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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.

Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified ‘scratch wound’ assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3−/− mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3−/− MEFs was less effective in promoting cell spreading and F-actin organisation or supporting ‘scratch wound’ closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

Vitamin D has been implicated in mammary gland development, however nothing is known about the role of the second 1,25-dihydroxyvitamin D3 (1,25D3) receptor MARRS and its effects on post-natal mammary gland development or interaction with 1,25D3 in this tissue. Therefore, the objectives of this thesis were to investigate these unknowns in a tissue-specific knockout mouse model. In study 1, MARRS was knocked out in murine mammary gland epithelial cells of 4-week old mice (n=30). Several markers of mammary gland (MG) growth were measured, including number of terminal end buds (TEB), ductal coverage of the fat pad, and ductal extension. It was found that the knockout animals had decreased numbers of TEBs (p=0.019), and decreased ductal extension (p=0.018). Study 2 aimed to expand the investigation to MARRS’s actions with varying vitamin D3 dietary dose. Abdominal MGs were collected from 6-week old MARRS knockout female mice (n=94) on diets of 10,000 IU/kg (replete), 1,000 IU/kg (sufficient) or 0 IU/kg (deficient) of D3. There was a significant interaction between genotype and diet regarding TEBs (p=0.001) and ductal coverage (p=0.03). Knockout mice on the sufficient diet had significantly fewer TEBs (p=0.001) compared to wildtypes on the same diet, but the opposite effect was seen in mice on the replete diet. The increased growth in knockout mice on replete diets was unexpected; therefore in Study 3, a novel double knockout mouse lacking both MARRS and VDR was created to study the actions of both pathways simultaneously. There was a significant overall interaction between genotype groups (p=0.004) regarding mean ductal coverage, yet number of TEBs remained similar across all genotypes. Overall, this thesis demonstrates that MARRS does indeed play a role in mammary gland development providing a growth-positive effect, and that effect can be altered depending on the amount of vitamin D3 available in the diet. Although neither MARRS nor VDR are essential for mammary gland growth, their signalling pathways and gene-diet interactions provide valuable information regarding dietary intakes during puberty and influences on tissue growth. These results now merit further studies in human populations to investigate dietary trends and long-term consequences in human adolescents. Natural Sciences and Engineering Research Council of Canada

Down regulation of the major histocompatibility class (MHC) I pathway plays an important role in tumour development, and can be achieved by suppression of HLA expression or mutations in the MHC peptide-binding pocket. The peptide-loading complex (PLC) loads peptides on the MHC-I molecule in a dynamic multi-step assembly process. The effects of cancer variants on ERp57 and tapasin components from the MHC-I pathway is less known, and they could have an impact on antigen presentation. Applying computational approaches, we analysed whether the ERp57-tapasin binding might be altered by missense mutations. The variants H408R(ERp57) and P96L, D100A, G183R(tapasin) at the protein&ndash, protein interface improved protein stability (&Delta, &Delta, G) during the initial screen of 14 different variants. The H408R(ERp57) and P96L(tapasin) variants, located close to disulphide bonds, were further studied by molecular dynamics (MD). Identifying intramolecular a-a' domain interactions, MD revealed open and closed conformations of ERp57 in the presence and absence of tapasin. In wild-type and mutant ERp57-tapasin complexes, residues Val97, Ser98, Tyr100, Trp405, Gly407(ERp57) and Asn94, Cys95, Arg97, Asp100(tapasin) formed common H-bond interactions. Moreover, comparing the H-bond networks for P96L and H408R with each other, suggests that P96L(tapasin) improved ERp57-tapasin binding more than the H408R(ERp57) mutant. During MD, the C-terminus domain (that binds MHC-I) in tapasin from the ERp57(H408R)-tapasin complex moved away from the PLC, whereas in the ERp57-tapasin(P96L) system was oppositely displaced. These findings can have implications for the function of PLC and, ultimately, for the presentation of MHC-I peptide complex on the tumour cell surface.

// Min Ho Choe 1 , Joong Won Min 1 , Hong Bae Jeon 2 , Dong-Hyung Cho 3 , Jeong Su Oh 4 , Hyun Gyu Lee 5 , Sang-Gu Hwang 1 , Sungkwan An 6 , Young-Hoon Han 1 and Jae-Sung Kim 1 1 Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Korea 2 Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea 3 Graduate School of East-West Medical Science, Kyung Hee University, Suwon, Korea 4 Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea 5 Department of Microbiology and Immunology, College of Medicine, Yonsei University, Seoul, Korea 6 Molecular-Targeted Drug Research Center and Korea Institute for Skin and Clinical Sciences, Konkuk University, Seoul, Korea Correspondence: Jae-Sung Kim, email: // Keywords : ERp57, STAT3, Mcl-1, Laryngeal cancer, Radioresistance Received : August 18, 2014 Accepted : December 12, 2014 Published : January 08, 2015 Abstract Although targeting radioresistant tumor cells is essential for enhancing the efficacy of radiotherapy, the signals activated in resistant tumors are still unclear. This study shows that ERp57 contributes to radioresistance of laryngeal cancer by activating STAT3. Increased ERp57 was associated with the radioresistant phenotype of laryngeal cancer cells. Interestingly, increased interaction between ERp57 and STAT3 was observed in radioresistant cells, compared to the control cells. This physical complex is required for the activation of STAT3 in the radioresistant cells. Among STAT3-regulatory genes, Mcl-1 was predominantly regulated by ERp57. Inhibition of STAT3 activity with a chemical inhibitor or siRNA-mediated depletion of Mcl-1 sensitized radioresistant cells to irradiation, suggesting that the ERp57-STAT3-Mcl-1 axis regulates radioresistance of laryngeal cancer cells. Furthermore, we observed a positive correlation between ERp57 and phosphorylated STAT3 or Mcl-1 and in vivo interactions between ERp57 and STAT3 in human laryngeal cancer. Importantly, we also found that increased ERp57-STAT3 complex was associated with poor prognosis in human laryngeal cancer, indicating the prognostic role of ERp57-STAT3 regulation. Overall, our data suggest that ERp57-STAT3 regulation functions in radioresistance of laryngeal cancer, and targeting the ERp57-STAT3 pathway might be important for enhancing the efficacy of radiotherapy in human laryngeal cancer.

ERp57 is a ubiquitous ER chaperone that has disulfide isomerase activity. Here, we found that both ERp57 and gastric H(+),K(+)-ATPase are expressed in a sample derived from the apical canalicular membranes of parietal cells. Overexpression of ERp57 in HEK293 cells stably expressing H(+),K(+)-ATPase significantly increased the ATPase activity without changing the expression level of H(+),K(+)-ATPase. Interestingly, overexpression of a catalytically inactive mutant of ERp57 (C57S/C60S/C406S/C409S) in the cells also increased H(+),K(+)-ATPase activity. In contrast, knockdown of endogenous ERp57 in H(+),K(+)-ATPase-expressing cells significantly decreased ATPase activity without changing the expression level of H(+),K(+)-ATPase. Overexpression and knockdown of ERp57 had no significant effect on the expression and function of Na(+),K(+)-ATPase. These results suggest that ERp57 positively regulates H(+),K(+)-ATPase activity apart from its chaperoning function.

There is no effective drug to treat EV71 infection yet. Traditional Chinese herbs are great resources for novel antiviral compounds. Here we showed that Oblongifolin M (OM), an active compound isolated from Garcinia oblongifolia, potently inhibited EV71 infection in a dose dependent manner. To identify its potential effectors in the host cells, we successfully identified 18 proteins from 52 differentially expressed spots by comparative proteomics studies. Further studies showed that knockdown of ERp57 inhibited viral replication through downregulating viral IRES (internal ribosome entry site) activities, whereas ectopic expression of ERp57 increased IRES activity and partly rescued the inhibitory effects of OM on viral replication. We demonstrated that OM is an effective antiviral agent; and that ERp57 is one of its cellular effectors against EV71 infection.

Background: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox-based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. Aim: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes – ERp57 in the regulation of platelet function. Methods/Results: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser-injury induced arterial thrombus formation in a murine model of thrombosis. Conclusions: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis.

Chromatin immunoprecipitation in M14 melanoma cells showed that the protein ERp57 (endoplasmic reticulum protein 57) binds to DNA in the proximity of STAT3 in a subset of STAT3-regulated genes. In the same cells, IL-6 induced a significant increase of the expression of one of these genes, i.e. CRP. Upon depletion of ERp57 by RNA interference, the phosphorylation of STAT3 on tyrosine 705 was decreased, and the IL-6-induced activation of CRP expression was completely suppressed. In vitro experiments showed that ERp57 is also required for the binding of STAT3 to its consensus sequence on DNA. Thus ERp57, previously shown to associate with STAT3 in the cytosol and in the nuclear STAT3-containing enhanceosome, is a necessary cofactor for the regulation of at least a subset of STAT3-dependent genes, probably intervening both at the site of STAT3 phosphorylation and at the nuclear level.

Calpains, calcium-dependent neutral cystein proteases, are involved in a variety of cellular processes. We have previously shown the characteristics of mitochondrial micro-calpain even though calpastatin, a specific endogenous inhibitor of cytosolic calpains, was not present in the mitochondria. This suggested that the regulatory system of mitochondrial calpains differs from that of cytosolic calpains, and endogenous regulatory molecule(s) must exist in the mitochondria. In this study, we have identified ERp57 in partially purified mitochondrial micro-calpain using peptide mass fingerprinting based on MALDI-TOFMS. ERp57 is a member of the protein-disulfide isomerase (PDI) family and functions as a molecular chaperone within the ER. We showed that ERp57 was present in the mitochondria and was associated with mitochondrial micro-calpain. PDI inhibitors, such as DTNB and PAO, caused a degradation of the mitochondrial mu-calpain large subunit. The release of apoptosis-inducing factor (AIF) from the mitochondrial inner membrane was inhibited by treatment of the isolated mitochondria with DTNB and immunoprecipitation of ERp57-associated mitochondrial mu-calpain. Mitochondrial micro-calpain band in casein zymography disappeared by treatment with anti-ERp57 antibody. Our results demonstrate that ERp57 forms complexes with mitochondrial mu-calpain, and ERp57-associated mitochondrial mu-calpain cleaves AIF to a truncated form.

U okviru ovog diplomskog rada istražena je interakcija između odabranih flavonoida (eupatorin, apigenin, apigenin-7-glukozid, kempferol, naringenin, 3-O-metilkvercetin, cijanidin, genistein, luteolin-7-glukozid) sa proteinom ERp57 koji spada u skupinu disulfid-izomeraza (PDI). Za procjenu vezanja flavonoida sa proteinom ERp57 korištena je fluorescencijska spektrofotometrija i učinak gašenja fluorescencije, dok je za ispitivanje utjecaja flavonoida na vezanje ERp57-DNA korištena migracijska elektroforeza (EMSA). Također je testiran utjecaj flavonoida na aktivnost i stabilnost proteina. Apigenin i eupatorin pokazali su najmanju konstantu disocijacije, odnosno najveći afinitet za ERp57 te ih to čini najboljim molekulama za vezanje na protein ERp57. Potvrđena je važnost hidroskilne skupine za interakciju flavonoida sa proteinom. In this thesis, interaction between a protein that belongs to the family of protein disulfide isomerases (PDI)- protein ERp57, and chosen flavonoids- eupatorin, apigenin, apigenin-7-glucoside, kaempferol, naringenin, 3-O-Methylquercetin, cyanidin, genistein and luteolin-7-glucoside was tested. Interaction between ERp57 and flavonoids was assessed using fluorescence quenching method, while electrophorectic mobility shift assay (EMSA) was used to evaluate flavonoid effect on ERp57-DNA interaction. Flavonoid influence on protein activity and protein stability was also tested. Apigenin and eupatorin showed the lowest dissociation constant and the highest binding affinity to ERp57, making them the best compounds to be used as selective inhibitors/modulators of ERp57 biological activities.

Glucose-regulated protein 58 (GRP58)-like immunoreactivity in rat liver obtained in the evening or after fasting underwent an electrophoretic band-shift, which disappeared after phosphatase-treatment. Since mass spectrometric analysis raised a possibility that Ser150 of GRP58 is phosphorylated, an antibody against the phosphoserine150 GRP58 was generated. Immunoreactivity to this antibody was increased in the evening and after fasting. Since GRP58 was shown to interact with signal transducer and activator of transduction 3 (STAT3), a leptin-related protein, the effect of leptin was examined. Immunoreactivity to the anti-phosphoGRP58 antibody was markedly elevated after the leptin injection, indicating that Ser150 of GRP58 is phosphorylated after fasting and leptin-treatment.

The selectivity and individual roles of the N-linked oligosaccharide chains of Sendai virus fusion protein (F protein) in the interaction with endoplasmic reticulum molecular chaperones were investigated by analyses of transient expression of single N-glycosylation mutants and sequential immunoprecipitation. We demonstrated differential interactions depending on the location of the N-linked oligosaccharide chain, and showed that these interactions were correlated with the folding and transport of F proteins. Moreover, mutant F proteins that lacked the specific N-linked oligosaccharide chains required for disulfide bond formation showed increased association with ERp57.

ERp57 participates in the regulation of calcium homeostasis. Although ERp57 modulates calcium flux across the plasma membrane and the endoplasmic reticulum membrane, its functions on mitochondria are largely unknown. Here, we found that ERp57 can regulate the expression of the mitochondrial calcium uniporter (MCU) and modulate mitochondrial calcium uptake. In ERp57-silenced HeLa cells, MCU was downregulated, and the mitochondrial calcium uptake was inhibited, consistent with the effect of MCU knockdown. When MCU was re-expressed in the ERp57 knockdown cells, mitochondrial calcium uptake was restored. Thus, ERp57 is a potent regulator of mitochondrial calcium homeostasis.

Parkinson's disease (PD) is a progressive neurodegenerative disorder whose etiology is still unclear in spite of extensive investigations. It has been hypothesized that 5-S-cysteinyldopamine (CysDA), a catechol-thioether metabolite of dopamine (DA), could be an endogenous parkinsonian neurotoxin. To gain further insight into its role in the neurodegenerative process, both CD1 mice and SH-SY5Y neuroblastoma cells were treated with CysDA, and the data were compared with those obtained by the use of 6-hydroxydopamine, a well-known parkinsonian mimetic. Intrastriatal injection of CysDA in CD1 mice caused a long-lasting depletion of DA, providing evidence of in vivo neurotoxicity of CysDA. Both in mice and in SH-SY5Y cells, CysDA treatment induced extensive oxidative stress, as evidenced by protein carbonylation and glutathione depletion, and affected the expression of two proteins, α-synuclein (α-Syn) and ERp57, whose levels are modulated by oxidative insult. Real-time PCR experiments support these findings, indicating an upregulation of both ERp57 and α-Syn expression. α-Syn aggregation was also found to be modulated by CysDA treatment. The present work provides a solid background sustaining the hypothesis that CysDA is involved in parkinsonian neurodegeneration by inducing extensive oxidative stress and protein aggregation.

U okviru ovog diplomskog rada istražena je interakcija između proteina ERp57 iz porodice disulfid-izomeraza (PDI) i flavonoida kvercitrina i izokvercitrina. Ispitivanje afiniteta vezanja odabranih flavonoida s proteinom ERp57 te oksidiranom i reduciranom domenom proteina D4 provedeno je primjenom fluorescencijske spektrofotometrije i učinka gašenja fluorescencije. Učinak flavonoida na disulfidno-reduktaznu aktivnost proteina je istražen koristeći di-FTC-inzulin i njegovu promjenu fluorescencije u interakciji s ERp57. Dobiveni rezultati su pokazali da testirani flavonoidi iz skupine flavonola imaju afinitet prema ERp57 te da stupajući u interakciju smanjuju aktivnost tog proteina. Dokazano je da se bolje vežu na oksidiranu formu D4 domene koja ima veću hidrofobnu izloženost. Kvercitrin je pokazao veći afinitet prema ERp57, dok je za izokvercitrin bolje inhibirao redoks aktivnost tog proteina.

Vitamin D3 spielt in der Knochenmineralisierung eine wichtige Rolle. Neben dem klassischen nukleären Vitamin D-Rezeptor (VDR) wurde ERp57, für 1,25-(OH)2 Vit D3 beschrieben. In dieser Arbeit wurde die Rolle von ERp57 in der Chondrozytendifferenzierung untersucht. Hierbei konnte gezeigt werden, dass die Matrixvesikel-assoziierte Form von ERp57 für die Entfernung des Rezeptors von der Chondrozytenoberfläche eine untergeordnete Rolle spielt. Zudem wurde eine knorpel-spezifischen ERp57 knock-out Maus (Erp57cart-/--Maus) untersucht. Die Erp57cart-/--Tiere waren lebensfähig, vital und hatten normale Lebenserwartung, zeigte jedoch gegenüber den WT-Mäusen ein geringeres Gewicht und kürzere Knochen. Dieser Unterschied ist im Alter von 4 Wochen und bei männlichen Tieren stärker als bei weiblichen Tieren ausgeprägt. Ursächlich hierfür scheint der später einsetzende Umbau der späten hypertrophen Zone der Wachstumsfuge durch Osteoklasten insbesondere beim pubertären Wachstumsschub zu sein.

Cell death by apoptosis is a fundamental biological process, requiring fine interactions with the immune system for the effective recognition and removal of the apoptotic cells. C1q, a complement molecule, essential in the innate immune system, has been shown to be strongly involved in the mechanism of recognition and elimination of apoptotic cells, mainly through its globular region (C1qGR). Recently, the translocation of calreticulin (CRT) at the surface of cancer cells has been identified as an eat-me signal, which can be immunogenic. Initially, the interaction between CRT and C1q was characterized on phagocytes surface and CRT was described as a receptor for the collagenous tails of C1q (C1qCLF). All these observations support a dual role of CRT at the surface of phagocytes and their targets. At first, using a FRET strategy we achieve to detect a direct interaction between CRT and C1qGR at the surface of early apoptotic HeLa cells. Second, the establishment of phagocytosis assays showed that calreticulin exposed at the surface of apoptotic cells could modulate phagocytosis: opposite effects were observed depending on the cellular model used (RNAi treated HeLa or MEF CRT-/-) and under certain conditions, a combined modulation of calreticulin and C1q was observed on the inflammatory response (cytokine production).; La mort cellulaire par apoptose est un processus biologique fondamental, nécessitant des interactions fines avec le système immunitaire pour une reconnaissance et une élimination efficace des cellules mortes. C'est ainsi que C1q, une molécule du complément, essentielle dans le système immunitaire inné, a été mise en évidence comme fortement impliquée dans le mécanisme de reconnaissance et d'élimination des cellules apoptotiques, notamment via sa région globulaire (C1qGR). Récemment, la translocation de la calréticuline (CRT) au niveau externe de la membrane plasmique des cellules cancéreuses a été identifiée comme un signal « eat-me » pouvant être immunogène. Initialement, l'interaction entre la CRT et C1q a été caractérisée à la surface des phagocytes et la CRT a été considérée comme un récepteur pour les queues collagènes de C1q (C1qCLF). L'ensemble de ces observations est en faveur d'un double rôle de la CRT, à la surface des phagocytes et des cellules apoptotiques. Dans un premier temps, l'élaboration d'une stratégie de FRET a permis de détecter une interaction directe entre la CRT et C1qGR à la surface de la cellule HeLa en apoptose précoce. Dans un second temps, la mise en place de tests de phagocytose a permis de montrer que la calréticuline exposée à la surface des cellules apoptotiques peut moduler la phagocytose : des effets opposés ont été observés selon le modèle cellulaire utilisé (HeLa traitées par des ARNi ou MEF CRT-/-) et dans certaines conditions, une modulation combinée de la calréticuline et de C1q a été observée sur la réponse inflammatoire (production de cytokines).

Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by a progressive and selective loss of dopaminergic neurons in the Substantia nigra. Recent studies indicate that 5-S-cysteinyldopamine (CysDA), a catechol-thioether metabolite of dopamine (DA), is strongly neurotoxic and may contribute to the onset and progression of this disease (1). To better understand CysDA contribution to neurodegeneration, it has been evaluated the modulation of the expression, aggregation and cellular localization of proteins known to be involved in the oxidative stress response. In particular, we focused our study on alpha-Synuclein (a-Syn), a protein considered the PD “hallmark” and on ERp57, an endoplasmic reticulum thiol oxidoreductase, known to be implicated in neurodegenerative disease (2). To achieve these goals, both CD1 mice and SH-SY5Y neuroblastoma cells were treated with CysDA and the data were compared to those obtained by the use of 6-hydroxydopamine (6-OHDA), a well known parkinsonian mimetic. Intrastriatal injection of CysDA in CD1 mice caused a long-lasting depletion of DA, without affecting serotonergic or noradrenergic systems, providing in vivo evidence of CysDA specific neurotoxicity at dopaminergic system level. Both in CD1 mice and in SH-SY5Y cells, CysDA treatment induced extensive oxidative stress, as evidenced by protein carbonylation and Glutathione (GSH) depletion. Moreover, CysDA treatment induced in CD1 mice an increase of α-Syn aggregated forms in the prefrontal cortex with respect to the striatum and hippocampus. These data are in agreement with previous findings which indicate that α-Syn is overexpressed in the prefrontal cortex in cognitive impairment (3). Conversely, ERp57 protein increase was observed in the striatum after CysDA treatment, the other brain regions were slightly affected. To assess α-Syn and ERp57 protein levels, aggregation and their cellular distribution, cytoplasm and nuclei fractions were obtained from SH-SY5Y cells treated with CysDA at different times. In the cytosol, α-Syn dimeric forms increased with incubation time, up to 24 hours, whereas the monomeric form was overexpressed within 8 hours, and decreased in prolonged incubations. In the nuclei, only the monomeric form was present. Concerning ERp57, the protein level was increased up to 24 hours in the cytosol, whereas in the nuclei it was transiently increased within 3 hours. Gene expression analyses have demonstrated that α-Syn and ERp57 mRNAs increased at 6 and 3 hours, respectively from neurotoxins administration. The overall data suggest that CysDA exerts neurotoxic effects similar to 6-OHDA, supporting the hypothesis that it may be an endogenous parkinsonian mimetic, able to induce α-Syn overexpression and aggregation and ERp57 upregulation. 1. Mosca et al (2006) Neurochem Int. 49:262 2.Coe et al (2010) Int J Biochem Cell Biol 42:796 3.Ferrer et al (2009) Progr Neurobiol 88:89

Dystroglycan (DG) is an extracellular receptor composed of two subunits, α-DG and β-DG, connected through the α-DG C-terminal domain and the β-DG N-terminal domain. We report an alanine scanning of all DG cysteine residues performed on DG-GFP constructs overexpressed in 293-Ebna cells, demonstrating that Cys-669 and Cys-713, both located within the β-DG N-terminal domain, are key residues for the DG precursor cleavage and trafficking, but not for the interaction between the two DG subunits. In addition, we have used immunprecipitation and confocal microscopy showing that ERp57, a member of the disulfide isomerase family involved in glycoprotein folding, is associated and colocalizes immunohistochemically with β-DG in the ER and at the plasma membrane of 293-Ebna cells. The β-DG–ERp57 complex also included α-DG. DG mutants, unable to undergo the precursor cleavage, were still associated to ERp57. β-DG and ERp57 were also co-immunoprecipitated in rat heart and kidney tissues. In vitro, a mutant ERp57, mimicking the reduced form of the wild-type protein, interacts directly with the recombinant N-terminal domain of both α-DG and β-DG with apparent dissociation constant values in the micromolar range. ERp57 is likely to be involved in the DG processing/maturation pathway, but its association to the mature DG complex might also suggest some further functional role that needs to be investigated., Highlights ► Cys-669 and Cys-713 are key residues for dystroglycan precursor cleavage. ► ERp57 is co-immunopurified with dystroglycan. ► ERp57 co-localizes with dystroglycan in the ER and at the plasma membrane. ► Recombinant ERp57 binds directly to dystroglycan recombinant domains.

The protein ERp57 (also known as PDIA3) is a widely distributed protein, mainly localized in the endoplasmic reticulum, where it acts as disulfide isomerase, oxidoreductase and chaperone, in concert with the lectins calreticulin (CRT) and calnexin. The ERp57/CRT complex has been detected on the cell surface and previous studies have suggested its involvement in programmed cell death. Although the ERp57-CRT complex has been characterized, little is known about its role in different cellular compartments as well as inhibitors of this interaction. We focused on the kinetic, extent and stability of the ERp57-CRT complex, using the surface plasmon resonance spectroscopy, investigating the possible role as inhibitor of the antibiotic vancomycin. Equilibrium thermodynamic data suggested that vancomycin may hinder the interaction between the two proteins and could interfere with the ERp57 conformational changes that stabilize the complex. Furthermore, by means of confocal microscopy, we evaluated the effect of the in vivo administration of vancomycin on the ERp57/CRT complex on the surface of HeLa cells. The model presented here could be used for the search of other specific inhibitors/interactors of ERp57, which can be extremely helpful to understand the biological pathways where the protein is involved and to modulate its activity.

Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane glycoprotein that localizes to myelin sheaths in the central nervous system. MOG has important implications in multiple sclerosis, as pathogenic anti-MOG antibodies have been detected in the sera of multiple sclerosis patients. As a membrane protein, MOG achieves its native structure in the endoplasmic reticulum where its folding is expected to be controlled by endoplasmic reticulum chaperones. Calnexin, calreticulin, and ERp57 are essential components of the endoplasmic reticulum quality control where they assist in the proper folding of newly synthesized glycoproteins. In this study, we show that expression of MOG is not affected by the absence of the endoplasmic reticulum quality control proteins calnexin, calreticulin, or ERp57. We also show that calnexin forms complexes with MOG and these interactions might be glycan-independent. Importantly, we show that cell surface targeting of MOG is not disrupted in the absence of the endoplasmic reticulum chaperones. This article is part of a special issue entitled: 11th European Symposium on Calcium.

Maurizio Paci, Giovanni Antonini, Nazareno Capitanio