RESUMEN
FK506-binding protein 9 (FKBP9) is involved in tumor malignancy by resistance to endoplasmic reticulum (ER) stress, and the up-regulation of FKBP9 is associated with patients' poor prognosis. The current knowledge of the molecular mechanisms is still limited. One previous study showed that FKBP9 could confer glioblastoma cell resistance to ER stress through ASK1-p38 signaling. However, the upstream regulatory mechanism of FKBP9 expression is still indistinct. In this study, we identified the FKBP9 binding proteins using co-immunoprecipitation followed by mass spectrometry. Results showed that FKBP9 interacted with the binding immunoglobulin protein (BiP). BiP bound directly to FKBP9 with high affinity. BiP prolonged the half-life of the FKBP9 protein and stabilized the FKBP9 protein. BiP and FKBP9 protein levels were positively correlated in patients with glioma, and patients with high expression of BiP and FKBP9 showed a worse prognosis. Further studies showed that FKBP9 knockout in genetically engineered mice inhibited intracranial glioblastoma formation and prolonged survival by decreasing cellular proliferation and ER stress-induced CHOP-related apoptosis. Moreover, normal cells may depend less on FKBP9, as shown by the absence of apoptosis upon FKBP9 knockdown in a non-transformed human cell line and overall normal development in homozygous knockout mice. These findings suggest an important role of BiP-regulated FKBP9-associated signaling in glioma progression and the BiP-FKBP9 axis may be a potential therapeutic target for glioma.
RESUMEN
During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Fibroblastos , Glucosa , Respuesta de Proteína Desplegada , Animales , Ratones , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Glucosa/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Ratones Noqueados , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Transducción de SeñalRESUMEN
BACKGROUND: Intestinal ischemia-reperfusion (II/R) injury is a common clinical emergency with high morbidity and mortality. Given the absence of efficacious prophylactic and therapeutic interventions and specific drugs, sustained efforts are essential to develop new targeted drugs. Corilagin, a naturally polyphenolic tannic acid widespread in longan, rambutan and many other edible economic crops with medicinal properties in China, is of interest due to its multiple bioactivities, including the potential to mitigate II/R injuries. Nevertheless, a clear understanding of its molecular targets and the intricate mechanisms against II/R injury remains obscure and requires further elucidation. OBJECTIVE: This study aimed to investigate corilagin's pharmacological impact and molecular mechanism for II/R injury. METHODS: An animal II/R model was established by clamping superior mesenteric artery (SMA), and the therapeutic efficacy of corilagin against II/R was evaluated by biochemical and pathological analysis. Next, integrated transcriptomic and proteomic analyses was performed to identify key targets. Moreover, endoplasmic reticulum stress (ERS) damage was respectively observed by transmission electron microscope (TEM), immunohistochemistry, TUNEL, flow cytometry and western blotting (WB). Finally, molecular docking, molecular dynamics (MD) simulation, cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) assays were utilized to assess the interaction between corilagin and binding immunoglobulin protein (Bip, Grp78 or Hspa5), and co-IP assay was conducted to investigate the interaction between Bip and its substrate proteins. RESULTS: Corilagin exhibited robust protection against II/R injuries, effectively alleviating intestinal tissue damage and oxidative stress induced by II/R. The modulation of ERS as a potential regulatory mechanism was investigated through an integrated transcriptomic and proteomic analysis, identifying Bip as a key target contributing to corilagin's protective effects. Further experimental evidence using molecular docking, MD simulation, CETSA, and DARTS assays confirmed the potentially direct interaction of corilagin with Bip. This interaction promoted the ubiquitin-dependent degradation of the Bip-substrate complex, thereby suppressing ERS-related signalling pathways, including the IRE1 branch, PERK branch, and ATF6 branch, to alleviate tissue damage. CONCLUSION: This study confirmed that corilagin could selectively bind to Bip, facilitating its ubiquitin-dependent recognition and degradation, thereby inhibiting severe endoplasmic reticulum stress signalling and alleviating II/R injury. A detailed mechanistic insight into the action mode of corilagin had been proposed, supporting its potential usage as an ERS inhibitor.
RESUMEN
Sex determination in animals is not only determined by karyotype but can also be modulated by environmental cues like temperature via unclear transduction mechanisms. Moreover, in contrast to earlier views that sex may exclusively be determined by either karyotype or temperature, recent observations suggest that these factors rather co-regulate sex, posing another mechanistic mystery. Here, we discovered that certain wild-isolated and mutant C. elegans strains displayed genotypic germline sex determination (GGSD), but with a temperature-override mechanism. Further, we found that BiP, an ER chaperone, transduces temperature information into a germline sex-governing signal, thereby enabling the coexistence of GGSD and temperature-dependent germline sex determination (TGSD). At the molecular level, increased ER protein-folding requirements upon increased temperatures lead to BiP sequestration, resulting in ERAD-dependent degradation of the oocyte fate-driving factor, TRA-2, thus promoting male germline fate. Remarkably, experimentally manipulating BiP or TRA-2 expression allows to switch between GGSD and TGSD. Physiologically, TGSD allows C. elegans hermaphrodites to maintain brood size at warmer temperatures. Moreover, BiP can also influence germline sex determination in a different, non-hermaphroditic nematode species. Collectively, our findings identify thermosensitive BiP as a conserved temperature sensor in TGSD, and provide mechanistic insights into the transition between GGSD and TGSD.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Células Germinativas , Procesos de Determinación del Sexo , Temperatura , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Masculino , Células Germinativas/metabolismo , Femenino , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genéticaRESUMEN
INTRODUCTION: Trophoblast homeostasis and differentiation require a proper endoplasmic reticulum (ER) function. The Krüppel-like factor-6 (KLF6) transcription factor modulates trophoblast migration, differentiation, and reactive oxygen species (ROS) production. Since ROS may impact on ER homeostasis, we assessed whether downregulation of KLF6 altered the unfolded protein response (UPR) and cellular process associated with ER homeostasis. MATERIALS AND METHODS: Protein and RNA expression were analyzed by Western blot and qRT-PCR, respectively, in extravillous trophoblast HTR-8/SVneo cells silenced for KLF6. Apoptosis was detected by flow cell cytometry using Annexin V Apoptosis Detection Kit. Protein trafficking was assessed by confocal microscopy of a reporter fluorescent protein whose release from the ER was synchronized. RESULTS: KLF6 downregulation reduced the expression of BiP, the master regulator of the UPR, at protein, mRNA, and pre-mRNA levels. Ire1α protein, XBP1 splicing, and DNAJB9 mRNA levels were also reduced in KLF6-silenced cells. Instead, PDI, Ero1α, and the p-eIF2α/eIF2α ratio as well as autophagy and proteasome dependent protein degradation remained unchanged while intracellular trafficking was increased. Under thapsigargin-induced stress, KLF6 silencing impaired BiP protein and mRNA expression increase, as well as the activation of the Ire1α pathway, but it raised the p-eIF2α/eIF2α ratio and CHOP protein levels. Nevertheless, apoptosis was not increased. DISCUSSION: Results provide the first evidence of KLF6 as a modulator of the UPR components. The increase in protein trafficking and protection from apoptosis, observed in KLF6-silenced cells, are consistent with its role in extravillous trophoblast migration and differentiation.
Asunto(s)
Apoptosis , Retículo Endoplásmico , Factor 6 Similar a Kruppel , Trofoblastos , Respuesta de Proteína Desplegada , Humanos , Trofoblastos/metabolismo , Trofoblastos/fisiología , Factor 6 Similar a Kruppel/metabolismo , Factor 6 Similar a Kruppel/genética , Retículo Endoplásmico/metabolismo , Respuesta de Proteína Desplegada/fisiología , Apoptosis/fisiología , Homeostasis , Línea Celular , Femenino , Estrés del Retículo Endoplásmico/fisiología , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Trofoblastos ExtravellososRESUMEN
BACKGROUND: Acetaminophen (APAP) overdose is a significant contributor to drug-induced liver injury worldwide. G-protein-coupled receptor 116 (GPR116) is an important homeostatic maintenance molecule in the body, but little is known about its role in APAP-induced liver injury (AILI). METHODS: GPR116 expression was determined in both human and mouse AILI models. Hepatic function and damage response were analyzed in hepatocyte-specific GPR116 deletion (GPR116â³HC) mice undergoing APAP challenge. RNA-sequencing, immunofluorescence confocal, and co-immunoprecipitation (CO-IP) were employed to elucidate the impact and underlying mechanisms of GPR116 in AILI. RESULTS: Intrahepatic GPR116 was upregulated in human and mice with AILI. GPR116â³HC mice were vulnerable to AILI compared to wild-type mice. Overexpression of GPR116 effectively mitigated AILI in wild-type mice and counteracted the heightened susceptibility of GPR116â³HC mice to APAP. Mechanistically, GPR116 inhibits the binding immunoglobulin protein (BiP), a critical regulator of ER function, through its interaction with ß-arrestin1, thereby mitigating ER stress during the early stage of AILI. Additionally, the activation of GPR116 by ligand FNDC4 has been shown to confer a protective effect against early hepatotoxicity caused by APAP in murine model. CONCLUSIONS: Upregulation of GPR116 on hepatocytes inhibits ER stress by binding to ß-arrestin1, protecting mice from APAP-induced hepatotoxicity. GPR116 may serve as a promising therapeutic target for AILI.
Asunto(s)
Acetaminofén , Enfermedad Hepática Inducida por Sustancias y Drogas , Estrés del Retículo Endoplásmico , Receptores Acoplados a Proteínas G , Animales , Humanos , Masculino , Ratones , Acetaminofén/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hígado/metabolismo , Hígado/patología , Hígado/efectos de los fármacos , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMEN
The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg2+ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.
Asunto(s)
Adenosina Trifosfato , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico , Estabilidad Proteica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Difosfato/metabolismo , Adenosina Difosfato/química , Unión Proteica , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/química , Dominios Proteicos , Magnesio/metabolismo , Magnesio/químicaRESUMEN
The AMP transferase, FICD, is an emerging drug target finetuning stress signaling in the endoplasmic reticulum (ER). FICD is a bi-functional enzyme, catalyzing both AMP addition (AMPylation) and removal (deAMPylation) from the ER resident chaperone BiP/GRP78. Despite increasing evidence linking excessive BiP/GRP78 AMPylation to human diseases, small molecules to inhibit pathogenic FICD variants are lacking. Using an in-vitro high-throughput screen, we identify two small-molecule FICD inhibitors, C22 and C73. Both molecules significantly inhibit FICD-mediated BiP/GRP78 AMPylation in intact cells while only weakly inhibiting BiP/GRP78 deAMPylation. C22 and C73 also efficiently inhibit pathogenic FICD variants and improve proinsulin processing in ß cells. Our study identifies and validates FICD inhibitors, highlighting a novel therapeutic avenue against pathologic protein AMPylation.
RESUMEN
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.
Asunto(s)
Chaperonas Moleculares , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/química , Retículo Endoplásmico/metabolismo , Animales , Adenosina Trifosfato/metabolismoRESUMEN
Adenosyl monophosphate (AMP)ylation (the covalent transfer of an AMP from Adenosine Triphosphate (ATP) onto a target protein) is catalyzed by the human enzyme Huntingtin Yeast Interacting Partner E (HYPE)/FicD to regulate its substrate, the heat shock chaperone binding immunoglobulin protein (BiP). HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the endoplasmic reticulum and mounting a unfolded protein response in times of proteostatic imbalance. Thus, manipulating HYPE's enzymatic activity is a key therapeutic strategy toward the treatment of various protein misfolding diseases, including neuropathy and early-onset diabetes associated with two recently identified clinical mutations of HYPE. Herein, we present an optimized, fluorescence polarization-based, high-throughput screening (HTS) assay to discover activators and inhibitors of HYPE-mediated AMPylation. After challenging our HTS assay with over 30,000 compounds, we discovered a novel AMPylase inhibitor, I2.10. We also determined a low micromolar IC50 for I2.10 and employed biorthogonal counter-screens to validate its efficacy against HYPE's AMPylation of BiP. Further, we report low cytotoxicity of I2.10 on human cell lines. We thus established an optimized, high-quality HTS assay amenable to tracking HYPE's enzymatic activity at scale, and provided the first novel small-molecule inhibitor capable of perturbing HYPE-directed AMPylation of BiP in vitro. Our HTS assay and I2.10 compound serve as a platform for further development of HYPE-specific small-molecule therapeutics.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Humanos , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Adenosina Monofosfato/análogos & derivados , Chaperón BiP del Retículo Endoplásmico/metabolismo , Células HEK293 , Proteínas de la Membrana , NucleotidiltransferasasRESUMEN
The human glucose-regulated protein GRP78 is a human chaperone that translocactes to the cell surface when cells are under stress. Theoretical studies suggested it could be involved in SARS-CoV-2 virus entry to cells. In this work, we used inâ vitro surface plasmon resonance-based assays to show that human GRP78 indeed binds to SARS-CoV-2 spike protein. We have designed and synthesised cyclic peptides based on the loop structure of amino acids 480-488 of the SARS-CoV-2 spike protein S1 domain from the Wuhan and Omicron variants and showed that both peptides bind to GRP78. Consistent with the greater infectiousness of the Omicron variant, the Omicron-derived peptide displays slower dissociation from the target protein. Both peptides significantly inhibit the binding of wild-type S1 protein to the human protein GRP78 suggesting that further development of these cyclic peptide motifs may provide a viable route to novel anti-SARS-CoV-2 agents.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico , Péptidos Cíclicos , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Unión Proteica , COVID-19/virología , COVID-19/metabolismoRESUMEN
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.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Pliegue de Proteína , Humanos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Nucleótidos , Adenosina Trifosfato/metabolismoRESUMEN
Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of acyl-coenzyme A oxidase 1 (ACOX1), which is the rate-limiting enzyme in the peroxisomal fatty acid ß-oxidation pathway, contributes to the development of fibrosis in renal allografts. ACOX1 deficiency leads to lipid accumulation and excessive oxidation of polyunsaturated fatty acids (PUFAs), which mediate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) reorganization respectively, thus causing fibrosis in renal allografts. Furthermore, activation of Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) signaling induced ACOX1 downregulation in a DNA methyltransferase 1 (DNMT1)-dependent manner. Overconsumption of PUFA resulted in endoplasmic reticulum (ER) stress, which played a vital role in facilitating ECM reorganization. Supplementation with PUFAs contributed to delayed fibrosis in a rat model of renal transplantation. The study provides a novel therapeutic approach that can delay chronic interstitial fibrosis in renal allografts by targeting the disorder of lipid metabolism.
Asunto(s)
Acil-CoA Oxidasa , Trasplante de Riñón , Riñón , Enfermedades Metabólicas , Animales , Ratas , Acil-CoA Oxidasa/metabolismo , Aloinjertos , Fibrosis , Riñón/patología , LípidosRESUMEN
Glucose-regulated protein 78 (GRP78), also termed HSPA5, was widely studied in cancer. It was recently approved that GRP78 has nuclear localization potential that sheds light on its role in cancer development. The inhibitor of DNA binding and differentiation 2 (ID2) is the nuclear component that associates with GRP78. The interaction between these two proteins is not understood clearly. In the current study, the binding pattern of GRP78/ID2 is predicted using computational methods. Protein-protein docking is used along with molecular dynamics simulation. The substrate binding domain ß of GRP78 can stably interact with the loop region (C42-S60) of ID2 as predicted in this study. This paves the way for a possible destabilizer for this association and cancer eradication.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico , Proteína 2 Inhibidora de la Diferenciación , Humanos , ADN , Chaperón BiP del Retículo Endoplásmico/metabolismo , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Neoplasias/metabolismo , ProteínasRESUMEN
A Borrelia miyamotoi gene with partial homology to bipA of relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae was identified by a GenBank basic alignment search analysis. We hypothesized that this gene product may be an immunogenic antigen as described for other relapsing fever Borrelia (RFB) and could serve as a serological marker for B. miyamotoi infections. The B. miyamotoi gene was a truncated version about half the size of the B. hermsii and B. turicatae bipA with a coding sequence of 894 base pairs. The gene product had a calculated molecular size of 32.7 kDa (including the signal peptide). Amino acid alignments with B. hermsii and B. turicatae BipA proteins and with other B. miyamotoi isolates showed conservation at the carboxyl end. We cloned the B. miyamotoi bipA-like gene (herein named bipM) and generated recombinant protein for serological characterization and for antiserum production. Protease protection analysis demonstrated that BipM was surface exposed. Serologic analyses using anti-B. miyamotoi serum samples from tick bite-infected and needle inoculated mice showed 94 % positivity against BipM. The 4 BipM negative serum samples were blotted against another B. miyamotoi antigen, BmaA, and two of them were seropositive resulting in 97 % positivity with both antigens. Serum samples from B. burgdorferi sensu stricto (s.s.)-infected mice were non-reactive against rBipM by immunoblot. Serum samples from Lyme disease patients were also serologically negative against BipM except for 1 sample which may have indicated a possible co-infection. A recently published study demonstrated that B. miyamotoi BipM was non-reactive against serum samples from B. hermsii, Borrelia parkeri, and B. turicatae infected animals. These results show that BipM has potential for a B. miyamotoi-infection specific and sensitive serodiagnostic to differentiate between Lyme disease and various RFB infections.
Asunto(s)
Infecciones por Borrelia , Borrelia , Enfermedad de Lyme , Fiebre Recurrente , Humanos , Animales , Ratones , Fiebre Recurrente/diagnóstico , Enfermedad de Lyme/diagnóstico , Infecciones por Borrelia/diagnóstico , AntígenosRESUMEN
Mammalian cells have three types of endoplasmic reticulum (ER) stress-sensing molecules: ATF6, IRE1, and PERK. Among these, ATF6 is unique in that it is processed in an ER-stress-specific manner and functions as a transcription factor for the activation of anti-ER stress genes (such as BiP). ATF6 is known to have two homologues, ATF6α and ATF6ß, and a greater understanding of their functions has been achieved through analyses using cultured cells. Physiological functions are also gradually being investigated in mice lacking ATF6α or ATF6ß. However, little is known about the effects on mouse organisms of the deletion of both the ATF6α and ATF6ß genes, since such double-knockout (DKO) mice suffer embryonic lethality at an early developmental stage. In this study, we generated and analyzed ATF6 DKO mice in which embryonic lethality was evaded by using Cre/loxP technology. Pancreatic ß cell-specific ATF6 DKO mice were born normally and lived without dysregulation of blood-glucose levels but had a reduced tolerance to glucose. Islets isolated from ATF6 DKO mice also showed low production and secretion of insulin and mild enhancement of IRE1 and PERK activity. We further examined the developmental abnormalities of systemic ATF6 DKO mice. The phenotypes of ATF6α-/-; ATF6ß-/- mice were similar to those previously reported, but ATF6α+/-; ATF6ß-/- and ATF6α-/-; ATF6ß+/- mice showed embryonic lethality at middle developmental stages, unlike those reported. Analysis of embryonic fibroblasts derived from these mice revealed that ATF6α and ATF6ß have a gene-dose-dependent functional redundancy and display distinct differences in their ability to induce BiP expression. (250 words).
Asunto(s)
Retículo Endoplásmico , Factores de Transcripción , Ratones , Animales , Retículo Endoplásmico/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , Estrés del Retículo Endoplásmico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Glucosa/metabolismo , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , MamíferosRESUMEN
This study aimed to examine the protective role of nebivolol (NEB) on liver tissue against the lipopolysaccharide (LPS)-induced sepsis model in rats by targeting endoplasmic reticulum (ER) stress-related binding immunoglobulin protein (Bip), CCAAT-enhancer-binding protein homologous protein (Chop) signaling pathways. Four groups, each comprising eight rats, were established: control, LPS, LPS + NEB, and NEB. Biochemical analyses included total oxidant status (TOS), serum aspartate transaminase (AST), and alanine aminotransferase (ALT) levels. Additionally, genetic assessments involved Chop and Bip/GRP78 mRNA expression levels, while histopathological examinations were conducted. Immunohistochemistry was used to determine interleukin-1 beta (IL-1 ß) and caspase-3 levels. The LPS group exhibited significantly higher AST, ALT, oxidative stress index, and TOS levels compared to the control group. Moreover, the LPS group demonstrated markedly increased Chop and Bip/GRP78 mRNA expression compared to the control group. Immunohistochemical analysis of the LPS group revealed significant upregulation in IL-1ß and caspase-3 expressions compared to the control group. Additionally, the LPS group showed significant hyperemia, mild hemorrhage, and inflammatory cell infiltrations. Comparatively, the LPS+NEB group exhibited a reversal of these alterations when compared to the LPS group. Collectively, our findings, suggest that NEB holds promise as a treatment in conditions where oxidative damage, inflammation, and ER stress-related apoptosis play significant roles in the pathogenesis.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Lipopolisacáridos , Hígado , Nebivolol , Estrés Oxidativo , Transducción de Señal , Factor de Transcripción CHOP , Animales , Lipopolisacáridos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Factor de Transcripción CHOP/metabolismo , Factor de Transcripción CHOP/genética , Masculino , Transducción de Señal/efectos de los fármacos , Nebivolol/farmacología , Nebivolol/uso terapéutico , Hígado/efectos de los fármacos , Hígado/patología , Hígado/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ratas , Inflamación/metabolismo , Inflamación/patología , Inflamación/prevención & control , Inflamación/tratamiento farmacológico , Chaperón BiP del Retículo Endoplásmico , Ratas Wistar , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Ratas Sprague-Dawley , Interleucina-1beta/metabolismoRESUMEN
The endoplasmic reticulum takes care of the folding, assembly, and quality control of thousands of proteins destined to the different compartments of the endomembrane system or to be secreted in the apoplast. Here we describe how these early events in the life of all these proteins can be followed biochemically by using velocity or isopycnic ultracentrifugation, metabolic labelling with radioactive amino acids, drug treatments, and immunoselection in various conditions and, in certain cases, predicted in silico by algorithms.
Asunto(s)
Algoritmos , Biosíntesis de Proteínas , Aminoácidos , Transporte Biológico , Retículo EndoplásmicoRESUMEN
The BiP co-chaperone DNAJC3 protects cells during ER stress. In mice, the deficiency of DNAJC3 leads to beta-cell apoptosis and the gradual onset of hyperglycemia. In humans, biallelic DNAJC3 variants cause a multisystem disease, including early-onset diabetes mellitus. Recently, hyperinsulinemic hypoglycemia (HH) has been recognized as part of this syndrome. This report presents a case study of an individual with HH caused by DNAJC3 variants and provides an overview of the metabolic phenotype of individuals with HH and DNAJC3 variants. The study demonstrates that HH may be a primary symptom of DNAJC3 deficiency and can persist until adolescence. Additionally, glycemia and insulin release were analyzed in young DNACJ3 knockout (K.O.) mice, which are equivalent to human infants. In the youngest experimentally accessible age group of 4-week-old mice, the in vivo glycemic phenotype was already dominated by a reduced total insulin secretion capacity. However, on a cellular level, the degree of insulin release of DNAJC3 K.O. islets was higher during periods of increased synthetic activity (high-glucose stimulation). We propose that calcium leakage from the ER into the cytosol, due to disrupted DNAJC3-controlled gating of the Sec61 channel, is the most likely mechanism for HH. This is the first genetic mechanism explaining HH solely by the disruption of intracellular calcium homeostasis. Clinicians should screen for HH in DNAJC3 deficiency and consider DNAJC3 variants in the differential diagnosis of congenital hyperinsulinism.
Asunto(s)
Hiperinsulinismo Congénito , Proteínas del Choque Térmico HSP40 , Adolescente , Animales , Humanos , Ratones , Calcio/metabolismo , Hiperinsulinismo Congénito/genética , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Insulina/metabolismo , Secreción de Insulina , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMEN
All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis, while DNA damage responses (DDRs) maintain genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primases pri-1 or pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like endoplasmic reticulum kinase (pek-1) branches of the (UPR-ER). Interestingly, activation of the (UPR-ER) output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators, ire-1 and X-box binding protein (xbp-1), and instead required the third branch of the (UPR-ER), activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the (UPR-ER), but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitizes animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause (UPR-ER) activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the (UPR-ER), but not other UPRs, responds to replication fork stress and that the (UPR-ER) is required to alleviate this stress.