Impaired O-Linked N-Acetylglucosaminylation in the Endoplasmic Reticulum by Mutated Epidermal Growth Factor (EGF) Domain-specific O-Linked N-Acetylglucosamine Transferase Found in Adams-Oliver Syndrome

Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the K_m value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 μm. Despite the relatively low K_m value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGT^R377Q were not affected. Importantly, the interaction between UDP-GlcNAc and EOGT^R377Q was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS.     

Crystal Structure of a Virus-Encoded Putative Glycosyltransferase

The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-Å resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs to glycosyltransferase family 4. The donor substrate was identified as GDP-mannose by isothermal titration calorimetry and was shown to bind into the cleft between the two domains in the protein. The active form of the enzyme is probably a dimer in which the active centers are separated by about 40 Å.Glycosyltransferases constitute a large family of enzymes that catalyze the transfer of sugar moieties from donor molecules to specific acceptor molecules. Unlike other enzyme families that usually share conserved features in their primary sequences, glycosyltransferases can have highly diversified sequences that have been grouped into more than 90 families (designated GTn, where n = 1, 2, …) (http://www.CAZy.org) (1, 15). However, two families, GT2 and GT4, account for about half of the total number of glycosyltransferases. Despite the large variation in the primary sequences of glycosyltransferases, their three-dimensional structures are usually conserved. There are two major glycosyltransferase structural types, named GT-A and GT-B. The GT-A members contain a single nucleotide-binding domain consisting of six parallel β-strands flanked by connecting α-helices (referred to as a “Rossmann fold” in most of the literature on these enzymes and herein). GT-A enzyme activities are usually metal ion dependent. The GT-B type glycosyltransferases have two Rossmann folds separated by a cleft that forms the substrate-binding site. Metal ions are normally not required for GT-B function. Based on their catalytic mechanism, glycosyltransferases are also classified as either retaining or inverting enzymes depending on the geometry between the sugar donor and the receptor in the product molecule (e.g., depending on whether the anomeric carbon atom is linked to the acceptor via its α or β position). If the anomeric carbon atom has the same configuration in the donor and in the product, the enzyme is classified as a retaining enzyme; if the configurations are different, the enzyme is considered to be an inverting enzyme (2).Many viruses, especially those that infect eukaryotic cells, have extensively glycosylated structural proteins. Glycans coating viral structural proteins serve multiple biological roles, e.g., they mimic host glycans to evade host cell immune reactions, aid in folding or assembly of viral structural proteins, function as a receptor recognized by cell surface proteins, or aid in stabilizing viral particles (see, e.g., reference 36).Typically, viruses use host-encoded glycosyltransferases and glycosidases located in the endoplasmic reticulum (ER) and Golgi apparatus to add and remove N-linked sugar residues from virus glycoproteins either during or shortly after translation of the protein. This posttranslational processing aids in protein folding and requires other host-encoded enzymes. After folding and assembly, virus glycoproteins are transported by host-sorting and membrane transport functions to virus-specified regions in host membranes, where they displace host glycoproteins. Progeny viruses then bud through these virus-specific target membranes, in what is usually the final step in the assembly of infectious virions (3, 14, 21, 36). Thus, nascent viruses become infectious only by budding through the target membrane, usually the plasma membrane, as they are released from the cell. Consequently, the glycan portion of virus glycoproteins is host specific. The theme that emerges is that virus glycoproteins are synthesized and glycosylated by the same mechanisms as host glycoproteins. Therefore, the only way to alter glycosylation of virus proteins is to either grow the virus in a different host or have a mutation in the virus protein that alters the protein glycosylation site.One explanation for this scenario is that, in general, viruses lack genes encoding glycosyltransferases. However, a few virus-encoded glycosyltransferases have been reported in recent years (see reference 17 for a review). Often these virus-encoded glycosyltransferases add sugars to compounds other than proteins. For instance, some phage-encoded glycosyltransferases modify virus DNA to protect it from host restriction endonucleases (see, e.g., reference 10), and a glycosyltransferase encoded by baculoviruses modifies a host insect ecdysteroid hormone, leading to its inactivation (22). Bovine herpesvirus 4 encodes a β-1,6-N-acetyl-glucosaminyltransferase that is localized in the Golgi apparatus and is probably involved in posttranslational modification of the virus structural proteins (32).One group of viruses differs from the scenario that viruses use the host machinery located in the ER and the Golgi apparatus to glycosylate their glycoproteins. These viruses are the large, plaque-forming, double-stranded DNA (dsDNA)-containing chloroviruses (family Phycodnaviridae) that infect eukaryotic algae (4, 34, 39, 40). The chloroviruses have up to 400 protein-encoding genes (or coding sequences [CDSs]). Annotation of six chlorovirus genomes showed that each virus encodes 3 to 6 putative glycosyltransferases (7-9, 16, 33). Three of these viruses, NY-2A, AR158, and the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1), infect Chlorella strain NC64A. Two of the viruses, MT325 and FR483, infect Chlorella Pbi, and one of them, Acanthocystis turfacea chlorella virus (ATCV-1), infects Chlorella SAG 3.83.Glycosylation of the PBCV-1 major capsid protein, Vp54, is at least partially performed by the viral glycosyltransferases (11, 20, 33, 38, 41). PBCV-1 encodes 5 putative glycosyltransferases. A previous structural study established that the N-terminal 211 amino acids of the A64R protein from PBCV-1 form a GT-A group glycosyltransferase that is a retaining enzyme belonging to the GT34 family and that UDP-glucose possibly serves as the donor sugar (41).Among the four additional PBCV-1 glycosyltransferase-encoding genes, gene a546l encodes a 396-amino-acid protein that resembles members in the GT4 family of glycosyltransferases, based on amino acid sequence comparison of members in the CAZy classification (1, 15). Homologs of this protein, A546L, are encoded by 3 other chloroviruses, NY-2A, AR158, and ATCV-1. Here, we report the crystal structure of one of these homologs, B736L, at 2.3-Å resolution.     

N-Linked Protein Glycosylation in the Endoplasmic Reticulum

The attachment of glycans to asparagine residues of proteins is an abundant and highly conserved essential modification in eukaryotes. The N-glycosylation process includes two principal phases: the assembly of a lipid-linked oligosaccharide (LLO) and the transfer of the oligosaccharide to selected asparagine residues of polypeptide chains. Biosynthesis of the LLO takes place at both sides of the endoplasmic reticulum (ER) membrane and it involves a series of specific glycosyltransferases that catalyze the assembly of the branched oligosaccharide in a highly defined way. Oligosaccharyltransferase (OST) selects the Asn-X-Ser/Thr consensus sequence on polypeptide chains and generates the N-glycosidic linkage between the side-chain amide of asparagine and the oligosaccharide. This ER-localized pathway results in a systemic modification of the proteome, the basis for the Golgi-catalyzed modification of the N-linked glycans, generating the large diversity of N-glycoproteome in eukaryotic cells. This article focuses on the processes in the ER. Based on the highly conserved nature of this pathway we concentrate on the mechanisms in the eukaryotic model organism Saccharomyces cerevisiae.The presence of glycans on proteins is known to influence their stability and solubility and the glycan core can contribute to folding processes (Shental-Bechor and Levy 2008; Hanson et al. 2009; Culyba et al. 2011). N-glycans also influence the function and activity of proteins (Skropeta 2009). The terminal residues of N-glycans play a key role in the quality control of protein folding in the ER. Ultimately the glycan signals whether a protein is correctly folded and can leave the ER to continue its maturation in the Golgi or whether the protein is not correctly folded and is degraded (Helenius and Aebi 2004; Aebi et al. 2010). It is therefore of great importance that the oligosaccharide to be transferred to proteins is complete. This “quality control” of the oligosaccharide is mediated by the substrate specificity of oligosaccharyltransferase.     

Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

Background

Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer.

Methodology

In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens.

Results

Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002).

Conclusion

Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer.     

O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation

Thrombospondin type 1 repeat (TSR) superfamily members regulate diverse biological activities ranging from cell motility to inhibition of angiogenesis. In this study, we verified that mouse protein O-fucosyltransferase-2 (POFUT2) specifically adds O-fucose to TSRs. Using two Pofut2 gene-trap lines, we demonstrated that O-fucosylation of TSRs was essential for restricting epithelial to mesenchymal transition in the primitive streak, correct patterning of mesoderm, and localization of the definitive endoderm. Although Pofut2 mutant embryos established anterior/posterior polarity, they underwent extensive mesoderm differentiation at the expense of maintaining epiblast pluripotency. Moreover, mesoderm differentiation was biased towards the vascular endothelial cell lineage. Localization of Foxa2 and Cer1 expressing cells within the interior of Pofut2 mutant embryos suggested that POFUT2 activity was also required for the displacement of the primitive endoderm by definitive endoderm. Notably, Nodal, BMP4, Fgf8, and Wnt3 expression were markedly elevated and expanded in Pofut2 mutants, providing evidence that O-fucose modification of TSRs was essential for modulation of growth factor signaling during gastrulation. The ability of Pofut2 mutant embryos to form teratomas comprised of tissues from all three germ layer origins suggested that defects in Pofut2 mutant embryos resulted from abnormalities in the extracellular environment. This prediction is consistent with the observation that POFUT2 targets are constitutive components of the extracellular matrix (ECM) or associate with the ECM. For this reason, the Pofut2 mutants represent a valuable tool for studying the role of O-fucosylation in ECM synthesis and remodeling, and will be a valuable model to study how post-translational modification of ECM components regulates the formation of tissue boundaries, cell movements, and signaling.     

O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.     

Experimental evolution and genome sequencing reveal variation in levels of clonal interference in large populations of bacteriophage φX174

Background

The first step of GPI anchor biosynthesis is catalyzed by PIG-A, an enzyme that transfers N -acetylglucosamine from UDP- N -acetylglucosamine to phosphatidylinositol. This protein is present in all eukaryotic organisms ranging from protozoa to higher mammals, as part of a larger complex of five to six 'accessory' proteins whose individual roles in the glycosyltransferase reaction are as yet unclear. The PIG-A gene has been shown to be an essential gene in various eukaryotes. In humans, mutations in the protein have been associated with paroxysomal noctural hemoglobuinuria. The corresponding PIG-A gene has also been recently identified in the genome of many archaeabacteria although genes of the accessory proteins have not been discovered in them. The present study explores the evolution of PIG-A and the phylogenetic relationship between this protein and other glycosyltransferases.

Results

In this paper we show that out of the twelve conserved motifs identified by us eleven are exclusively present in PIG-A and, therefore, can be used as markers to identify PIG-A from newly sequenced genomes. Three of these motifs are absent in the primitive eukaryote, G. lamblia. Sequence analyses show that seven of these conserved motifs are present in prokaryote and archaeal counterparts in rudimentary forms and can be used to differentiate PIG-A proteins from glycosyltransferases. Using partial least square regression analysis and data involving presence or absence of motifs in a range of PIG-A and glycosyltransferases we show that (i) PIG-A may have evolved from prokaryotic glycosyltransferases and lipopolysaccharide synthases, members of the GT4 family of glycosyltransferases and (ii) it is possible to uniquely classify PIG-A proteins versus glycosyltransferases.

Conclusion

Besides identifying unique motifs and showing that PIG-A protein from G. lamblia and some putative PIG-A proteins from archaebacteria are evolutionarily closer to glycosyltransferases, these studies provide a new method for identification and classification of PIG-A proteins.     

Loss of all three calreticulins, CRT1, CRT2 and CRT3, causes enhanced sensitivity to water stress in Arabidopsis

Key message

The calreticulin triple knockout mutant shows growth defects in response to abiotic stress.

Abstract

The endoplasmic reticulum (ER) is an essential organelle that is responsible for the folding and maturation of proteins. During ER stress, unfolded protein aggregates accumulate in the cell, leading to the unfolded protein response (UPR). The UPR up-regulates the expression of ER-stress-responsive genes encoding calreticulin (CRT), an ER-localized Ca²⁺-binding protein. To understand the function of plant CRTs, we generated a triple knockout mutant, t123, which lacks CRT1, CRT2 and CRT3 and examined the roles of calreticulins in abiotic stress tolerance. A triple knockout mutant increased sensitivity to water stress which implies that calreticulins are involved in the Arabidopsis response to water stress. We identified that the cyclophilin AtCYP21-2, which is located in the ER, was specifically enhanced in the t123 mutants. Seed germination of the atcyp21-1 mutant was retarded by water stress. Taken together, these results suggest that regulatory proteins that serve to protect plants from water stress are folded properly in part with the help of calreticulins. The AtCYP21-2 may also participate in this protein-folding process in association with calreticulins.     

Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

The low density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the “SimpleCell” O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position −1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.     

Folding, quality control, and secretion of pancreatic ribonuclease in live cells

Although bovine pancreatic RNase is one of the best characterized proteins in respect to structure and in vitro refolding, little is known about its synthesis and maturation in the endoplasmic reticulum (ER) of live cells. We expressed the RNase in live cells and analyzed its folding, quality control, and secretion using pulse-chase analysis and other cell biological techniques. In contrast to the slow in vitro refolding, the protein folded almost instantly after translation and translocation into the ER lumen (t_½ < 3 min). Despite high stability of the native protein, only about half of the RNase reached a secretion competent, monomeric form and was rapidly transported from the rough ER via the Golgi complex (t_½ = 16 min) to the extracellular space (t_½ = 35 min). The rest remained in the ER mainly in the form of dimers and was slowly degraded. The dimers were most likely formed by C-terminal domain swapping since mutation of Asn¹¹³, a residue that stabilizes such dimers, to Ser increased the efficiency of secretion from 59 to 75%. Consistent with stringent ER quality control in vivo, the secreted RNase in the bovine pancreas was mainly monomeric, whereas the enzyme present in the cells also contained 20% dimers. These results suggest that the efficiency of secretion is not only determined by the stability of the native protein but by multiple factors including the stability of secretion-incompetent side products of folding. The presence of N-glycans had little effect on the folding and secretion process.     

Dolichol phosphate mannose synthase: a Glycosyltransferase with Unity in molecular diversities

N-glycans provide structural and functional stability to asparagine-linked (N-linked) glycoproteins, and add flexibility. Glycan biosynthesis is elaborative, multi-compartmental and involves many glycosyltransferases. Failure to assemble N-glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N-glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol-linked tetra-decasaccharide (Glc₃Man₉GlcNAc₂-PP-Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O- and C-mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT-A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP-dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C-terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C-terminus, DPMS is still classified as Type I and Type II enzyme. Because of an apparent bio-sensing ability, extracellular signaling and microenvironment regulate DPMS catalytic activity. In this review, we highlight some important features and the molecular diversities of DPMS.     

Recombinant human hemoglobin: Expression and refolding of β-globin fromEscherichia coli

A plasmid analogous to the one described by Nagai and Thogersen (Nature,309, 810–812, 1984) has been constructed for the expression of globins inE. coli. Induction with nalidixic acid produces high yields of a fusion protein, NS1-FX-β-globin, where NS1 represents 81 residues of a flu virus protein and FX represents a blood-clotting Factor Xa recognition sequence, Ile-Glu-Gly-Arg. This fusion protein is readily solubilized in 50 mM NaOH and remains in solution when thepH is adjusted to 8.6. Under these conditions, the fusion protein is hydrolyzed by activated Factor X, giving authentic β-globin which can be folded in the presence of cyanohemin and native α-chains to produce a tetrameric hemoglobin with the functional properties of natural human hemoglobin.     

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.The endoplasmic reticulum (ER)² is the first organelle in the secretory pathway of eukaryotic cells and provides an optimum environment for maturation of newly synthesized secretory and membrane proteins. Protein folding/assembly in the ER is aided by molecular chaperones and folding enzymes. Molecular chaperones in the ER assist folding of newly synthesized proteins and prevent them from premature misfolding and/or aggregate formation (1, 2). Protein folding in the ER is often associated with formation of disulfide bonds, which contribute to stabilization of native, functional states of proteins. Disulfide bond formation could be a rate-limiting step of protein folding both in vitro and in vivo (3, 4), and the ER has a set of folding enzymes including protein-disulfide isomerase (PDI) and its homologs that catalyze disulfide bond formation (5, 6).In parallel, protein folding/assembly in the ER relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER. Misfolded or aberrant proteins are retained in the ER for refolding by ER-resident chaperones, whereas terminally misfolded proteins are degraded by the mechanism known as ER-associated degradation (ERAD). The ERAD consists of recognition and processing of aberrant substrate proteins, retrotranslocation across the ER membrane, and subsequent proteasome-dependent degradation in the cytosol. More than 20 different components have been identified to be involved in this process in yeast and mammals (7).The majority of proteins synthesized in the ER are glycoproteins, in which N-linked glycans are not only important for folding but also crucial for their ERAD if they fail in folding. Specifically, trimming of one or more mannose residues of Man₉GlcNAc₂ oligosaccharide and recognition of the modified mannose moiety represent a key step for selection of terminally misfolded proteins for disposal (8). A mannosidase I-like protein, Mnl1p/Htm1p (yeast), and EDEM (mammals, ER degradation enhancing α-mannosidase-like protein) were identified as candidates for lectins that recognize ERAD substrates with modified mannose moieties (9–11). Both Mnl1p and EDEM contain an N-terminal mannosidase homology domain (MHD), which lacks cysteine residues conserved among α1,2-mannosidase family members and is proposed to function in recognition of mannose-trimmed carbohydrate chains (supplemental Fig. S1). However, whether Mnl1p or EDEM indeed functions as an ERAD-substrate-binding lectin or has a mannosidase activity is still in debate (11–15), and Yos9p was suggested to take the role of ERAD-substrate binding lectin (14, 16–18). Mnl1p, but not EDEM, has a large C-terminal extension, which does not show any homology to known functional domains and is conserved only among fungal Mnl1p homologs (supplemental Fig. S1).After recognition of the modified mannose signal for degradation, aberrant proteins are maintained or converted to be retrotranslocation competent by ER chaperones including BiP (19). PDI was also indicated to be involved in these steps in the ERAD by, for example, its possible chaperone-like functions (20–23). The yeast PDI, Pdi1p, contains four thioredoxin-like domains, two of which have a CGHC motif as active sites, followed by a C-terminal extension containing the ER retention signal. During its catalytic cycle, PDI transiently forms a mixed disulfide intermediate with its substrate through an intermolecular disulfide bond between the cysteine residues of the active site of PDI and the substrate molecule.Here we report identification of PDI as an Mnl1p-interacting protein. Stable interactions between the C-terminal domain of Mnl1p and PDI involve intermolecular disulfide bonds. Stably interacting PDI is required for formation of the functionally essential intramolecular disulfide bond in the MHD of Mnl1p, which in turn stabilizes and prolongs the Mnl1p-PDI interactions. Possible roles for those stable interactions between Mnl1p and PDI in the ERAD will be discussed.     

Role of the tumor suppressor IQGAP2 in metabolic homeostasis: possible link between diabetes and cancer

Deficiency of IQGAP2, a scaffolding protein expressed primarily in liver leads to rearrangements of hepatic protein compartmentalization and altered regulation of enzyme functions predisposing development of hepatocellular carcinoma and diabetes. Employing a systems approach with proteomics, metabolomics and fluxes characterizations, we examined the effects of IQGAP2 deficient proteomic changes on cellular metabolism and the overall metabolic phenotype. Iqgap2 ^?/?mice demonstrated metabolic inflexibility, fasting hyperglycemia and obesity. Such phenotypic characteristics were associated with aberrant hepatic regulations of glycolysis/gluconeogenesis, glycogenolysis, lipid homeostasis and futile cycling corroborated with corresponding proteomic changes in cytosolic and mitochondrial compartments. IQGAP2 deficiency also led to truncated TCA-cycle, increased anaplerosis, increased supply of acetyl-CoA for de novo lipogenesis, and increased mitochondrial methyl-donor metabolism necessary for nucleotides synthesis. Our results suggest that changes in metabolic networks in IQGAP2 deficiency create a hepatic environment of a ‘pre-diabetic’ phenotype and a predisposition to non-alcoholic fatty liver disease which has been linked to the development of hepatocellular carcinoma.     

Endoplasmic Reticulum Stress Induces Different Molecular Structural Alterations in Human Dilated and Ischemic Cardiomyopathy

Background

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for the synthesis and folding of proteins as well as for signalling and calcium storage, that has been linked to the contraction-relaxation process. Perturbations of its homeostasis activate a stress response in diseases such as heart failure (HF). To elucidate the alterations in ER molecular components, we analyze the levels of ER stress and structure proteins in human dilated (DCM) and ischemic (ICM) cardiomyopathies, and its relationship with patient''s functional status.

Methods and Results

We examined 52 explanted human hearts from DCM (n = 21) and ICM (n = 21) subjects and 10 non-failing hearts as controls. Our results showed specific changes in stress (IRE1, p<0.05; p-IRE1, p<0.05) and structural (Reticulon 1, p<0.01) protein levels. The stress proteins GRP78, XBP1 and ATF6 as well as the structural proteins RRBP1, kinectin, and Nogo A and B, were upregulated in both DCM and ICM patients. Immunofluorescence results were concordant with quantified Western blot levels. Moreover, we show a novel relationship between stress and structural proteins. RRBP1, involved in procollagen synthesis and remodeling, was related with left ventricular function.

Conclusions

In the present study, we report the existence of alterations in ER stress response and shaping proteins. We show a plausible effect of the ER stress on ER structure in a suitable sample of DCM and ICM subjects. Patients with higher values of RRBP1 had worse left ventricular function.     

The Golgi apparatus mediates the transport of phytohemagglutinin to the protein bodies in bean cotyledons

When developing cotyledons of Phaseolus vulgaris L. were labeled with [³H]fucose, fucose-labeled phytohemagglutinin (PHA) was found in organelles with average densities of 1.13 g cm^-3 and 1.22 g cm^-3. The position of these organelles on isopycnic sucrose gradients was independent of the presence of MgCl₂ and ethylenediaminetetraacetate in the media, indicating that the fucose-labeled PHA was not associated with the rough endoplasmic reticulum (ER). The organelles with a density of 1.13 g cm^-3 were identified as membranes of the Golgi apparatus on the basis of the similarity of their sedimentation properties and those of the Golgi marker enzyme, inosine diphosphatase, in both isopycnic and rate-zonal sucrose gradients. The organelles with a density of 1.22 g cm^-3 were identified as small (0.1–0.4 μm), electron-dense vesicles with a protein content similar to that of the protein bodies. Pulsechase experiments with [³H]fucose indicated that fucose-labeled PHA first appeared in the Golgi-apparatus-derived membranes and later in the dense vesicles. Fucose-labeled PHA chased out of the Golgi apparatus first, then out of the dense vesicles, and accumulated in the soluble portion of the homogenate which contained the contents of the broken protein bodies. Fucose-labeled PHA chased out of the two types of organelles with a t _1/2 of 20–30 min, a rate three to four times faster than newly synthesized PHA chases out of the bulk of the ER (Chrispeels, M.J., Bollini, R., 1982, Plant Physiol. 70, 1425–1428). This result indicates that the Golgi apparatus is a much smaller compartment than the ER in the storage parenchyma cells. The sodium ionophore, monensin, which interferes with the function of the Golgi apparatus of animal cells, blocks the biosynthesis and—or transport of fucose- and galactose-labeled macromolecules to the cotyledon cell walls. Monensin also blocks the transport of labeled PHA out of the Golgi apparatus and into the protein bodies. These results provide the first biochemical evidence that a specific storage protein which accumulates in seeds is modified in, and passes through, the Golgi apparatus on its way to the protein bodies.     

Protein O-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7⁺/MyoD⁻ cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.