The structural principles underlying molybdenum insertase complex assembly.

Within the cell, the trace element molybdenum (Mo) is only biologically active when complexed either within the nitrogenase-specific FeMo cofactor or within the molybdenum cofactor (Moco). Moco consists of an organic part, called molybdopterin (MPT) and an inorganic part, that is, the Mo-center. The enzyme which catalyzes the Mo-center formation is the molybdenum insertase (Mo-insertase). Mo-insertases consist of two functional domains called G- and E-domain. The G-domain catalyzes the formation of adenylated MPT (MPT-AMP), which is the substrate for the E-domain, that catalyzes the actual molybdate insertion reaction. Though the functions of E- and G-domain have been elucidated to great structural and mechanistic detail, their combined function is poorly characterized. In this work, we describe a structural model of the eukaryotic Mo-insertase Cnx1 complex that was generated based on cross-linking mass spectrometry combined with computational modeling. We revealed Cnx1 to form an asymmetric hexameric complex which allows the E- and G-domain active sites to align in a catalytic productive orientation toward each other.

Antibodies to calnexin and mutated calreticulin are common in human sera.

PURPOSE OF THE STUDY: Calreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homolog calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloproliferative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for myeloproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin. PATIENTS AND METHODS: We analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay. RESULTS: Antibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high correlation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin. CONCLUSION: Collectively, these findings indicate that calreticulin mutations may be common and not necessarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloproliferative neoplasm.

[Calnexin (CNX) enhances the killing effect of CD8+ T cells on colorectal cancer cells by promoting MHC I expression].

Objective To investigate the killing effect and molecular mechanism of aberrant expression of calnexin (CNX) in the colorectal cancer (CRC) on the CD8+ T immune cells. Methods Immunohistochemistry was used to detect CNX protein level in 102 pairs of CRC cancer and adjacent non-cancerous tissues. Western blotting was employed to examine the protein expression of MHC I in the HCT-15 cells overexpressed with CNX or in the SW480 cells whose CNX expressions were knockdown by siRNA. Murine CD8+ T cells isolated from the spleen were cocultured with CT-26 murine CRC cells infected with lentivirus-mediated CNX overexpression. The killing effect of CD8+ T cells on CT-26 cells was determined by cytotoxicity kit. The secretion of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) in the culture medium were examined by ELISA. Results The protein level of CNX in colorectal cancer tissues were significantly lower than that in non-cancerous tissues. CNX overexpressed in HCT-15 cells was upregulated and CNX knockdown in SW480 cells downregulated the MHC I expression in these cells. Furthermore, the overexpression of CNX could not only enhance the killing effect of CD8+ T cells on CT-26 cells, but also promote the secretion of IFN-γ and TNF-α from these cells. Conclusion CNX can enhance the killing potential of CD8+ T cells on tumor cells through upregulating the MHC I expression in colorectal cancer cells.

Glycan structure-based perspectives on the entry and release of glycoproteins in the calnexin/calreticulin cycle.

N-glycans are attached to newly synthesised polypeptides and are involved in the folding, secretion, and degradation of N-linked glycoproteins. In particular, the calnexin/calreticulin cycle, which is the central mechanism of the entry and release of N-linked glycoproteins depending on the folding sates, has been well studied. In addition to biological studies on the calnexin/calreticulin cycle, several studies have revealed complementary roles of in vitro chemistry-based research in the structure-based understanding of the cycle. In this mini-review, we summarise chemistry-based results and highlight their importance for further understanding of the cycle.

Two calcium-binding chaperones from the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause.

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin-type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N-globular domain, a P-arm domain, and a highly charged C-terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause-related expression analysis in the whole body revealed an upregulation of both genes by post-diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause-related expression pattern in the entire body for both genes. Fat body-specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post-diapause. This study suggests that calcium-binding chaperones play similar and possibly gender-specific roles during diapause.

PDI Family Members as Guides for Client Folding and Assembly.

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.

Standing on the Shoulders of Viruses.

My coworkers and I have used animal viruses and their interaction with host cells to investigate cellular processes difficult to study by other means. This approach has allowed us to branch out in many directions, including membrane protein characterization, endocytosis, secretion, protein folding, quality control, and glycobiology. At the same time, our aim has been to employ cell biological approaches to expand the fundamental understanding of animal viruses and their pathogenic lifestyles. We have studied mechanisms of host cell entry and the uncoating of incoming viruses as well as the synthesis, folding, maturation, and intracellular movement of viral proteins and molecular assemblies. I have had the privilege to work in institutions in four different countries. The early years in Finland (the University of Helsinki) were followed by 6 years in Germany (European Molecular Biology Laboratory), 16 years in the United States (Yale School of Medicine), and 16 years in Switzerland (ETH Zurich).

Calnexin cycle - structural features of the ER chaperone system.

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.

SMAR1 favors immunosurveillance of cancer cells by modulating calnexin and MHC I expression.

Down-regulation or loss of MHC class I expression is a major mechanism used by cancer cells to evade immunosurveillance and increase their oncogenic potential. MHC I mediated antigen presentation is a complex regulatory process, controlled by antigen processing machinery (APM) dictating immune response. Transcriptional regulation of the APM that can modulate gene expression profile and their correlation to MHC I mediated antigen presentation in cancer cells remain enigmatic. Here, we reveal that Scaffold/Matrix-Associated Region 1- binding protein (SMAR1), positively regulates MHC I surface expression by down-regulating calnexin, an important component of antigen processing machinery (APM) in cancer cells. SMAR1, a bonafide MAR binding protein acts as a transcriptional repressor of several oncogenes. It is down-regulated in higher grades of cancers either through proteasomal degradation or through loss of heterozygosity (LOH) at the Chr.16q24.3 locus where the human homolog of SMAR1 (BANP) has been mapped. It binds to a short MAR region of the calnexin promoter forming a repressor complex in association with GATA2 and HDAC1. A reverse correlation between SMAR1 and calnexin was thus observed in SMAR1-LOH cells and also in tissues from breast cancer patients. To further extrapolate our findings, influenza A (H1N1) virus infection assay was performed. Upon viral infection, the levels of SMAR1 significantly increased resulting in reduced calnexin expression and increased MHC I presentation. Taken together, our observations establish that increased expression of SMAR1 in cancers can positively regulate MHC I surface expression thereby leading to higher chances of tumor regression and elimination of cancer cells.

N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding.

Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.

Mapping the ER Interactome: The P Domains of Calnexin and Calreticulin as Plurivalent Adapters for Foldases and Chaperones.

The lectin chaperones calreticulin (CRT) and calnexin (CNX) contribute to the folding of glycoproteins in the ER by recruiting foldases such as the protein disulfide isomerase ERp57 and the peptidyl prolyl cis-trans isomerase CypB. Recently, CRT was shown to interact with the chaperone ERp29. Here, we show that ERp29 directly binds to the P domain of CNX. Crystal structures of the D domain of ERp29 in complex with the P domains from CRT and calmegin, a tissue-specific CNX homolog, reveal a commonality in the mechanism of binding whereby the tip of the P domain functions as a plurivalent adapter to bind a variety of folding factors. We show that mutation of a single residue, D348 in CNX, abrogates binding to ERp29 as well as ERp57 and CypB. The structural diversity of the accessory factors suggests that these chaperones became specialized for glycoprotein folding through convergent evolution of their P-domain binding sites.

Fatty acid binding protein (Fabp) 5 interacts with the calnexin cytoplasmic domain at the endoplasmic reticulum.

Calnexin is a type 1 integral endoplasmic reticulum membrane molecular chaperone with an endoplasmic reticulum luminal chaperone domain and a highly conserved C-terminal domain oriented to the cytoplasm. Fabp5 is a cytoplasmic protein that binds long-chain fatty acids and other lipophilic ligands. Using a yeast two-hybrid screen, immunoprecipitation, microscale thermophoresis analysis and cellular fractionation, we discovered that Fabp5 interacts with the calnexin cytoplasmic C-tail domain at the endoplasmic reticulum. These observations identify Fabp5 as a previously unrecognized calnexin binding partner.

PDI family protein ERp29 recognizes P-domain of molecular chaperone calnexin.

Endoplasmic reticulum (ER) resident lectin chaperone calnexin (CNX) and calreticulin (CRT) assist folding of nascent glycoproteins. Their association with ERp57, a member of PDI family proteins (PDIs) which promote disulfide bond formation of unfolded proteins, has been well documented. Recent studies have provided evidence that other PDIs may also interact with CNX and CRT. Accordingly, it seems possible that the ER provides a repertoire of CNX/CRT-PDI complexes, in order to facilitate refolding of various glycoproteins. In this study, we examined the ability of PDIs to interact with CNX. Among them ERp29 was shown to interact with CNX, similarly to ERp57. Judging from the dissociation constant, its ability to interact with CNX was similar to that of ERp57. Results of further analyses by using a CNX mutant imply that ERp29 and ERp57 recognize the same domain of CNX, whereas the mode of interaction with CNX might be somewhat different between them.

Structure of human promyeloperoxidase (proMPO) and the role of the propeptide in processing and maturation.

Myeloperoxidase (MPO) is synthesized by neutrophil and monocyte precursor cells and contributes to host defense by mediating microbial killing. Although several steps in MPO biosynthesis and processing have been elucidated, many questions remained, such as the structure-function relationship of monomeric unprocessed proMPO versus the mature dimeric MPO and the functional role of the propeptide. Here we have presented the first and high resolution (at 1.25 Å) crystal structure of proMPO and its solution structure obtained by small-angle X-ray scattering. Promyeloperoxidase hosts five occupied glycosylation sites and six intrachain cystine bridges with Cys-158 of the very flexible N-terminal propeptide being covalently linked to Cys-319 and thereby hindering homodimerization. Furthermore, the structure revealed (i) the binding site of proMPO-processing proconvertase, (ii) the structural motif for subsequent cleavage to the heavy and light chains of mature MPO protomers, and (iii) three covalent bonds between heme and the protein. Studies of the mutants C158A, C319A, and C158A/C319A demonstrated significant differences from the wild-type protein, including diminished enzymatic activity and prevention of export to the Golgi due to prolonged association with the chaperone calnexin. These structural and functional findings provide novel insights into MPO biosynthesis and processing.

Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor.

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.

The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER "sheet" proliferation after overexpression of tubular junction-forming proteins.

Interaction Between HIV-1 Nef and Calnexin: From Modeling to Small Molecule Inhibitors Reversing HIV-Induced Lipid Accumulation.

OBJECTIVE: HIV-infected patients are at an increased risk of developing atherosclerosis, in part because of downmodulation and functional impairment of ATP-binding cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an ER chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting the translational impact of this finding. APPROACH AND RESULTS: Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that the interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nmol/L measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with an affinity of 9.4 nmol/L. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the National Cancer Institute library of compounds, we identified a compound, 1[(7-oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages. CONCLUSION: This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects.

Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin.

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non-palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.

Dimerization-dependent folding underlies assembly control of the clonotypic αßT cell receptor chains.

In eukaryotic cells, secretory pathway proteins must pass stringent quality control checkpoints before exiting the endoplasmic reticulum (ER). Acquisition of native structure is generally considered to be the most important prerequisite for ER exit. However, structurally detailed protein folding studies in the ER are few. Furthermore, aberrant ER quality control decisions are associated with a large and increasing number of human diseases, highlighting the need for more detailed studies on the molecular determinants that result in proteins being either secreted or retained. Here we used the clonotypic αß chains of the T cell receptor (TCR) as a model to analyze lumenal determinants of ER quality control with a particular emphasis on how proper assembly of oligomeric proteins can be monitored in the ER. A combination of in vitro and in vivo approaches allowed us to provide a detailed model for αßTCR assembly control in the cell. We found that folding of the TCR α chain constant domain Cα is dependent on αß heterodimerization. Furthermore, our data show that some variable regions associated with either chain can remain incompletely folded until chain pairing occurs. Together, these data argue for template-assisted folding at more than one point in the TCR α/ß assembly process, which allows specific recognition of unassembled clonotypic chains by the ER chaperone machinery and, therefore, reliable quality control of this important immune receptor. Additionally, it highlights an unreported possible limitation in the α and ß chain combinations that comprise the T cell repertoire.

Calnexin functions in antibacterial immunity of Marsupenaeus japonicus.

Calnexin (Cnx) is an endoplasmic reticulum membrane-bound lectin chaperone that comprises a dedicated maturation system with another lectin chaperone calreticulin (Crt). This maturation system is known as the Cnx/Crt cycle. The main functions of Cnx are Ca(2+) storage, glycoprotein folding, and quality control of synthesis. Recent studies have shown that Cnx is important in phagocytosis and in optimizing dendritic cell immunity. However, the functions of Cnx in invertebrate innate immunity remain unclear. In this research, we characterized Cnx in the kuruma shrimp Marsupenaeus japonicus (designated as MjCnx) and detected its function in shrimp immunity. The expression of MjCnx was upregulated in several tissues challenged with Vibrio anguillarum. Recombinant MjCnx could bind to bacteria by binding polysaccharides. MjCnx protein existed in the cytoplasm and on the membrane of hemocytes and was upregulated by bacterial challenge. The recombinant MjCnx enhanced the clearance of V. anguillarum in vivo, and the clearance effects were impaired after silencing MjCnx with RNA interference assay. Recombinant MjCnx promoted phagocytosis efficiency of hemocytes. These results suggest that MjCnx functions as one of the pattern recognition receptors and has crucial functions in shrimp antibacterial immunity.