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PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3. [ABSTRACT FROM AUTHOR]

It is believed that DNA double-strand breaks induced by Zika virus (ZIKV) infection in pregnant women is a main reason of brain damage (e.g. microcephaly, severe brain malformation, and neuropathy) in newborn babies [1,2], but its underlying mechanism is poorly understood. In this study, we report that the depletion of ERp57, a member of the protein disulphide isomerase (PDI) family, leads to the limited production of ZIKV in nerve cells. ERp57 knockout not only suppresses viral induced reactive oxygen species (ROS) mediated host DNA damage, but also decreases apoptosis. Strikingly, DNA damage depends on ERp57-bridged complex formation of viral protein NS2B/NS3. LOC14, an ERp57 inhibitor, restricts ZIKV infection and virus-induced DNA damage. Our work reveals an important role of ERp57 in both ZIKV propagation and virus-induced DNA damage, suggesting a potential target against ZIKV infection.

Abstract Hostile microenvironment of cancer cells provoke a stressful condition for endoplasmic reticulum (ER) and stimulate the expression and secretion of ER chaperones, leading to tumorigenic effects. However, the molecular mechanism underlying these effects is largely unknown. In this study, we reveal that the last four residues of ER chaperones, which are recognized by KDEL receptor (KDELR), is required for cell proliferation and migration induced by secreted chaperones. By combining proximity-based mass spectrometry analysis, split venus imaging and membrane yeast two hybrid assay, we present that EGF receptor (EGFR) may be a co-receptor for KDELR on the surface. Prior to ligand addition, KDELR spontaneously oligomerizes and constantly undergoes recycling near the plasma membrane. Upon KDEL ligand binding, the interactions of KDELR with itself and with EGFR increase rapidly, leading to augmented internalization of KDELR and tyrosine phosphorylation in the C-terminus of EGFR. STAT3, which binds the phosphorylated tyrosine motif on EGFR, is subsequently activated by EGFR and mediates cell growth and migration. Taken together, our results suggest that KDELR serves as a bona fide cell surface receptor for secreted ER chaperones and transactivates EGFR-STAT3 signaling pathway.

PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3.

Hostile microenvironment of cancer cells provoke a stressful condition for endoplasmic reticulum (ER) and stimulate the expression and secretion of ER chaperones, leading to tumorigenic effects. However, the molecular mechanism underlying these effects is largely unknown. In this study, we reveal that the last four residues of ER chaperones, which are recognized by KDEL receptor (KDELR), is required for cell proliferation and migration induced by secreted chaperones. By combining proximity-based mass spectrometry analysis, split venus imaging and membrane yeast two hybrid assay, we present that EGF receptor (EGFR) may be a co-receptor for KDELR on the surface. Prior to ligand addition, KDELR spontaneously oligomerizes and constantly undergoes recycling near the plasma membrane. Upon KDEL ligand binding, the interactions of KDELR with itself and with EGFR increase rapidly, leading to augmented internalization of KDELR and tyrosine phosphorylation in the C-terminus of EGFR. STAT3, which binds the phosphorylated tyrosine motif on EGFR, is subsequently activated by EGFR and mediates cell growth and migration. Taken together, our results suggest that KDELR serves as a bona fide cell surface receptor for secreted ER chaperones and transactivates EGFR-STAT3 signaling pathway. [ABSTRACT FROM AUTHOR]

The protein disulfide isomerase A3 (PDIA3) is directly or indirectly involved in various physiopathological processes and participates in cancer initiation, progression and chemosensitivity. However, little is known about its involvement in glioblastoma. To obtain specific information, we performed cellular experiments in the T98G and U−87 MG glioblastoma cell lines to evaluate the role of PDIA3. The loss of PDIA3 functions, either through inhibition or silencing, reduced glioblastoma cells spreading by triggering cytotoxic phenomena. PDIA3 inhibition led to a redistribution of PDIA3, resulting in the formation of protein aggregates visualized through immunofluorescence staining. Concurrently, cell cycle progression underwent arrest at the G1/S checkpoint. After PDIA3 inhibition, ROS-independent DNA damage and the activation of the repair system occurred, as evidenced by the phosphorylation of H2A.X and the overexpression of the Ku70 protein. We also demonstrated through a clonogenic assay that PDIA3 inhibition could increase the chemosensitivity of T98G and U-87 MG cells to the approved glioblastoma drug temozolomide (TMZ). Overall, PDIA3 inhibition induced cytotoxic effects in the analyzed glioblastoma cell lines. Although further in vivo studies are needed, the results suggested PDIA3 as a novel therapeutic target that could also be included in already approved therapies. [ABSTRACT FROM AUTHOR]

Thiol isomerases, including PDI, ERp57, ERp5, and ERp72, play important and distinct roles in cancer progression, cancer cell signaling, and metastasis. We recently discovered that zafirlukast, an FDA‐approved medication for asthma, is a pan‐thiol isomerase inhibitor. Zafirlukast inhibited the growth of multiple cancer cell lines with an IC50 in the low micromolar range, while also inhibiting cellular thiol isomerase activity, EGFR activation, and downstream phosphorylation of Gab1. Zafirlukast also blocked the procoagulant activity of OVCAR8 cells by inhibiting tissue factor‐dependent Factor Xa generation. In an ovarian cancer xenograft model, statistically significant differences in tumor size between control vs treated groups were observed by Day 18. Zafirlukast also significantly reduced the number and size of metastatic tumors found within the lungs of the mock‐treated controls. When added to a chemotherapeutic regimen, zafirlukast significantly reduced growth, by 38% compared with the mice receiving only the chemotherapeutic treatment, and by 83% over untreated controls. Finally, we conducted a pilot clinical trial in women with tumor marker‐only (CA‐125) relapsed ovarian cancer, where the rate of rise of CA‐125 was significantly reduced following treatment with zafirlukast, while no severe adverse events were reported. Thiol isomerase inhibition with zafirlukast represents a novel, well‐tolerated therapeutic in the treatment of ovarian cancer. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by GBA1 mutations resulting in defective glucocerebrosidase (GCase) and consequent accumulation of its substrates ß-glucosylceramide (ß-GlcCer). We reported progranulin (PGRN), a secretary growth factorlike molecule and an intracellular lysosomal protein was a crucial co-factor of GCase. PGRN binds to GCase and recruits Heat Shock Protein 70 (Hsp70) to GCase through its C-terminal Granulin (Grn) E domain, termed as ND7. In addition, both PGRN and ND7 are therapeutic against GD. Herein we found that both PGRN and its derived ND7 still displayed significant protective effects against GD in Hsp70 deficient cells. To delineate the molecular mechanisms underlying PGRN's Hsp70-independent regulation of GD, we performed a biochemical co-purification and mass spectrometry with His-tagged PGRN and His-tagged ND7 in Hsp70 deficient cells, which led to the identification of ERp57, also referred to as protein disulfide isomerase A3 (PDIA3), as a protein that binds to both PGRN and ND7. Within type 2 neuropathic GD patient fibroblasts L444P, bearing GBA1 L444P mutation, deletion of ERp57 largely abolished the therapeutic effects of PGRN and ND7, as manifested by loss of effects on lysosomal storage, GCase activity, and ß-GlcCer accumulation. Additionally, recombinant ERp57 effectively restored the therapeutic effects of PGRN and ND7 in ERp57 knockout L444P fibroblasts. Collectively, this study reports ERp57 as a previously unrecognized binding partner of PGRN that contributes to PGRN regulation of GD. [ABSTRACT FROM AUTHOR]

Abstract The ERp57/PDIA3 protein is a pleiotropic member of the PDIs family and, although predominantly located in the endoplasmic reticulum (ER), has indeed been found in other cellular compartments, such as the nucleus or the cell membrane. ERp57/PDIA3 is an important research target considering it can be found in various subcellular locations. This protein is involved in many different physiological and pathological processes, and our review describes new data on its functions and summarizes some ligands identified as PDIA3-specific inhibitors.

The protein disulfide isomerase A3 (PDIA3) is directly or indirectly involved in various physiopathological processes and participates in cancer initiation, progression and chemosensitivity. However, little is known about its involvement in glioblastoma. To obtain specific information, we performed cellular experiments in the T98G and U−87 MG glioblastoma cell lines to evaluate the role of PDIA3. The loss of PDIA3 functions, either through inhibition or silencing, reduced glioblastoma cells spreading by triggering cytotoxic phenomena. PDIA3 inhibition led to a redistribution of PDIA3, resulting in the formation of protein aggregates visualized through immunofluorescence staining. Concurrently, cell cycle progression underwent arrest at the G1/S checkpoint. After PDIA3 inhibition, ROS-independent DNA damage and the activation of the repair system occurred, as evidenced by the phosphorylation of H2A.X and the overexpression of the Ku70 protein. We also demonstrated through a clonogenic assay that PDIA3 inhibition could increase the chemosensitivity of T98G and U-87 MG cells to the approved glioblastoma drug temozolomide (TMZ). Overall, PDIA3 inhibition induced cytotoxic effects in the analyzed glioblastoma cell lines. Although further in vivo studies are needed, the results suggested PDIA3 as a novel therapeutic target that could also be included in already approved therapies.

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid‐β peptide (Aβ) in the form of senile plaques. Aβ can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aβ peptides, suggesting that it may be a carrier protein which prevents aggregation of Aβ. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aβ with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aβ25−35 fragment in vitro with high affinity via two in silico‐predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short‐term Aβ25−35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aβ25−35 and restores cellular viability, by preventing Aβ25−35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aβ toxicity and highlights it as a possible therapeutic tool in the treatment of AD. [ABSTRACT FROM AUTHOR]

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that affects motoneurons. Mutations in superoxide dismutase 1 (SOD1) have been described as a causative genetic factor for ALS. Mice overexpressing ALS-linked mutant SOD1 develop ALS symptoms accompanied by histopathological alterations and protein aggregation. The protein disulfide isomerase family member ERp57 is one of the main up-regulated proteins in tissue of ALS patients and mutant SOD1 mice, whereas point mutations in ERp57 were described as possible risk factors to develop the disease. ERp57 catalyzes disulfide bond formation and isomerization in the endoplasmic reticulum (ER), constituting a central component of protein quality control mechanisms. However, the actual contribution of ERp57 to ALS pathogenesis remained to be defined. Here, we studied the consequences of overexpressing ERp57 in experimental ALS using mutant SOD1 mice. Double transgenic SOD1G93A/ERp57WT animals presented delayed deterioration of electrophysiological activity and maintained muscle innervation compared to single transgenic SOD1G93A littermates at early-symptomatic stage, along with improved motor performance without affecting survival. The overexpression of ERp57 reduced mutant SOD1 aggregation, but only at disease end-stage, dissociating its role as an anti-aggregation factor from the protection of neuromuscular junctions. Instead, proteomic analysis revealed that the neuroprotective effects of ERp57 overexpression correlated with increased levels of synaptic and actin cytoskeleton proteins in the spinal cord. Taken together, our results suggest that ERp57 operates as a disease modifier at early stages by maintaining motoneuron connectivity.

It is believed that DNA double-strand breaks induced by Zika virus (ZIKV) infection in pregnant women is a main reason of brain damage (e.g. microcephaly, severe brain malformation, and neuropathy) in newborn babies [1,2], but its underlying mechanism is poorly understood. In this study, we report that the depletion of ERp57, a member of the protein disulphide isomerase (PDI) family, leads to the limited production of ZIKV in nerve cells. ERp57 knockout not only suppresses viral induced reactive oxygen species (ROS) mediated host DNA damage, but also decreases apoptosis. Strikingly, DNA damage depends on ERp57-bridged complex formation of viral protein NS2B/NS3. LOC14, an ERp57 inhibitor, restricts ZIKV infection and virus-induced DNA damage. Our work reveals an important role of ERp57 in both ZIKV propagation and virus-induced DNA damage, suggesting a potential target against ZIKV infection.

The ERp57/PDIA3 protein is a pleiotropic member of the PDIs family and, although predominantly located in the endoplasmic reticulum (ER), has indeed been found in other cellular compartments, such as the nucleus or the cell membrane. ERp57/PDIA3 is an important research target considering it can be found in various subcellular locations. This protein is involved in many different physiological and pathological processes, and our review describes new data on its functions and summarizes some ligands identified as PDIA3-specific inhibitors. [ABSTRACT FROM AUTHOR]

The vitamin D hormone, 1,25dihydroxyvitamin D3 (1,25(OH)2D3), and related compounds derived from vitamin D3 or lumisterol as a result of metabolism via the enzyme CYP11A1, have been shown, when applied 24 hours before or immediately after UV irradiation, to protect human skin cells and skin from DNA damage due to UV exposure, by reducing both cyclobutane pyrimidine dimers (CPD) and oxidative damage in the form of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐OHdG). We now report that knockdown of either the vitamin D receptor or the endoplasmic reticulum protein ERp57 by small, interfering RNA (siRNA) abolished the reductions in UV‐induced DNA damage with 20‐hydroxyvitamin D3 or 24‐hydroxylumisterol3, as previously shown for 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced oxygen consumption rates in UV‐exposed and sham‐exposed human keratinocytes and reduced phosphorylation of cyclic AMP response binding element protein (CREB). Both these actions have been shown to inhibit skin carcinogenesis after chronic UV exposure, consistent with the anticarcinogenic activity of 1,25(OH)2D3. The requirement for a vitamin D receptor for the photoprotective actions of 1,25(OH)2D3 and of naturally occurring CYP11A1‐derived vitamin D–related compounds may explain why mice lacking the vitamin D receptor in skin are more susceptible to UV‐induced skin cancers, whereas mice lacking the 1α‐hydroxylase and thus unable to make 1,25(OH)2D3 are not more susceptible. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Abstract Background Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA’s immunogenicity, and many efforts have been made to stabilize the expressed HA proteins. Methods In this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA. Results The percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells. Conclusion ERp57 could improve the stability and immunogenicity of H3N2 HA.

ABSTRACT The vitamin D hormone, 1,25dihydroxyvitamin D3 (1,25(OH)2D3), and related compounds derived from vitamin D3 or lumisterol as a result of metabolism via the enzyme CYP11A1, have been shown, when applied 24 hours before or immediately after UV irradiation, to protect human skin cells and skin from DNA damage due to UV exposure, by reducing both cyclobutane pyrimidine dimers (CPD) and oxidative damage in the form of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐OHdG). We now report that knockdown of either the vitamin D receptor or the endoplasmic reticulum protein ERp57 by small, interfering RNA (siRNA) abolished the reductions in UV‐induced DNA damage with 20‐hydroxyvitamin D3 or 24‐hydroxylumisterol3, as previously shown for 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced oxygen consumption rates in UV‐exposed and sham‐exposed human keratinocytes and reduced phosphorylation of cyclic AMP response binding element protein (CREB). Both these actions have been shown to inhibit skin carcinogenesis after chronic UV exposure, consistent with the anticarcinogenic activity of 1,25(OH)2D3. The requirement for a vitamin D receptor for the photoprotective actions of 1,25(OH)2D3 and of naturally occurring CYP11A1‐derived vitamin D–related compounds may explain why mice lacking the vitamin D receptor in skin are more susceptible to UV‐induced skin cancers, whereas mice lacking the 1α‐hydroxylase and thus unable to make 1,25(OH)2D3 are not more susceptible. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The protein ERp57 is a stress-responsive protein, mainly exists in the endoplasmic reticulum (ER), and a small amount in the cell membrane, cytoplasm, nucleus and mitochondria, which is involved in the signal transduction from the cell surface, the regulation process that occurs in the nucleus, and the formation of polymer protein complexes involved in DNA repair. Various degrees of ERp57 dysregulation has been observed in many types of non-communicable diseases especially in cancers. Previous studies showed that the expression of ERp57 could play a key role in occurrence and development of cancers such as breast cancer, gastric cancer, ovarian cancer, etc.; in addition, it has been suggested to play a pivotal role in disease progression of non-cancerous diseases such as neurodegeneration, liver disease, kidney disease, intestinal irritability syndrome and airway hypersensitivity. Thus, abnormal expression of ERp57 could be used as promising biomarker for cancer diagnosis and prognosis based on the previous studies. In this regard, current study was aimed to review the literature, which have been elucidate the role of ERp57 protein expression in both non-cancer and cancer disease. Overall, most studies have shown that inhibiting/knocking out of ERp57 could inhibit the cell proliferation and also induce apoptosis in both human cancerous and non-cancerous cells. Also, it has been suggested that the overexpression of ERp57 could intensify the cancer development. Therefore, it could be hypothesized that targeting of ERp57 might be a potential treatment in cancerous and non-cancerous diseases. [ABSTRACT FROM AUTHOR]

Essential thrombocythemia (ET) is a clonal disorder of a multipotent hematopoietic progenitor cell. In most patients, a driving mutation of Janus kinase 2 gene, calreticulin gene or myeloproliferative leukemia virus oncogene is detected. The occurrence of thrombotic and/or bleeding complications is very typical in manifestations of ET, with many cases of both occurring in the same patient. The thrombotic or bleeding phenotype can be a consequence of the coexistence of driving and non-driving molecular mutations and polymorphisms, affecting the platelet number and function. This paper discusses the nature of this disease, paying special attention to calreticulin gene function. [ABSTRACT FROM AUTHOR]

Abstract Background ERp57 dysfunction has been shown to contribute to tumorigenesis in multiple malignances. However, the role of ERp57 in clear cell renal carcinoma (ccRCC) remains unclear. Methods Cell proliferation ability was measured by MTT and colony forming assays. Western blotting and quantitative real-time PCR (qRT-PCR) were performed to measure protein and mRNA expression. Co-immunoprecipitation (CoIP) and proximity ligation assay (PLA) were performed to detect protein-protein interaction. Chromatin immunoprecipitation (ChIP), ribonucleoprotein immunoprecipitation (RIP), and oligo pull-down were used to confirm DNA–protein and RNA–protein interactions. Promoter luciferase analysis was used to detect transcription factor activity. Results Here we found ERp57 was overexpressed in ccRCC tissues, and the higher levels of ERp57 were correlated with poor survival in patients with ccRCC. In vivo and in vitro experiments showed that ccRCC cell proliferation was enhanced by ERp57 overexpression and inhibited by ERp57 deletion. Importantly, we found ERp57 positively regulated ILF3 expression in ccRCC cells. Mechanically, ERp57 was shown to bind to STAT3 protein and enhance the STAT3-mediated transcriptional activity of ILF3. Furthermore, ILF3 levels were increased in ccRCC tissues and associated with poor prognosis. Interestingly, we revealed that ILF3 could bind to ERp57 and positively regulate its expression by enhancing its mRNA stability. Furthermore, ccRCC cell proliferation was moderated via the ERp57/STAT3/ILF3 feedback loop. Conclusions In summary, our results indicate that the ERp57/STAT3/ILF3 feedback loop plays a key role in the oncogenesis of ccRCC and provides a potential therapeutic target for ccRCC treatment.

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that affects motoneurons. Mutations in superoxide dismutase 1 (SOD1) have been described as a causative genetic factor for ALS. Mice overexpressing ALS-linked mutant SOD1 develop ALS symptoms accompanied by histopathological alterations and protein aggregation. The protein disulfide isomerase family member ERp57 is one of the main up-regulated proteins in tissue of ALS patients and mutant SOD1 mice, whereas point mutations in ERp57 were described as possible risk factors to develop the disease. ERp57 catalyzes disulfide bond formation and isomerization in the endoplasmic reticulum (ER), constituting a central component of protein quality control mechanisms. However, the actual contribution of ERp57 to ALS pathogenesis remained to be defined. Here, we studied the consequences of overexpressing ERp57 in experimental ALS using mutant SOD1 mice. Double transgenic SOD1G93A/ERp57WT animals presented delayed deterioration of electrophysiological activity and maintained muscle innervation compared to single transgenic SOD1G93A littermates at early-symptomatic stage, along with improved motor performance without affecting survival. The overexpression of ERp57 reduced mutant SOD1 aggregation, but only at disease end-stage, dissociating its role as an anti-aggregation factor from the protection of neuromuscular junctions. Instead, proteomic analysis revealed that the neuroprotective effects of ERp57 overexpression correlated with increased levels of synaptic and actin cytoskeleton proteins in the spinal cord. Taken together, our results suggest that ERp57 operates as a disease modifier at early stages by maintaining motoneuron connectivity. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease-19), represents a far more serious threat to public health than SARS and MERS coronaviruses, due to its ability to spread more efficiently than its predecessors. Currently, there is no worldwide-approved effective treatment for COVID-19, urging the scientific community to intense efforts to accelerate the discovery and development of prophylactic and therapeutic solutions against SARS-CoV-2 infection. In particular, effective antiviral drugs are urgently needed. With few exceptions, therapeutic approaches to combat viral infections have traditionally focused on targeting unique viral components or enzymes; however, it has now become evident that this strategy often fails due to the rapid emergence of drug-resistant viruses. Targeting host factors that are essential for the virus life cycle, but are dispensable for the host, has recently received increasing attention. The spike glycoprotein, a component of the viral envelope that decorates the virion surface as a distinctive crown ("corona") and is essential for SARS-CoV-2 entry into host cells, represents a key target for developing therapeutics capable of blocking virus invasion. This review highlights aspects of the SARS-CoV-2 spike biogenesis that may be amenable to host-directed antiviral targeting. [ABSTRACT FROM AUTHOR]

Aggregates of α-synuclein contribute to the etiology of Parkinson’s Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson’s Disease. The protective role of PDI in Parkinson’s Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a central step in celiac disease (CD) pathogenesis. Microbial transglutaminase (mTG) mimics the enzymatic function of TG2 and might play a role in CD. TG2 is inhibited by endogenous oxidative endoplasmic reticulum-resident protein 57 (ERp57), but data about mTG are lacking. We investigated the localization of ERp57 in duodenal biopsies and examined inhibition of TG2, and mTG by competitive, and oxidative molecules. Localization of ERp57 was investigated in duodenal biopsies from CD, and control patients by electron microcopy. Inhibition of TG2 and mTG was analyzed on an in vitro level using a photometric assay. ERp57 was observed within the lamina propria and its abundance within the endoplasmic reticulum (ER) was reduced in CD patients. TG2 was oxidatively inhibited by up to 95% by PX12 (p < 0.001) and L-cystine (p < 0.001), whereas mTG remained unaffected. The reduced presence of ERp57 within the ER of CD biopsies suggests a regulatory function of this protein within CD pathogenesis. PX12 and L-cystine oxidatively inhibit TG2 and might serve as treatment options in CD. mTG is poorly regulated and could contribute to the accumulation of immunogenic peptides within the gut with potential pathogenic effects.

The endoplasmic reticulum (ER) is the major folding compartment for secreted and membrane proteins and is the site of a specific chaperone system, the calnexin cycle, for folding N‐glycosylated proteins. Recent structures of components of the calnexin cycle have deepened our understanding of quality control mechanisms and protein folding pathways in the ER. In the calnexin cycle, proteins carrying monoglucosylated glycans bind to the lectin chaperones calnexin and calreticulin, which recruit a variety of function‐specific chaperones to mediate protein disulfide formation, proline isomerization, and general protein folding. Upon trimming by glucosidase II, the glycan without an inner glucose residue is no longer able to bind to the lectin chaperones. For proteins that have not yet folded properly, the enzyme UDP‐glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by adding a glucose back to the N‐glycan. This allows the misfolded proteins to re‐associate with calnexin and calreticulin for additional rounds of chaperone‐mediated refolding and prevents them from exiting the ERs. Here, we review progress in structural studies of the calnexin cycle, which reveal common features of how lectin chaperones recruit function‐specific chaperones and how UGGT recognizes misfolded proteins. [ABSTRACT FROM AUTHOR]

Background: Hemagglutinin (HA), as the surface immunogenic protein, is the most important component of influenza viruses. Previous studies showed that the stability of HA was significant for HA's immunogenicity, and many efforts have been made to stabilize the expressed HA proteins. Methods: In this study, the protein disulfide isomerases (PDIs) were investigated for the ability to improve the stability of HA protein. Two members of the PDIs family, PDI and ERp57, were over-expressed or down-expressed in 293 T cells. The expression of H3 HA and PDIs were investigated by real-time qPCR, western-blot, immunofluorescence assay, and flow cytometry. The stability of HA was investigated by western-blot under non-reducing condition. Moreover, BALB/c mice were immunized subcutaneously twice with the vaccine that contained HA proteins from the ERp57-overexpressed and conventional 293 T cells respectively to investigate the impact of ERp57 on the immunogenicity of H3N2 HA. Results: The percentage of the disulfide-bonded HA trimers increased significantly in the PDIs-overexpressed 293 T cells, and ERp57 was more valid to the stability of HA than PDI. The knockdown of ERp57 by small interfering RNA significantly decreased the percentage of the disulfide-bonded HA trimers. HA proteins from ERp57-overexpressed 293 T cells stimulated the mice to generate significantly higher HA-specific IgG against H1N1 and H3N2 viruses than those from the conventional cells. The mice receiving H3 HA from ERp57-overexpressed 293 T cells showed the better resistance against H1N1 viruses and the higher survival rate than the mice receiving H3 HA from the conventional cells. Conclusion: ERp57 could improve the stability and immunogenicity of H3N2 HA. [ABSTRACT FROM AUTHOR]

Therapy-induced presentation of cell surface calreticulin (CRT) is a pro-phagocytic immunogen beneficial for invoking anti-tumor immunity. Here, we characterized the roles of ERp57 and α-integrins as CRT-interacting proteins that coordinately regulate CRT translocation from the ER to the surface during immunogenic cell death. Using T-lymphoblasts as a genetic cell model, we found that drug-induced surface CRT is dependent on ERp57, while drug-induced surface ERp57 is independent of CRT. Differential subcellular immunostaining assays revealed that ERp57−/− cells have minimal cytosolic CRT, indicating that ERp57 is indispensable for extra-ER accumulation of CRT. Stimulation of integrin activity, with either cell adhesion or molecular agonists, resulted in decreased drug-induced surface CRT and ERp57 levels. Similarly, surface CRT and ERp57 was reduced in cells expressing GFFKR, a conserved α-integrin cytosolic motif that binds CRT. Drug-induced surface ERp57 levels were consistently higher in CRT−/− cells, suggesting integrin inhibition of surface ERp57 is an indirect consequence of α-integrin binding to CRT within the CRT-ERp57 complex. Furthermore, β1−/− cells with reduced expression of multiple α-integrins, exhibit enhanced levels of drug-induced surface CRT and ERp57. Our findings highlight the coordinate involvement of plasma membrane integrins as inhibitors, and ERp57 originating from the ER as promoters, of CRT translocation from the ER to the cell surface.

ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+.

Therapy-induced presentation of cell surface calreticulin (CRT) is a pro-phagocytic immunogen beneficial for invoking anti-tumor immunity. Here, we characterized the roles of ERp57 and α-integrins as CRT-interacting proteins that coordinately regulate CRT translocation from the ER to the surface during immunogenic cell death. Using T-lymphoblasts as a genetic cell model, we found that drug-induced surface CRT is dependent on ERp57, while drug-induced surface ERp57 is independent of CRT. Differential subcellular immunostaining assays revealed that ERp57−/− cells have minimal cytosolic CRT, indicating that ERp57 is indispensable for extra-ER accumulation of CRT. Stimulation of integrin activity, with either cell adhesion or molecular agonists, resulted in decreased drug-induced surface CRT and ERp57 levels. Similarly, surface CRT and ERp57 was reduced in cells expressing GFFKR, a conserved α-integrin cytosolic motif that binds CRT. Drug-induced surface ERp57 levels were consistently higher in CRT−/− cells, suggesting integrin inhibition of surface ERp57 is an indirect consequence of α-integrin binding to CRT within the CRT-ERp57 complex. Furthermore, β1−/− cells with reduced expression of multiple α-integrins, exhibit enhanced levels of drug-induced surface CRT and ERp57. Our findings highlight the coordinate involvement of plasma membrane integrins as inhibitors, and ERp57 originating from the ER as promoters, of CRT translocation from the ER to the cell surface. [ABSTRACT FROM AUTHOR]

Background/Aims: ER-Stress and activation of unfolded protein response belong to the major factors involved in chemoresistance in cancer cells. In this study we investigated the effect of shikonin on the survival of acute myeloid leukemia cells and the role of ER-stress protein ERP57, a protein disulfide isomerase, in improvement of chemotherapy. Methods: Using MTT assay we studied cytotoxic effects of shikonin on HL-60 cells. The flow cytometry was adopted to examine the shikonin induced mode of cell death in HL-60 cells. The overall protein expression alteration resulting from shikonin treatment was investigated using proteomics methods. Western blotting was performed to quantify the alteration in protein expression in HL-60 after shikonin treatment. Silencing and overexpression studies were carried out to highlight the therapeutic role of ERP57 in shikonin effect on AML cells. Results: Shikonin induces apoptosis in HL-60 cells without significant effect on Primary cells from healthy volunteers. The apoptotic effect was dose and time dependent and was accompanied by strong alteration in cell proteome. Among the proteins targeted by shikonin, ERP57 was significantly downregulated in HL-60 after treatment. Compared to healthy control ERP57 was found to be highly expressed in AML cell line HL60 and was downregulated after shikonin treatment. Overexpression of ERP57 protected HL-60 from shikonin induced apoptosis, whereas knockdown of ERP57 expression resulted in increase in shikonin induced apoptosis. Conclusions: Our results demonstrate that ERP57 plays a crucial role in resistance towards shikonin induced apoptosis in AML cells. Targeting of ERP57 might offer a new therapeutic option for the treatment of acute myeloid leukemia.

The pathogenesis of glaucoma, a common neurodegenerative disease, involves an immunologic component. Changes in the natural autoantibody profile of glaucoma patients were detected, showing not only up-regulated but also down-regulated immunoreactivities. In recent studies we were able to demonstrate that the antibody changes have a large influence on protein profiles of neuroretinal cells. Furthermore we could demonstrate neuroprotective potential of one of the down-regulated antibodies (γ-synuclein antibody). Anti-GFAP antibody is another antibody found down-regulated in glaucoma patients. Since GFAP expression is intensified in glaucomatous retina, the aim of this study was to detect the effect of GFAP antibodies on neuroretinal cells. This is realized with a viability-test as well as proteomic analysis of cells incubated with GFAP antibodies. Furthermore, possible interaction partners of the GFAP antibody in neuroretinal cells were identified by western blot, mass spectrometry and indirect immunofluorescence staining. We found that the GFAP antibody is able to protect cells from oxidative stress, which is due to changed protein expressions of the actin cytoskeleton. Furthermore we detected a cross-reaction of the antibody to endoplasmic reticulum resident protein 57 on the cell membrane, which seems to lead to a changed signaling in the cells triggering the protective effects.

Many AB toxins contain an enzymatic A moiety that is anchored to a cell-binding B moiety by a disulfide bridge. After receptor-mediated endocytosis, some AB toxins undergo retrograde transport to the endoplasmic reticulum (ER) where reduction of the disulfide bond occurs. The reduced A subunit then dissociates from the holotoxin and enters the cytosol to alter its cellular target. Intoxication requires A chain separation from the holotoxin, but, for many toxins, it is unclear if reduction alone is sufficient for toxin disassembly. Here, we examined the link between reduction and disassembly for several ER-translocating toxins. We found disassembly of the reduced Escherichia coli heat-labile enterotoxin (Ltx) required an interaction with one specific ER-localized oxidoreductase: protein disulfide isomerase (PDI). In contrast, the reduction and disassembly of ricin toxin (Rtx) and Shiga toxin 1 (Stx1) were coupled events that did not require PDI and could be triggered by reductant alone. PDI-deficient cells accordingly exhibited high resistance to Ltx with continued sensitivity to Rtx and Stx1. The distinct structural organization of each AB toxin thus appears to determine whether holotoxin disassembly occurs spontaneously upon disulfide reduction or requires the additional input of PDI., Competing Interests: The authors declare no conflict of interest.

Porcine reproductive and respiratory syndrome virus (PRRSV), a virulent pathogen of swine, suppresses the innate immune response and induces persistent infection. One mechanism used by viruses to evade the immune system is to cripple the antigen-processing machinery in monocyte-derived dendritic cells (MoDCs). In this study, we show that MoDCs infected by PRRSV express lower levels of the major histocompatibility complex (MHC)-peptide complex proteins TAP1 and ERp57 and are impaired in their ability to stimulate T cell proliferation and increase their production of CD83. Neutralization of sCD83 removes the inhibitory effects of PRRSV on MoDCs. When MoDCs are incubated with exogenously added sCD83 protein, TAP1 and ERp57 expression decreases and T lymphocyte activation is impaired. PRRSV nonstructural protein 1α (Nsp1α) enhances CD83 promoter activity. Mutations in the ZF domain of Nsp1α abolish its ability to activate the CD83 promoter. We generated recombinant PRRSVs with mutations in Nsp1α and the corresponding repaired PRRSVs. Viruses with Nsp1α mutations did not decrease levels of TAP1 and ERp57, impair the ability of MoDCs to stimulate T cell proliferation, or increase levels of sCD83. We show that the ZF domain of Nsp1α stimulates the secretion of CD83, which in turn inhibits MoDC function. Our study provides new insights into the mechanisms of immune suppression by PRRSV. IMPORTANCE PRRSV has a severe impact on the swine industry throughout the world. Understanding the mechanisms by which PRRSV infection suppresses the immune system is essential for a robust and sustainable swine industry. Here, we demonstrated that PRRSV infection manipulates MoDCs by interfering with their ability to produce proteins in the MHC-peptide complex. The virus also impairs the ability of MoDCs to stimulate cell proliferation, due in large part to the enhanced release of soluble CD83 from PRRSV-infected MoDCs. The viral nonstructural protein 1 (Nsp1) is responsible for upregulating CD83 promoter activity. Amino acids in the ZF domain of Nsp1α (L5-2A, rG45A, G48A, and L61-6A) are essential for CD83 promoter activation. Viruses with mutations at these sites no longer inhibit MoDC-mediated T cell proliferation. These findings provide novel insights into the mechanism by which the adaptive immune response is suppressed during PRRSV infection. [ABSTRACT FROM AUTHOR]

The protein disulfide isomerase ERp57 (GRp58/PDIA3/1,25D3-MARRS) has been implicated in a multitude of signaling pathways throughout the entire body. Most thoroughly studied for its protein-folding role, ERp57 has also been found to have multiple binding partners, and have significant effects on cellular growth. ERp57 has been studied n the context of several neurodegenerative disorders, metabolic conditions, and can be used as a prognosis marker in certain cancers. One role, as an alternate vitamin D binding receptor, has prompted research in tissues with known vitamin D activity, such as the intestine and bone. Vitamin D has been studied in relation to mammary gland growth and development, but it is not yet known if ERp57 plays an independent role in this tissue. In this study, ERp57 was knocked out in murine mammary gland epithelial cells of 30 4-week old mice. Several markers of mammary gland growth were measured, including number of terminal end buds (TEB), ductal coverage of the fat pad, and ductal extension. It was found the knockout animals had decreased numbers of TEBs (p = 0.019), and decreased ductal extension (p = 0.018) compared to wildtype animals, with no differences in gross body weight. Immunohistochemistry analysis of mammary glands showed ERp57 localized to the apical side of alveolar branches, and on leading edges of TEBs. These results provide further evidence for ERp57 functioning separately to the VDR, and further insights into the roles of ERp57. [ABSTRACT FROM AUTHOR]

Stress of cultured species is a common problem, affecting the host health, in aquatic farming. It occurs when interactions among the three elements – host, environment and pathogens, are not in balance, particularly under intensive culturing conditions (de la Vega et al., 2006). Hosts respond to stress by triggering cellular Ca 2 + signaling to activate many downstream pathways for their functions in order to mitigate the stress effect. Calreticulin (CRT), Calnexin (CNX) and Endoplasmic reticulum protein 57 (ERp57) are involved in Ca 2 + signaling in stress response mechanisms and are essential for survival and viability of organisms under stress conditions. In this study, the full length cDNA of CNX and ERp57 in the giant tiger shrimp ( Penaeus monodon ) were identified. Amino acid sequences deduced from both cDNA showed significantly high similarity with those present in other crustaceans. One and two forms of PmCNX and PmERp57 transcripts were found, respectively. For PmERp57 , its genomic sequence was additionally identified. It spanned 8257 base pairs (bp), composing of 10 exons (84–226 bp long) and 9 introns (93–2787 bp long). Both genes were expressed in all examined tissues of both juvenile and broodstock P. monodon . Effects of Vibrio harveyi and abotic stress commonly found in aquaculture on expression of PmCRT , PmCNX and PmERp57 were examined in hemocytes by quantitative real-time PCR. High temperature, hypo- and hyper-osmosis and V. harveyi infection induced expression all of the three genes ( P < 0.05). In contrast, analysis of hypoxia effect on expression of the Ca 2 + signaling-related genes, which was firstly studied in aquatic species, showed to suppress their expression ( P < 0.05). Results from this study suggested that PmCRT , PmCNX and PmERp57 are related to stress response mechanisms and immune system of P. monodon . Their expression is therefore a potential candidate for markers to detect or monitor the early sign of shrimp's stress and immunity changes in farming. [ABSTRACT FROM AUTHOR]

Study question: Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction?Summary answer: ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content.What is known already: A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins.Study design, size, duration: The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed.Participants/materials, setting, methods: Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays.Main results and the role of chance: Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa.Large scale data: N/A.Limitations, reasons for caution: The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted.Wider implications of the findings: Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction.Study funding/competing interest(s): This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests. [ABSTRACT FROM AUTHOR]

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by GBA1 mutations resulting in defective glucocerebrosidase (GCase) and consequent accumulation of its substrates β-glucosylceramide (β-GlcCer). We reported progranulin (PGRN), a secretary growth factor-like molecule and an intracellular lysosomal protein was a crucial co-factor of GCase. PGRN binds to GCase and recruits Heat Shock Protein 70 (Hsp70) to GCase through its C-terminal Granulin (Grn) E domain, termed as ND7. In addition, both PGRN and ND7 are therapeutic against GD. Herein we found that both PGRN and its derived ND7 still displayed significant protective effects against GD in Hsp70 deficient cells. To delineate the molecular mechanisms underlying PGRN's Hsp70-independent regulation of GD, we performed a biochemical co-purification and mass spectrometry with His-tagged PGRN and His-tagged ND7 in Hsp70 deficient cells, which led to the identification of ERp57, also referred to as protein disulfide isomerase A3 (PDIA3), as a protein that binds to both PGRN and ND7. Within type 2 neuropathic GD patient fibroblasts L444P, bearing GBA1 L444P mutation, deletion of ERp57 largely abolished the therapeutic effects of PGRN and ND7, as manifested by loss of effects on lysosomal storage, GCase activity, and β-GlcCer accumulation. Additionally, recombinant ERp57 effectively restored the therapeutic effects of PGRN and ND7 in ERp57 knockout L444P fibroblasts. Collectively, this study reports ERp57 as a previously unrecognized binding partner of PGRN that contributes to PGRN regulation of GD.

Background/Aims: ER-Stress and activation of unfolded protein response belong to the major factors involved in chemoresistance in cancer cells. In this study we investigated the effect of shikonin on the survival of acute myeloid leukemia cells and the role of ER-stress protein ERP57, a protein disulfide isomerase, in improvement of chemotherapy. Methods: Using MTT assay we studied cytotoxic effects of shikonin on HL-60 cells. The flow cytometry was adopted to examine the shikonin induced mode of cell death in HL-60 cells. The overall protein expression alteration resulting from shikonin treatment was investigated using proteomics methods. Western blotting was performed to quantify the alteration in protein expression in HL-60 after shikonin treatment. Silencing and overexpression studies were carried out to highlight the therapeutic role of ERP57 in shikonin effect on AML cells. Results: Shikonin induces apoptosis in HL-60 cells without significant effect on Primary cells from healthy volunteers. The apoptotic effect was dose and time dependent and was accompanied by strong alteration in cell proteome. Among the proteins targeted by shikonin, ERP57 was significantly downregulated in HL-60 after treatment. Compared to healthy control ERP57 was found to be highly expressed in AML cell line HL60 and was downregulated after shikonin treatment. Overexpression of ERP57 protected HL-60 from shikonin induced apoptosis, whereas knockdown of ERP57 expression resulted in increase in shikonin induced apoptosis. Conclusions: Our results demonstrate that ERP57 plays a crucial role in resistance towards shikonin induced apoptosis in AML cells. Targeting of ERP57 might offer a new therapeutic option for the treatment of acute myeloid leukemia. [ABSTRACT FROM AUTHOR]

Disturbance of endoplasmic reticulum ( ER) proteostasis is a common feature of amyotrophic lateral sclerosis ( ALS). Protein disulfide isomerases ( PDIs) are ER foldases identified as possible ALS biomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized four ALS-linked mutations recently identified in two major PDI genes, PDIA1 and PDIA3/ ERp57. Phenotypic screening in zebrafish revealed that the expression of these PDI variants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutant PDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of these PDI mutants. Finally, targeting ERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifies ER proteostasis imbalance as a risk factor for ALS, driving initial stages of the disease. [ABSTRACT FROM AUTHOR]

As a nongenomic action, 1,25-dihydroxyvitamin D 3 (1,25D 3 ) induces L-type Ca 2+ channel-mediated extracellular Ca 2+ influx in human aortic smooth muscle cells (HASMCs), which activates a disintegrin and metalloprotease 10 (ADAM10) to cleave and shed the ectodomain of tumor necrosis factor receptor 1 (TNFR1). In this study, we examined the potencies of other vitamin D 3 and D 2 analogs to stimulate the ectodomain shedding of TNFR1 in HASMCs. 25-Hydroxyvitamin D 3 (25D 3 ), a precursor of 1,25D 3 , and elocalcitol, an analog of 1,25D 3 , caused ectodomain shedding of TNFR1 within 30 min, whereas 1,25-dihydroxyvitamin D 2 (1,25D 2 ) and paricalcitol, a derivative of 1,25D 2 , did not. Both 25D 3 and elocalcitol rapidly induced extracellular Ca 2+ influx and markedly increased intracellular Ca 2+ , while 1,25D 2 and paricalcitol caused only small increases in intracellular Ca 2+ . 25D 3 - and elocalcitol-induced TNFR1 ectodomain sheddings were abolished by verapamil and in Ca 2+ -free media. Both 25D 3 and elocalcitol caused the translocation of ADAM10 to the cell surface, which was inhibited by verapamil, while 1,25D 2 and paricalcitol did not cause ADAM10 translocation. When ADAM10 was depleted by ADAM10-siRNA, 25D 3 and elocalcitol could not induce ectodomain shedding of TNFR1. The plasma membrane receptor, endoplasmic reticulum stress protein 57 (ERp57), but not the classic vitamin D receptor, mediated the nongenomic action of vitamin D to induce ectodomain shedding of TNFR1. In summary, like 1,25D 3 , 25D 3 and elocalcitol caused ADAM10-mediated ectodomain shedding of TNFR1, whereas 1,25D 2 and paricalcitol did not. The difference may depend on their affinities to ERp57 through which extracellular Ca 2+ influx is induced. [ABSTRACT FROM AUTHOR]

Evidences suggest that ERp57 and PGK-1 signaling lead to cancer cell proliferation and migration. We hypothesized that ERp57 and PGK-1 down-regulation may inactivate matrix metalloproteinase (MMP)-2, -9 expressions and inhibit hepatocellular carcinoma (HCC) migration. Antrodia cinnamomea is widely prescribed as an adjuvant to treat HCC in Taiwan. We aimed to investigate if ethanol extract of fruiting bodies of Antrodia cinnamomea (EEAC) and its active ingredients (i.e., zhankuic acid A, cordycepin, and adenosine) can modulate HCC cancer cells migration through ERp57 and PGK-1 and other molecular pathways such as PI3K/Akt and MAPK. ERp57 and PGK-1 siRNA were transfected into HCC to determine effects on MMP-2/-9 expressions and cell migration. We then examined the inhibitory effects of EEAC and its active ingredients on HCC migration and its related mechanisms including ERp57, PGK-1, PI3K/Akt, and MAPK signaling pathways. Down-regulation of ERp57 and PGK-1 by siRNA decreased MMP-2, -9 expressions and Transwell cell migration in HCC. Nontoxic EEAC markedly inhibited migration of HCC, and significantly inhibited activities and protein expressions of MMP-2 and -9, while the expression of the endogenous inhibitors (TIMP-1 and TIMP-2) of these proteins increased. Nontoxic EEAC and its active ingredients decreased ERp57, GLUD-1, GST-pi, and PGK-1 protein expressions. Finally, nontoxic EEAC inhibited the phosphorylated FAK, PI3K/Akt, and MAPK signaling. Our findings first indicate that EEAC and its ingredients effectively suppress HCC migration. Additionally, the molecular mechanisms appear to be mediated, in part, through the down-regulation of ERp57, PGK-1, MAPK, and PI3K/Akt. [ABSTRACT FROM AUTHOR]

Human leukocyte antigen (HLA) class I molecules are ligands for antigen receptors of cytotoxic T cells (CTL) and inhibitory receptors of natural killer (NK) cells. The high degree of HLA class I polymorphism allows for the selection of distinct and diverse sets of antigenic peptide ligands for presentation to CTL. The extensive polymorphisms of the HLA class I genes also result in large variations in their intracellular folding and assembly characteristics. Recent findings indicate that North American HLA-B variants differ significantly in the stabilities of their peptide-deficient forms and in the requirements for the endoplasmic reticulum (ER)-resident factor tapasin for proper assembly. In HIV-infected individuals, the presence of tapasin-independent HLA-B allotypes links to more rapid progression to death. Further studies are important to better understand how the intrinsic structural characteristics of HLA class I folding intermediates affect immune responses mediated by CTL and NK cells. [ABSTRACT FROM AUTHOR]