Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have expressed in COS-1 cells mutants of neprilysin (neutral endopeptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was either substituted for V42-T-M-I or inserted after T38 in the signal peptide/membrane anchor (SA) domain. These mutations were introduced in full-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP delta cyto(H1) and NEP delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP delta cyto(H1), NEP(H2), and NEP delta cyto(H2) were secreted. Furthermore, carbonate treatment of isolated intracellular membranes suggested that cleavage of the SA domain was performed in the endoplasmic reticulum, presumably by signal peptidase. Sequencing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala46 but also at the carboxy side of Ala41 in NEP(H2) and NEP delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of the cleavage site and, in the case of NEP(H1) and NEP(H2), the efficiency of cleavage of the SA domain.
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PMID:Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion. 798 81

It was previously shown that estrogen induces a membrane glycoprotein (molecular mass, 95 kDa) in the chicken oviducts, which exhibits several properties similar to transferrin receptors (Poola, I., and Lucas, J. J. (1988) J. Biol. Chem. 263, 19137-19146). In the present study, we have further investigated its molecular and transferrin binding properties. We have sequenced several internal peptides isolated from the purified protein by endopeptidase Lys-C. We have found that it has a high degree of sequence homologies with those of chicken heat-shock protein (cHsp108), mouse endoplasmic reticulum protein (mERp99), hamster glucose-regulated protein (hagrp94), and human tumor rejection antigen (hTRAgp96), all of which are shown to be highly homologous to each other and to yeast hsp90. We demonstrate here that the [35S]methionine-labeled immunoaffinity-purified estrogen-inducible membrane glycoprotein binds to the transferrin affinity columns similar to iron-modulated transferrin receptors. Indirect immunofluorescence microscopic studies indicate that it is an intracellular glycoprotein unlike transferrin receptors. We have isolated two molecular forms of the protein, with molecular masses of 116 and 104 kDa, by immunoaffinity column purification, immunoprecipitation, Western blotting, and pulse-chase labeling analyses. Both 116-and 104-kDa species bind transferrin. This protein can be induced by heat-shocking the oviduct cells at 45 degrees C for 3h and recovering at 37 degrees C for 2-3 h. It is also expressed in the human breast cancer cell lines, MCF-7 and T-47D. All these properties taken together strongly suggest that the estrogen-inducible membrane glycoprotein is a novel transferrin-binding protein, structurally related to the stress-regulated proteins.
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PMID:The estrogen-inducible transferrin receptor-like membrane glycoprotein is related to stress-regulated proteins. 806 20

A processing protease for the human immunodeficiency virus type I (HIV-I) envelope glycoprotein gp160 precursor has been purified to homogeneity from the post-nuclear membrane fraction of a human T4+ lymphocyte clone. Most of the processing activity was found to be present in the fractions of endoplasmic reticulum and Golgi apparatus of the cells. The purified enzyme has a monomeric structure with a molecular mass of 26 +/- 3 kDa, as judged by gel-permeation liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The purified enzyme converted gp160 to gp120 and gp41, showing a pH optimum of 6.5-7.0. Direct amino acid sequencing of the amino terminus of the product gp41 revealed that the cleavage site of gp160 was between Arg511 and Ala512. The enzyme activity was inhibited by trypsin-type protease inhibitors, but was not affected by CaCl2, MgCl2 or chelating agents. The properties of the purified enzyme are clearly distinct from those of processing proteases reported previously. Although the significance of the enzyme in vivo is not currently certain, judging from its cleavage specificity and subcellular localization, this endopeptidase appears to be a processing enzyme for the human immunodeficiency virus type I gp160 precursor protein in human T cells.
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PMID:Processing protease for gp160 human immunodeficiency virus type I envelope glycoprotein precursor in human T4+ lymphocytes. Purification and characterization. 809 9

Rabbit neutral endopeptidase-24.11 is a type II transmembrane protein with a 27-amino acid residue positively charged NH2-terminal cytoplasmic domain, a 23-amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. In order to study the mechanism of membrane anchoring of neutral endopeptidase-24.11, we created mutants in which the cytoplasmic tail was deleted. Expression of these mutants in COS-1 cells resulted in the secretion of approximately 10-20% of the protein into the culture medium, due possibly to the cleavage of part or all of the signal peptide/membrane anchor domain by the rough endoplasmic reticulum signal peptidase. In a second set of mutants, a hydrophilic sequence (GSQNS) was inserted midway in the signal peptide/membrane anchor domain of neutral endopeptidase-24.11. When this hydrophilic sequence was introduced into the full-length neutral endopeptidase-24.11, approximately 20% of the enzyme activity was recovered in the culture medium. This proportion increased to 93% when the cytosolic tail was deleted. Sequencing of the [3H]tyrosine- or [3H]isoleucine-labeled secreted protein indicated that proteolysis, possibly by signal peptidase, occurred on the COOH-terminal side of the signal peptide/membrane anchor domain. We conclude that the efficient cleavage of the signal peptide/membrane anchor domain and secretion of the protein require both the deletion of the cytosolic domain and the presence of a hydrophilic sequence.
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PMID:Transformation of the signal peptide/membrane anchor domain of a type II transmembrane protein into a cleavable signal peptide. 842 44

Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has alpha-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268. 2699-2704; Yang. Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382-386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the alpha-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have alpha-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-I cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (I) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) alpha-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the alpha-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulse chase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.
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PMID:Secretion of a type II integral membrane protein induced by mutation of the transmembrane segment. 907 81

A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.
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PMID:A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein. 908 76

Human obesity has an inherited component, but in contrast to rodent obesity, precise genetic defects have yet to be defined. A mutation of carboxypeptidase E (CPE), an enzyme active in the processing and sorting of prohormones, causes obesity in the fat/fat mouse. We have previously described a women with extreme childhood obesity (Fig. 1), abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism and elevated plasma proinsulin and pro-opiomelanocortin (POMC) concentrations but a very low insulin level, suggestive of a defective prohormone processing by the endopeptidase, prohormone convertase 1 (PC1; ref. 4). We now report this proband to be a compound heterozygote for mutations in PC1. Gly-->Arg483 prevents processing of proPC1 and leads to its retention in the endoplasmic reticulum (ER). A-->C+4 of the intro-5 donor splice site causes skipping of exon 5 leading to loss of 26 residues, a frameshift and creation of a premature stop codon within the catalytic domain. PC1 acts proximally to CPE in the pathway of post-translational processing of prohormones and neuropeptides. In view of the similarity between the proband and the fat/fat mouse phenotype, we infer that molecular defects in prohormone conversion may represent a generic mechanism for obesity, common to humans and rodents.
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PMID:Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene. 920 82

To understand the mechanism of the maturation of various proteins in protein-storage vacuoles, we purified a 48-kDa aspartic endopeptidase composed of 32-kDa and 16-kDa subunits from castor bean. Immunocytochemical and cell fractionation analyses of the endosperm of maturing castor bean seed showed that the aspartic endopeptidase was localized in the matrix of the protein-storage vacuoles, where a variety of seed storage proteins were also present. The amount of the aspartic endopeptidase increased at the mid-maturation stage of the seeds before accumulation of the storage proteins. To determine how the aspartic endopeptidase is responsible for maturation of seed proteins in concert with the vacuolar processing enzyme, we prepared 35S-labeled proproteins of seed proteins from the endoplasmic reticulum fraction of pulse-labeled maturing endosperm and used the authentic proproteins as substrates for in vitro processing experiments. The purified aspartic endopeptidase was unable to convert any of three endosperm proproteins, pro2S albumin, proglobulin, and proricin, into their mature sizes, while the purified vacuolar processing enzyme could convert all three proproteins. We further examined the activity of aspartic endopeptidase on the cleavage of an internal propeptide of Arabidopsis pro2S albumin, which is known to be removed post-translationally. The aspartic endopeptidase cleaved the propeptide at three sites under acidic conditions. These results suggest that aspartic endopeptidase cannot directly convert pro2S albumin into the mature form, but it may play a role in trimming the C-terminal propeptides from the subunits that are produced by the action of the vacuolar processing enzyme.
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PMID:An aspartic endopeptidase is involved in the breakdown of propeptides of storage proteins in protein-storage vacuoles of plants. 921 Apr 75

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene in Cpe(fat)/Cpe(fat) mice affects proinsulin processing. Cell lines derived from the pancreatic beta-cells of Cpe(fat)/Cpe(fat) mice were generated by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD mice expressing the simian virus 40 large T oncogene under the control of the rat insulin II promoter. Two cell lines, designated NIT-2 and NIT-3, were cultured from adenomatous islets obtained from F2 littermates and were compared with the NIT-1 cell line previously developed from mice with wild-type CPE. Electron microscopy of the cultured NIT-2 and -3 cells showed increased numbers of enlarged and electron-lucent granules compared with NIT-1 cells. Pro-CPE, but not the mature form of CPE, is present in NIT-2 and -3 cells, and neither pro-CPE nor CPE are secreted into the medium. Immunocytochemistry shows the pro-CPE to be localized to an endoplasmic reticulum-like structure in NIT-3 cells. Proinsulin is less extensively processed in NIT-2 and -3 cells than in NIT-1 cells, indicating that the Cpe(fat) mutation affects both the endopeptidase and carboxypeptidase reactions. The secretion of insulin/proinsulin from NIT-2 and -3 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.
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PMID:Beta-cell lines derived from transgenic Cpe(fat)/Cpe(fat) mice are defective in carboxypeptidase E and proinsulin processing. 934 19

The mammalian endopeptidase furin is a type 1 integral membrane protein that is predominantly localized to the TGN and is degraded in lysosomes with a t1/2 = 2-4 h. Whereas the localization of furin to the TGN is largely mediated by sorting signals in the cytosolic tail of the protein, we show here that targeting of furin to lysosomes is a function of the luminal domain of the protein. Inhibition of lysosomal degradation results in the accumulation of high molecular weight aggregates of furin; aggregation is also dependent on the luminal domain of furin. Temperature and pharmacologic manipulations suggest that furin aggregation occurs in the TGN and thus precedes delivery to lysosomes. These findings are consistent with a model in which furin becomes progressively aggregated in the TGN, an event that leads to its transport to lysosomes. Our observations indicate that changes in the aggregation state of luminal domains can be potent determinants of biosynthetic targeting to lysosomes and suggest the possible existence of quality control mechanisms for disposal of aggregated proteins in compartments of the secretory pathway other than the endoplasmic reticulum.
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PMID:Aggregation as a determinant of protein fate in post-Golgi compartments: role of the luminal domain of furin in lysosomal targeting. 941 68


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