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Signal peptide peptidase-like 2c (SPPL2c) is a testis-specific aspartyl intramembrane protease that contributes to male gamete function both by catalytic and non-proteolytic mechanisms. Here, we provide an unbiased characterisation of the in vivo interactome of SPPL2c identifying the ER chaperone calnexin as novel binding partner of this enzyme. Recruitment of calnexin specifically required the N-glycosylation within the N-terminal protease-associated domain of SPPL2c. Importantly, mutation of the single glycosylation site of SPPL2c or loss of calnexin expression completely prevented SPPL2c-mediated intramembrane proteolysis of all tested substrates. By contrast and despite rather promiscuous binding of calnexin to other SPP/SPPL proteases, expression of the chaperone was exclusively required for SPPL2c-mediated proteolysis. Despite some impact on the stability of SPPL2c most presumably due to assistance in folding of the luminal domain of the protease, calnexin appeared to be recruited rather constitutively to the protease thereby boosting its catalytic activity. In summary, we describe a novel, highly specific mode of intramembrane protease regulation, highlighting the need to systematically approach control mechanisms governing the proteolytic activity of other members of the aspartyl intramembrane protease family. [ABSTRACT FROM AUTHOR]

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood–brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood–brain barrier and is primarily responsible for preventing activated T-cells from entering the brain. [ABSTRACT FROM AUTHOR]

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER. [ABSTRACT FROM AUTHOR]

Environmental changes can trigger endoplasmic reticulum (ER) stress and misfolded protein accumulation, potentially leading to pre-eclampsia (PE). Amyloid-β (Aβ) is a crucial misfolded protein that can overactivate autophagy. Our study assessed the expression of Aβ1−42 and autophagic activity in PE placental tissues and trophoblasts under ER stress. Placental tissues were surgically collected from normal pregnant women (NP) and pregnant women with late-onset PE (LOPE) delivering through cesarean section. The expression levels of Aβ1−42 were detected in both PE and NP placental tissues, as well as in tunicamycin (TM)-induced HTR-8/SVneo cells. Autophagy-related proteins, such as Beclin-1, the ratio of LC3-II to LC3-I, ATG5, and SQSTM1/p62 in the placental tissues and HTR-8/SVneo cells were measured by Western blot. The number and morphology of autophagosomes were observed using transmission electron microscopy (TEM). Potential targets associated with the unfolded protein response (UPR) in the placental tissues of NP and PE cases were screened using PCR Arrays. The misfolded protein was significantly upregulated in the PE group. In both PE placental tissues and TM-induced HTR-8/SVneo cells, not only was Aβ1−42 upregulated, but also Beclin-1, ATG5, and LC3BII/I were significantly increased, accompanied by an increase in autophagosome count, while SQSTM1/P62 was downregulated. A total of 17 differentially expressed genes (DEGs) associated with the UPR were identified, among which elevated calnexin (CANX) was validated in the placenta from both PE and TM-induced HTR-8/SVneo cells. Autophagy is significantly upregulated in PE cases due to ER stress-induced Aβ1−42 accumulation, likely mediated by autophagy-related proteins involved in the UPR. [ABSTRACT FROM AUTHOR]

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood–brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood–brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.

Abstract Background TP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer. Methods We analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-β1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy. Results Mutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner. Conclusions The mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies.

Richard Lerner was a visionary polymath of the 20th century; a medical doctor, chemist, immunologist, inventor, and founder of new scientific fields. As a tribute to his life and scientific achievements, we discuss his connection with Oxford University and the Glycobiology Institute and we discuss our research data on the host glycoprotein folding pathway and how its inhibition can lead to broad spectrum antiviral drugs. This host targeting approach for developing antiviral therapies has the advantage of being effective against many viruses, therefore can be used to prevent new epidemics to become pandemics. [ABSTRACT FROM AUTHOR]

The scavenger receptor cysteine‐rich (SRCR) domain is a key constituent in diverse proteins. N‐glycosylation is important in protein expression and function. In the SRCR domain of different proteins, N‐glycosylation sites and functionality vary substantially. In this study, we examined the importance of N‐glycosylation site positions in the SRCR domain of hepsin, a type II transmembrane serine protease involved in many pathophysiological processes. We analysed hepsin mutants with alternative N‐glycosylation sites in the SRCR and protease domains using three‐dimensional modelling, site‐directed mutagenesis, HepG2 cell expression, immunostaining, and western blotting. We found that the N‐glycan function in the SRCR domain in promoting hepsin expression and activation on the cell surface cannot be replaced by alternatively created N‐glycans in the protease domain. Within the SRCR domain, the presence of an N‐glycan in a confined surface area was essential for calnexin‐assisted protein folding, endoplasmic reticulum (ER) exiting, and zymogen activation of hepsin on the cell surface. Hepsin mutants with alternative N‐glycosylation sites on the opposite side of the SRCR domain were trapped by ER chaperones, resulting in the activation of the unfolded protein response in HepG2 cells. These results indicate that the spatial N‐glycan positioning in the SRCR domain is a key determinant in the interaction with calnexin and subsequent cell surface expression of hepsin. These findings may help to understand the conservation and functionality of N‐glycosylation sites in the SRCR domains of different proteins. [ABSTRACT FROM AUTHOR]

Background: TP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer. Methods: We analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-β1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy. Results: Mutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner. Conclusions: The mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

Abstract Background Under abiotic stress conditions, cotton growth is inhibited and yield losses are severe. Identification of calnexin family members and function analysis under abiotic stress laid the foundation for the screening of stress-related candidate genes. Results A total of 60 CNX family members have been identified in Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii, and they were divided into two categories: CNX and CRT genes. Through the construction of a phylogenetic tree, they were subdivided into three classes. Further analysis of chromosome localization, conserved promoters, gene structure and selection under pressure showed that the family members were highly conserved in the evolution process. Analysis of cis-acting elements in the promoter regions showed that CNX family genes contain regulatory elements for growth and development, anaerobic, drought, defense and stress response, and plant hormones. Using RNA-seq data to study the expression pattern of GhCNX genes under cold, hot, salt stress and Polyethylene glycol, it was observed that the gene expression levels changed by different degrees under different stress conditions, indicating that GhCNX members were involved in the regulation of multiple biological stresses. Conclusion This study provides an insight into the members of cotton CNX genes. The results of this study suggested that CNX family members play a role in defense against adversity and provide a foundation for the discovery of stress-related genes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID- 19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stresslike responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or humanspecific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARSCoV- 2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse. [ABSTRACT FROM AUTHOR]

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome. Synopsis: Calnexin is a membrane‐integral chaperone that acts on glycosylated proteins. This study shows that its transmembrane domain is also involved in substrate binding, defines how intramembrane substrate recognition occurs, and reveals that this additional binding mode supports chaperoning. The mass‐spectrometric interactome of calnexin contains many non‐glycosylated membrane proteins.Calnexin can bind membrane protein substrates in the lipid bilayer via its own transmembrane domain.A systematic analysis reveals how calnexin recognizes particular features within its clients in the membrane.Intra‐membrane client recognition synergizes with sugar‐based recognition of calnexin clients to protect labile chaperone substrates. [ABSTRACT FROM AUTHOR]

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC‐II molecules. The pathways leading to endogenous MHC‐II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy‐dependent and ‐independent endogenous presentation of HIV‐ and HCMV‐derived peptides. By studying the immunopeptidome of MHC‐II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC‐II‐loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC‐II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC‐II‐restricted endogenous presentation pathway, and we propose one potential mechanism of action. Synopsis: The autophagy receptor T6BP (TAX1BP1) stabilizes the invariant chain (CD74) and regulates MHC‐II trafficking, thereby favoring the presentation of high‐affinity peptides to virus‐specific CD4+ T cells. T6BP loss prevents the activation of HIV‐ and HCMV‐specific CD4+ T cell clones.T6BP expression affects the quality and the diversity of peptides presented by MHC‐II molecules.T6BP interacts with the cytoplasmic tail of calnexin to favor MHC‐II molecule chaperoning by the invariant chain (CD74).Calnexin loss replicates T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. [ABSTRACT FROM AUTHOR]

Dihydroartemisinin (DHA) may inhibit the migration and invasion of liver cancer cells by reducing ATP synthase production (specifically ATP1A1 and ATP5H) through the calcium/calmodulin dependent protein kinase kinase 2/solute carrier family 8 member B1 signaling pathway. However, it is unclear whether DHA regulates ATP synthase activity by modulating other calcium ion signals to inhibit the energy metabolism and the transfer of hepatocellular carcinoma (HCC) cells. Using the Gene Expression Profiling Interactive Analysis database, a search for specific expression genes in liver cancer tissues was performed. Human HCC HuH-7 and Li-7 cells were used to produce CANX overexpression and small interfering RNA cell models. The study assessed changes in cell proliferation, apoptosis, migration and invasion. Reactive oxygen species production, ATP production, mitochondrial membrane potential (JC-1), NAD+/NADH ratio and mitochondrial fluorescence were also evaluated. Western blotting was used to assess changes in CANX, ATP6V1 domain (ATP6V1F) and V0 domain (ATP6V0B) protein expression levels. The results demonstrated that CANX is highly expressed in liver cancer tissues. Furthermore, CANX regulated malignant biological behavior, mediated mitochondrial function and energy metabolism. However, these effects were inhibited by DHA, which decreased the expression of CANX, ATP6V0B and ATP6V1F. The findings of the present study underscore the central role of CANX in affecting the malignant biological behavior of liver cancer cells by regulating mitochondrial function and energy metabolism. Additionally, they indicate that DHA serves an anticancer role by inhibiting CANX expression. [ABSTRACT FROM AUTHOR]

Da-Ming Xu,1,&ast; Ze-Wen Zhang,2,&ast; Jing-Xing Yi,3 Long Xie,3 Wen-Jun Yu,2 Jin-Feng Qiu,4 Cheng-Wei Xu,5 Chun-Ling He,6 Xian-Ru Xu,7 Jun Yin3 1Division of Urological Surgery, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China; 2Division of Hematology, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China; 3Department of Clinical Laboratory Medicine, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China; 4Division of Respirology, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China; 5Department of Blood Purification, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China; 6Department of Pathology, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China; 7Division of Inventional Ultrasonic Therapeutics, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong Province, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jun Yin, Department of Clinical Laboratory Medicine, The Second Affiliated Hospital of Shantou University Medical College, Dongxia Road North, Shantou, Guangdong Province, 515041, People’s Republic of China, Tel +86 754 8891 5950, Email jyin@stu.edu.cnBackground: Adult chronic immune thrombocytopenia (chronic ITP) is a common autoimmune hemorrhagic disease characterized by decreased platelet production and increased platelet destruction, leading to thrombocytopenia. In this study, Ca2+, calnexin (CNX) and calreticulin (CRT) within platelets from adult patients with chronic ITP were investigated.Methods: Platelets were isolated from blood specimen collected from 20 adult patients with chronic ITP and 20 healthy volunteers. Ca2+, CNX and CRT were determined by flow cytometry, and the results were analyzed with EXPO32 ADC software.Results: Flow cytometry showed the expressions of Ca2+ (74.19± 19.40% vs 22.79± 10.47%) was elevated (P< 0.05). However, CNX (15.10± 7.32% vs 41.79± 14.45%) and CRT (25.11± 12.66% vs 38.58± 12.02%) were decreased markedly in platelets from adult patients with chronic ITP (P< 0.05 compared with healthy volunteers).Conclusion: Based on enhanced expression of Ca2+ and attenuated expression of CNX and CRT in patients with chronic ITP, Ca2+ concentration and its associated down-regulated proteins may be important regulatory signals in the pathogenesis of chronic ITP.Keywords: platelets, chronic immune thrombocytopenia, Ca2+, calnexin, calreticulin

N‐linked glycans are specifically attached to asparagine residues in a N‐X‐S/T motif of secretory pathway glycoproteins. N‐glycosylation of newly synthesized glycoproteins directs their folding via the lectin chaperones calnexin and calreticulin that are associated with protein‐folding enzymes and glycosidases of the endoplasmic reticulum (ER). Misfolded glycoproteins are retained in the ER by the same lectin chaperones. The work by Sun et al. (FEBS J 2023, 10.1111/febs.16757) in this issue focusses on hepsin, a serine protease on the surface of liver and other organs. The authors deduce that spatial positioning of N‐glycans on one side of a conserved domain of hepsin, known as the scavenger receptor‐rich cysteine domain, regulates calnexin selection for hepsin maturation and transport through the secretory pathway. If N‐glycosylation is elsewhere on hepsin, then it is misfolded and has a prolonged accumulation with calnexin and BiP. This association coincides with the engagement of stress response pathways that sense glycoprotein misfolding. The topological considerations of N‐glycosylation dissected by Sun et al. may help unravel how key sites of N‐glycosylation sites required for protein folding and transport have evolved to select the lectin chaperone calnexin pathway for folding and quality control. [ABSTRACT FROM AUTHOR]

Keywords: Antigen presentation; autophagy receptors; calnexin; HLA-DM; immunopeptidome; interactome; invariant chain; MHC; T cells; TAX1BP1 EN Antigen presentation autophagy receptors calnexin HLA-DM immunopeptidome interactome invariant chain MHC T cells TAX1BP1 2153 2155 3 06/27/23 20230701 NES 230701 CD4 SP + sp T lymphocytes play a major role in establishing and maintaining antiviral immunity. In this punctum, we will focus on MHC-II-restricted endogenous antigen presentation and the involvement of TAX1BP1 in the biology of MHC-II molecules. The characterization of the MHC-II immunopeptidome, meaning the repertoire of peptides presented by MHC-II molecules, revealed that macroautophagy (herein referred to as autophagy) contributes to the processing of cytoplasmic and nuclear antigens. [Extracted from the article]

Tightly regulated cell surface expression of NTRK2/TrkB provides a mechanism for fine-tuning cellular responses to the neurotrophic factor BDNF. Recently, the degradation of NTRK2 by reticulophagy has been identified as a mechanism to limit its availability for trafficking to the cell membrane. The ER-chaperone CANX (calnexin) delivers NTRK2 to the reticulophagy receptor RETREG1/Fam134b for lysosomal degradation. Upon phosphorylation of CANX, NTRK2 is released from this complex, which facilitates its cell surface transport. These results identify a novel role for CANX in regulating the cell surface expression of NTRK2 and imply a function for reticulophagy that goes beyond regulating the degradation of misfolded proteins within the ER. [ABSTRACT FROM AUTHOR]

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and β-human chorionic gonadotropin (β-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a β-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of β-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development. [ABSTRACT FROM AUTHOR]

The antioxidant role of mitochondrial uncoupling protein 3 (UCP3) is controversial. This work aimed to investigate the effects of UCP3 on the heart of mice housed at thermoneutral temperature, an experimental condition that avoids the effects of thermoregulation on mitochondrial activity and redox homeostasis, preventing the alterations related to these processes from confusing the results caused by the lack of UCP3. WT and KO UCP3 mice were acclimatized at 30 °C for 4 weeks and hearts were used to evaluate metabolic capacity and redox state. Tissue and mitochondrial respiration, the activities of the mitochondrial complexes, and the protein expression of mitochondrial complexes markers furnished information on mitochondrial functionality. The levels of lipid and protein oxidative damage markers, the activity of antioxidant enzymes, the reactive oxygen species levels, and the susceptibility to in vitro Fe-ascorbate-induced oxidative stress furnished information on redox state. UCP3 ablation reduced tissue and mitochondrial respiratory capacities, not affecting the mitochondrial content. In KO UCP3 mice, the mitochondrial complexes activities were lower than in WT without changes in their content. These effects were accompanied by an increase in the level of oxidative stress markers, ROS content, and in vitro susceptibility to oxidative stress, notwithstanding that the activities of antioxidant enzymes were not affected by UCP3 ablation. Such modifications are also associated with enhanced activation/phosphorylation of EIF2α, a marker of integrated stress response and endoplasmic reticulum stress (GRP778 BIP). The lack of UCP3 makes the heart more prone to oxidative insult by reducing oxygen consumption and increasing ROS. Our results demonstrate that UCP3 helps the cell to preserve mitochondrial function by mitigating oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Adult chronic immune thrombocytopenia (chronic ITP) is a common autoimmune hemorrhagic disease characterized by decreased platelet production and increased platelet destruction, leading to thrombocytopenia. In this study, Ca2+, calnexin (CNX) and calreticulin (CRT) within platelets from adult patients with chronic ITP were investigated. Methods: Platelets were isolated from blood specimen collected from 20 adult patients with chronic ITP and 20 healthy volunteers. Ca2+, CNX and CRT were determined by flow cytometry, and the results were analyzed with EXPO32 ADC software. Results: Flow cytometry showed the expressions of Ca2+ (74.19± 19.40% vs 22.79± 10.47%) was elevated (P< 0.05). However, CNX (15.10± 7.32% vs 41.79± 14.45%) and CRT (25.11± 12.66% vs 38.58± 12.02%) were decreased markedly in platelets from adult patients with chronic ITP (P< 0.05 compared with healthy volunteers). Conclusion: Based on enhanced expression of Ca2+ and attenuated expression of CNX and CRT in patients with chronic ITP, Ca2+ concentration and its associated down-regulated proteins may be important regulatory signals in the pathogenesis of chronic ITP. [ABSTRACT FROM AUTHOR]

Calnexin (CNX) is one of the major calcium-binding proteins in endoplasmic reticulum (ER) and plays a crucial role in regulating Ca2+ homeostasis and unfolded protein response (UPR). In the present study, PyCNX was identified in Yesso Scallop Patinopecten yessoensis. The open reading frame (ORF) of PyCNX was of 1794 bp encoding a putative polypeptide of 598 amino acid residues with an N-terminal signal peptide, a typical calreticulin (CRT) domain and a C-terminal transmembrane domain. The deduced amino acid sequence of PyCNX shared 47.91%-70.16% identities with CNXs from other species. The mRNA transcripts of PyCNX were constitutively expressed in all the examined tissues, including gills, gonad, hepatopancreas, mantle and haemocytes, with the highest expression level in gills. The mRNA transcripts of PyCNX in gills were significantly up-regulated at 1 h (p < 0.01), down-regulated to similar level of Blank group at 3 h (p > 0.05) and 6 h (p > 0.05), and decreased significantly from 12 to 48 h (p < 0.05) after acute high temperature stress (25 °C). PyCNX was mainly located in the ER of haemocytes. The expression profiles of PyATF6, PyIRE1 and PyGRP78 in the gills of scallops after acute high temperature stress were investigated using quantitative real-time PCR. The mRNA transcripts of PyATF6 increased significantly at 1 h, 3 h and 6 h after acute high temperature stress (p < 0.01), then down-regulated to similar level of Blank group at 12 h (p > 0.05) and 24 h (p > 0.05), and finally significantly up-regulated again at 48 h (p < 0.05). Similar to PyATF6, the mRNA transcripts of PyIRE1 were significantly up-regulated at 1 h (p < 0.05), 3 h (p < 0.01), 6 h (p < 0.05) and 48 h (p < 0.05) after acute high temperature stress. The mRNA transcripts of PyGRP78 were significantly up-regulated at 3 h (p < 0.05), reached the highest level at 6 h (p < 0.01) after acute high temperature stress, and kept significant higher level at 12-48 h (p < 0.05). These results indicated that PyCNX was involved in the response upon the acute high temperature stress of scallops probably by regulating UPR.

The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and β-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another β-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications. [ABSTRACT FROM AUTHOR]

The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and β-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another β-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications.

Proper function of the endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site has been linked to pathophysiological states, including metabolic diseases. Although the ER and mitochondria play distinct cellular roles, these organelles also form physical interactions with each other at sites defined as mitochondria-associated ER membranes (MAMs), which are essential for calcium, lipid and metabolite exchange. Here we show that in the liver, obesity leads to a marked reorganization of MAMs resulting in mitochondrial calcium overload, compromised mitochondrial oxidative capacity and augmented oxidative stress. Experimental induction of ER-mitochondria interactions results in oxidative stress and impaired metabolic homeostasis, whereas downregulation of PACS-2 or IP3R1, proteins important for ER-mitochondria tethering or calcium transport, respectively, improves mitochondrial oxidative capacity and glucose metabolism in obese animals. These findings establish excessive ER-mitochondrial coupling as an essential component of organelle dysfunction in obesity that may contribute to the development of metabolic pathologies such as insulin resistance and diabetes.

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and β-human chorionic gonadotropin (β-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a β-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of β-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development.

Immunization with recombinant glycoprotein-based vaccines is a promising approach to induce protective immunity against viruses. However, the complex biosynthetic maturation requirements of these glycoproteins typically necessitate their production in mammalian cells to support their folding and post-translational modification. Despite these clear advantages, the incumbent costs and infrastructure requirements with this approach can be prohibitive in developing countries, and the production scales and timelines may prove limiting when applying these production systems to the control of pandemic viral outbreaks. Plant molecular farming of viral glycoproteins has been suggested as a cheap and rapidly scalable alternative production system, with the potential to perform post-translational modifications that are comparable to mammalian cells. Consequently, plant-produced glycoprotein vaccines for seasonal and pandemic influenza have shown promise in clinical trials, and vaccine candidates against the newly emergent severe acute respiratory syndrome coronavirus-2 have entered into late stage preclinical and clinical testing. However, many other viral glycoproteins accumulate poorly in plants, and are not appropriately processed along the secretory pathway due to differences in the host cellular machinery. Furthermore, plant-derived glycoproteins often contain glycoforms that are antigenically distinct from those present on the native virus, and may also be under-glycosylated in some instances. Recent advances in the field have increased the complexity and yields of biologics that can be produced in plants, and have now enabled the expression of many viral glycoproteins which could not previously be produced in plant systems. In contrast to the empirical optimization that predominated during the early years of molecular farming, the next generation of plant-made products are being produced by developing rational, tailor-made approaches to support their production. This has involved the elimination of plant-specific glycoforms and the introduction into plants of elements of the biosynthetic machinery from different expression hosts. These approaches have resulted in the production of mammalian N-linked glycans and the formation of O-glycan moieties in planta. More recently, plant molecular engineering approaches have also been applied to improve the glycan occupancy of proteins which are not appropriately glycosylated, and to support the folding and processing of viral glycoproteins where the cellular machinery differs from the usual expression host of the protein. Here we highlight recent achievements and remaining challenges in glycoengineering and the engineering of glycosylation-directed folding pathways in plants, and discuss how these can be applied to produce recombinant viral glycoproteins vaccines.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) 1 and 2 are central hubs in the chaperone network of the endoplasmic reticulum (ER), acting as gatekeepers to the early secretory pathway, yet little is known about their cellular clients. These two quality control sensors control lectin chaperone binding and glycoprotein egress from the ER. A quantitative glycoproteomics strategy was deployed to identify cellular substrates of the UGGTs at endogenous levels in CRISPR-edited HEK293 cells. The 71 UGGT substrates identified were mainly large multidomain and heavily glycosylated proteins when compared to the general N-glycoproteome. UGGT1 was the dominant glucosyltransferase with a preference toward large plasma membrane proteins whereas UGGT2 favored the modification of smaller, soluble lysosomal proteins. This study sheds light on differential specificities and roles of UGGT1 and UGGT2 and provides insight into the cellular reliance on the carbohydrate-dependent chaperone system to facilitate proper folding and maturation of the cellular N-glycoproteome.

The lectin chaperones calnexin (CNX) and calreticulin (CRT) localized in the endoplasmic reticulum play important roles in glycoprotein quality control. Although the interaction between these lectin chaperones and ERp57 is well known, it has been recently reported that endoplasmic reticulum protein 29 (ERp29), a member of PDI family, interacts with CNX and CRT. The biochemical function of ERp29 is unclear because it exhibits no ERp57-like redox activity. In this study, we addressed the possibility that ER chaperones CNX and CRT are connected via ERp29, based on our observation that ERp29 exists as a dimer. As a result, we showed that CNX dimerizes through ERp29. These results endorse the hypothesis that ERp29 serves as a bridge that links two molecules of CNX. Also, we showed that similar complexes such as CNX–CRT were formed via ERp29. • ER chaperone calnexins form dimer complex via ERp29. • ERp29 serves as a bridge that links two molecules of CNX. • Hetero complexes such as CNX–CRT are also formed via ERp29. [ABSTRACT FROM AUTHOR]

Immunization with recombinant glycoprotein-based vaccines is a promising approach to induce protective immunity against viruses. However, the complex biosynthetic maturation requirements of these glycoproteins typically necessitate their production in mammalian cells to support their folding and post-translational modification. Despite these clear advantages, the incumbent costs and infrastructure requirements with this approach can be prohibitive in developing countries, and the production scales and timelines may prove limiting when applying these production systems to the control of pandemic viral outbreaks. Plant molecular farming of viral glycoproteins has been suggested as a cheap and rapidly scalable alternative production system, with the potential to perform post-translational modifications that are comparable to mammalian cells. Consequently, plant-produced glycoprotein vaccines for seasonal and pandemic influenza have shown promise in clinical trials, and vaccine candidates against the newly emergent severe acute respiratory syndrome coronavirus-2 have entered into late stage preclinical and clinical testing. However, many other viral glycoproteins accumulate poorly in plants, and are not appropriately processed along the secretory pathway due to differences in the host cellular machinery. Furthermore, plant-derived glycoproteins often contain glycoforms that are antigenically distinct from those present on the native virus, and may also be under-glycosylated in some instances. Recent advances in the field have increased the complexity and yields of biologics that can be produced in plants, and have now enabled the expression of many viral glycoproteins which could not previously be produced in plant systems. In contrast to the empirical optimization that predominated during the early years of molecular farming, the next generation of plant-made products are being produced by developing rational, tailor-made approaches to support their production. This has involved the elimination of plant-specific glycoforms and the introduction into plants of elements of the biosynthetic machinery from different expression hosts. These approaches have resulted in the production of mammalian N-linked glycans and the formation of O-glycan moieties in planta. More recently, plant molecular engineering approaches have also been applied to improve the glycan occupancy of proteins which are not appropriately glycosylated, and to support the folding and processing of viral glycoproteins where the cellular machinery differs from the usual expression host of the protein. Here we highlight recent achievements and remaining challenges in glycoengineering and the engineering of glycosylation-directed folding pathways in plants, and discuss how these can be applied to produce recombinant viral glycoproteins vaccines. [ABSTRACT FROM AUTHOR]

We previously showed that calnexin (Canx)‐deficient mice are desensitized to experimental autoimmune encephalomyelitis (EAE) induction, a model that is frequently used to study inflammatory demyelinating diseases, due to increased resistance of the blood‐brain barrier to immune cell transmigration. We also discovered that Fabp5, an abundant cytoplasmic lipid‐binding protein found in brain endothelial cells, makes protein‐protein contact with the cytoplasmic C‐tail domain of Canx. Remarkably, both Canx‐deficient and Fabp5‐deficient mice commonly manifest resistance to EAE induction. Here, we evaluated the importance of Fabp5/Canx interactions on EAE pathogenesis and on the patency of a model blood‐brain barrier to T‐cell transcellular migration. The results demonstrate that formation of a complex comprised of Fabp5 and the C‐tail domain of Canx dictates the permeability of the model blood‐brain barrier to immune cells and is also a prerequisite for EAE pathogenesis. [ABSTRACT FROM AUTHOR]

The protein folding and lipid moiety status of glycosylphosphatidylinositol- anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation. [ABSTRACT FROM AUTHOR]

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]

The group A scavenger receptor cysteine-rich (SRCR) domain is a conserved module present in numerous proteins involved in diverse biological processes. Hepsin, a hepatic protease implicated in many cancers, consists of a cytoplasmic tail, a transmembrane domain and an extracellular regions with a group A SRCR domain and a serine protease domain. Like in many SRCR-containing proteins, the SRCR domain in hepsin has an N-glycosylation site, but its functional significance is unknown. In this study, we confirmed N-glycosylation at Asn112 in hepsin by glycosidase digestion and site-directed mutagenesis in human hepatoma cells. In Western blotting, fluorogenic substrate assay, flow cytometry, and protein-chase experiments, we found that Asn112 to Gln (N112Q) mutation inhibited hepsin intracellular trafficking, cell surface expression, and zymogen activation. By immunofluorescent staining, we found that the N112Q mutant was more abundant than wild-type hepsin in the endoplasmic reticulum (ER). Further co-immunoprecipitation studies indicated increased binding of the N112Q mutant to calnexin and binding-immunoglobulin protein (BiP), two ER chaperones. Our results indicate that the N-glycan in the SRCR domain of hepsin promotes intracellular trafficking and cell surface expression, possibly by a calnexin-dependent mechanism in facilitating ER exiting. [ABSTRACT FROM AUTHOR]

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2 -deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain. [ABSTRACT FROM AUTHOR]