Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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In this study we report that human platelets display neutral (nSMase) and acid sphingomyelinase (aSMase) as well as acid ceramidase (aCerase) activity. Cell activation by thrombin resulted in a marked decrease of intracellular aSMase activity, accompanied by the release of enzyme into the medium. In contrast, thrombin treatment did not affect aCerase activity. Two major protein bands of 73 and 70 kDa were recognized by aSMase antibodies in resting platelet lysates and in the medium of stimulated cells. Phorbol esters together with the calcium ionophore A23187 fully reproduced thrombin action on aSMase release. The secreted enzymatic activity was insensitive to digestion with endoglycosidase H but it was stimulated by Zn2+, although to a limited extent compared to aSMase constitutively released by murine endothelial cells. Taken together, these data suggest that secreted aSMase does not originate from the lysosomal compartment but rather from other platelet vesicles.
Mol Cell Biochem 2000 Feb
PMID:Characterization of sphingomyelinase activity released by thrombin-stimulated platelets. 1082 24

Wolfram (DIDMOAD) syndrome is an autosomal recessive neurodegenerative disorder accompanied by insulin-dependent diabetes mellitus and progressive optic atrophy. Recent positional cloning led to identification of the WFS1 (Wolfram syndrome 1) gene, a member of a novel gene family of unknown function. In this study, we generated a specific antibody against the C-terminus of the WFS1 protein and investigated its subcellular localization in cultured cells. We also studied its distribution in the rat brain. Biochemical studies indicated the WFS1 protein to be an integral, endoglycosidase H-sensitive membrane glycoprotein that localizes primarily in the endoplasmic reticulum (ER). Consistent with this, immunofluorescence cell staining of overexpressed WFS1 showed a characteristic reticular pattern over the cytoplasm and overlapped with the ER marker staining. No co-localization of WFS1 with mitochondria argues against an earlier clinical hypothesis that Wolfram syndrome is a mitochondria-mediated disorder. In the rat brain, at both the protein and mRNA level, WFS1 was found to be present predominantly in selected neurons in the hippocampus CA1, amygdaloid areas, olfactory tubercle and superficial layer of the allocortex. These expression sites, i.e. components of the limbic system or structures closely associated with this system, may be involved in the psychiatric, behavioral and emotional abnormalities characteristic of this syndrome. ER localization of WFS1 suggests that this protein plays an as yet undefined role in membrane trafficking, protein processing and/or regulation of ER calcium homeostasis. These studies represent a first step toward the characterization of WFS1 protein, which presumably functions to maintain certain populations of neuronal and endocrine cells.
Hum Mol Genet 2001 Mar 01
PMID:WFS1 (Wolfram syndrome 1) gene product: predominant subcellular localization to endoplasmic reticulum in cultured cells and neuronal expression in rat brain. 1118 71

PHEX is homologous to the M13 zinc metallopeptidases, a class of type II membrane glycoproteins. Although more than 140 mutations in the PHEX gene have been identified in patients with X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets, the molecular consequences of disease-causing PHEX mutations have not yet been investigated. We examined the effect of PHEX missense mutations on cellular trafficking of the recombinant protein. Four mutant PHEX cDNAs were generated by PCR mutagenesis: C85R, G579R and S711R, identified in XLH patients, and E581V, previously engineered in neutral endopeptidase 24.11, where it abolished catalytic activity but not plasma membrane targeting. Wild-type and mutant PHEX cDNAs were transfected in HEK(293) cells and PHEX protein expression was characterized. In contrast to the wild-type and E581V PHEX proteins, the C85R, G579R and S711R mutants were completely sensitive to endoglycosidase H digestion, indicating that they were not fully glycosylated. Sequestration of the disease-causing mutant proteins in the endoplasmic reticulum (ER) and plasma membrane localization of wild-type and E581V PHEX proteins was demonstrated by immunofluorescence and cell surface biotinylation. Of the three mutant PHEX proteins, the S711R was the least stable and the only one that could be rescued from the ER to the plasma membrane in cells grown at 26 degrees C. The chemical chaperone glycerol failed to correct defective targeting of all three mutant proteins. Our data provide a mechanism for loss of PHEX function in XLH patients expressing the C85R, G579R and S711R mutations.
Hum Mol Genet 2001 Jul 15
PMID:Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. 1146 71

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, inhibits the secretion of proteins and causes the redistribution of resident Golgi proteins into the endoplasmic reticulum (ER). In this study, the effect of NDGA on lipoprotein lipase (LPL) secretion was investigated in 3T3-L1 adipocytes, and compared with those of brefeldin A (BFA), a well-known fungal metabolite that exhibits similar ER-Golgi redistribution. Both BFA and NDGA blocked secretions of LPL. In the presence of BFA, the active and dimeric LPL was accumulated in adipocytes. After endoglycosidase H (endo H) digestion, the proportion of LPL subunits with partially endo H-sensitive oligosaccharide was significantly increased with BFA. However, in the presence of NDGA, the cellular LPL became inactive, and only the endo H-sensitive fraction of the LPL subunit was observed. An increase of the aggregated forms was observed in the fractions of the sucrose-density gradient ultracentrifugation. These properties of LPL in the NDGA-treated cells were similar to those of LPL that is retained in ER, and the effects of NDGA could not be reversed by BFA. These results indicate that the inhibitory mechanism of NDGA on the LPL secretion is functionally different from the ER-Golgi redistribution that is induced by BFA.
J Biochem Mol Biol 2002 Sep 30
PMID:Effect of nordihydroguaiaretic acid on the secretion of lipoprotein lipase. 1235 96

Rigid spine muscular dystrophy and the classical form of multiminicore disease are caused by mutations in SEPN1 gene, leading to a new clinical entity referred to as SEPN1-related myopathy. SEPN1 codes for selenoprotein N, a new member of the selenoprotein family, the function of which is still unknown. In a previous study, two isoforms were deduced from SEPN1 transcript analyses. Using polyclonal antibodies directed against SEPN1 and cDNA constructs encoding for the two isoforms, we show that the main SEPN1 gene product corresponds to a 70 kDa protein, containing a single selenocysteine residue. Subcellular fractionation experiments and endoglycosidase H sensitivity indicate that SEPN1 is a glycoprotein-localized within the endoplasmic reticulum. Immunofluorescence analyses confirm this subcellular localization and green fluorescent protein fusion experiments demonstrate the presence of an endoplasmic reticulum-addressing and -retention signal within the N-terminus. SEPN1 is present at a high level in several human fetal tissues and at a lower level in adult ones, including skeletal muscle. Its high expression in cultured myoblasts is also down-regulated in differentiating myotubes, suggesting a role for SEPN1 in early development and in cell proliferation or regeneration.
Hum Mol Genet 2003 May 01
PMID:Selenoprotein N: an endoplasmic reticulum glycoprotein with an early developmental expression pattern. 1270 Jan 73

GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini ( approximately 450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-Delta1-239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor's efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.
Mol Endocrinol 2003 Nov
PMID:Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling. 1292 Feb 37

We compared the tissue mRNA prevalence and protein maturation of two splice variants of mouse ADAM33, a metalloprotease implicated in airway hyperresponsiveness. These variant cDNAs, designated 914 (alpha) and 906 (beta), encode membrane-bound forms that differ primarily in 26 residues (exon 17) between the cysteine-rich and epidermal growth factor-like domains. Proteins of approximately 120 and 103 kD, detectable by anti-ADAM33 antibodies, were expressed in 914-transfected HEK293 cells. The time-dependent appearance of the approximately 100-kD form and its inhibition by a peptidyl chloromethylketone, or the calcium ionophore, A23187, indicated that this was mature ADAM33, which was processed by a furin-like convertase. One form, approximately 110 kD, was detected in 906-transfected cell lysates. Trypsin and biotinylation treatment of transfected cells demonstrated that all of the mature approximately 100-kD, a minority of the approximately 120-kD pro-form, and none of the 906-expressed 110-kD form localized to the cell surface. The mature form was resistant to endoglycosidase H(f). The approximately 110-kD form was endoglycosidase H(f)-sensitive, indicating retention proximal to the trans-Golgi, consistent with a lack of maturation. Quantitation of transcripts demonstrated that those containing exon 17 predominate, whereas those lacking exon 17 are negligible in the mouse lung, although detectable at low levels in mouse testis, heart, and brain. Thus, potential dominant-negative effects exerted by the nonprocessed 906-encoded beta splice variant are unlikely to occur in mouse lung.
Am J Respir Cell Mol Biol 2004 Apr
PMID:Mouse ADAM33: two splice variants differ in protein maturation and localization. 1297 1

A splice variant of human lutropin (LH)/choriogonadotropin (CG)-receptor [hLHR(exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a normal menstrual cycle. Supported by a detergent-soluble binding assay and a receptor biotinylation experiment, the receptor binding assay shows hLHR(exon 9) is neither expressed at the cell surface nor has the capability of binding to hCG. In addition, hLHR(exon 9) was confirmed in the endoplasmic reticulum (ER) by endoglycosidase H treatment. A coimmunoprecipitation experiment clearly showed that hLHR(exon 9) and constitutively inactivate mutant-LHRs, which stay in the ER, form an association with the human follitropin (FSH)-receptor (hFSHR). This suggests that in the presence of mutant-LHR, hFSHR, which is trapped in the ER and associated with hLHR(exon 9), is unable to come up to the plasma membrane. This phenomenon is specific among gonadotropin receptors because human TSH receptor failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the hFSHR receptor protein level within the cells, which impaired cAMP production. To elucidate the mechanism underlying the decrease in hFSHR protein by this receptor complex, we performed a Percoll fractionation experiment, which indicated that the receptor complex drove hFSHR to the lysosome instead of the plasma membrane. These results reveal a novel mechanism of FSHR expression regulation.
Mol Endocrinol 2005 Aug
PMID:Association of human follitropin (FSH) receptor with splicing variant of human lutropin/choriogonadotropin receptor negatively controls the expression of human FSH receptor. 1589 Jun 74

Ferroportin (FPN) is the main iron export protein in mammals. The actual structure of FPN in vivo and the pathogenesis of ferroportin-related disease are unknown. We aimed at studying the structure and biochemical properties of FPN in mouse tissues that are key for iron homeostasis during various iron manipulations in vivo. We performed glycosylation and oligomerization studies in spleen and liver extracts from mice fed a standard, iron-deprived or iron-enriched diet for 5 months. Purification by affinity chromatography and sucrose gradient show that FPN is not part of a large multiprotein complex. Dietary manipulations did not affect the monomeric status of the native or denatured protein. The glycosylation studies showed that ferroportin is digested by peptide: N-glycosidase F but not by endoglycosidase H. The same results were obtained using protein extracts from iron-deficient or iron-loaded mice. In conclusion, our studies indicate that mouse FPN, regardless of the tissue iron status, is glycosylated but not enriched in mannose residues, and that exists mainly in monomeric form. The latter finding may have important implications for understanding the pathogenesis of the disease due to ferroportin mutations.
Blood Cells Mol Dis
PMID:Ferroportin is a monomer in vivo in mice. 1638 Feb 75

The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect alpha-amylases, but not of plant alpha-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, alpha-amylase inhibitor (alphaAl) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies). Purified alphaAl can be resolved by SDS-PAGE into five bands (M(r) 15,000-19,000), four of which have covalently attached glycans. These bands represent glycoforms of two different polypeptides. All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it (LM Hoffman [1984] J Mol Appl Genet 2: 447-453; J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889). The primary translation product of alphaAl is a polypeptide of M(r) 28,000. Immunologically cross-reacting glycopolypeptides of M(r) 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M(r) 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that alphaAl is a typical bean lectin-type protein that is synthesized on the rough endoplasmlc reticulum, modified in the Golgi, and transported to the protein storage vacuoles.
 ...
PMID:Characterization of alpha-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris. 1666 38


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