngeal carcinoma (NPC) cells. This work aims to investigate the effect of SN in NPC growth and exploring the mechanism of action. STUDY DESIGN: Applying in vivo study, in vitro study and in silico study to indicate the mechanism of SN in inhibiting NPC growth. METHODS: Saucerneol (SN) toxicity was measured with MTT assay. NPC proliferation was measured with EdU and colony formation assays, cell cycle was detected with flow cytometry. NPC migration and invasion were measured with scratch assay and matrigel transwell method. Further, human NPC xenograft tumor models were established in nude mice to evaluate the therapeutic efficacy of SN in vivo. Toxicological analysis was performed on H&E staining and IHC. Quantitative real-time PCR and Western blot analyses were used to evaluate the expression levels of key molecules in PI3K/AKT/mTOR, MAPK, NF-κB, and HIF-1α signal pathways. Target predicting was conducted using computational method, and target identification was carried out by ATPase assay and TSA. RESULTS: SN, a potent NPC inhibitor that was previously isolated from Saururus chinensis in our lab, is proven to inhibit the proliferation and metastasis of HONE1 cell lines and inhibit the growth of human NPC xenografts in nude mice. Moreover, we further articulate the molecular mechanism of action for SN and, reveal that SN promotes the expression of cell cycle-dependent kinase inhibitory protein p21 Waf1/Cip1 through targeting Grp94 and then inhibiting PI3K/AKT signaling pathway as well as up-regulating p53 to disrupt the progression of HONE1 cells. CONCLUSION: SN significantly inhibits NPC cells proliferation and metastasis in vitro and in vivo via selectively inhibit Grp94 and then blocking PI3K/AKT/mTOR/HIF-1α signaling pathway. This study firstly provides a novel selective Grp94 inhibitor as a NPC candidate.
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Furanos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Lignanas/farmacologia , Proteínas de Membrana/metabolismo , Neoplasias Nasofaríngeas , Fosfatidilinositol 3-Quinases , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
How immune tolerance is lost to pancreatic ß-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin chaperone glucose-regulated protein (GRP) 94 from the endoplasmic reticulum (ER) leads to mishandling of proinsulin, ER stress, and activation of the immunoproteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered ß-cell major histocompatibility complex (MHC) class-I bound peptidome and inflammasome activation, sensitizing ß-cells to immune attack. We used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20 µM), or exposed to proinflammatory cytokines interleukin (IL)-1ß or interferon gamma (IFNγ) (15 pg/mL and 10 ng/mL, respectively) for 24 h. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC), followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing protein (NLRP1), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1ß expression and secretion were investigated by Western blotting. GRP94 KO increased RT1.A expression in ß-cells, as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptide diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NLRP1 expression was upregulated in GRP94 deficient cells along with decreased IκBα content while proIL-1ß cellular levels declined, coupled with increased secretion of mature IL-1ß. Our results suggest that limiting ß-cell proinsulin chaperoning enhances RT1.A expression alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress associated with impeding proinsulin handling may sensitize ß-cells to immune-attack.
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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.
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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
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.
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Neoplasias da Mama , Citoproteção , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular , Retículo Endoplasmático/metabolismo , Feminino , HumanosRESUMO
HSP70 and HSP90 are two powerful chaperone machineries involved in survival and proliferation of tumor cells. Residing in various cellular compartments, HSP70 and HSP90 perform specific functions. Concurrently, HSP70 and HSP90 homologs may also translocate from their primary site under various stress conditions. Herein, we address the current literature on the role of HSP70 and HSP90 chaperone networks in cancer. The goal is to provide a comprehensive review on the functions of cytosolic, mitochondrial and endoplasmic reticulum HSP70 and HSP90 homologs in cancer. Given that high expression of HSP70 and HSP90 enhances tumor development and associates with tumor aggressiveness, further understanding of HSP70 and HSP90 chaperone networks may provide clues for the discoveries of novel anti-cancer therapies.
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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.
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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
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancers and is not eligible for hormone and anti-HER2 therapies. Identifying therapeutic targets and associated biomarkers in TNBC is a clinical challenge to improve patients' outcome and management. High infiltration of CD206+ M2-like macrophages in the tumor microenvironment (TME) indicates poor prognosis and survival in TNBC patients. As we previously showed that membrane expression of GRP94, an endoplasmic reticulum chaperone, was associated with the anti-inflammatory profile of human PBMC-derived M2 macrophages, we hypothesized that intra-tumoral CD206+ M2 macrophages expressing GRP94 may represent innovative targets in TNBC for theranostic purposes. We demonstrate in a preclinical model of 4T1 breast tumor-bearing BALB/c mice that (i) CD206-expressing M2-like macrophages in the TME of TNBC can be specifically detected and quantified using in vivo SPECT imaging with 99mTc-Tilmanocept, and (ii) the inhibition of GRP94 with the chemical inhibitor PU-WS13 induces a decrease in CD206-expressing M2-like macrophages in TME. This result correlated with reduced tumor growth and collagen content, as well as an increase in CD8+ cells in the TME. 99mTc-Tilmanocept SPECT imaging might represent an innovative non-invasive strategy to quantify CD206+ tumor-associated macrophages as a biomarker of anti-GRP94 therapy efficacy and TNBC tumor aggressiveness.
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Receptor de Manose/genética , Glicoproteínas de Membrana/genética , Neoplasias de Mama Triplo Negativas/genética , Microambiente Tumoral/genética , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Dextranos/farmacologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Mananas/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Transdução de Sinais/efeitos dos fármacos , Pentetato de Tecnécio Tc 99m/análogos & derivados , Pentetato de Tecnécio Tc 99m/farmacologia , Tomografia Computadorizada de Emissão de Fóton Único , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
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
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.