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1.
Bimodal targeting of the endoplasmic reticular protein, cytochrome P4501A1 (CYP1A1), to mitochondria involves activation of a cryptic mitochondrial targeting signal through endoprotease processing of the protein. Here, we characterized the endoprotease that regulates mitochondrial targeting of CYP1A1. The endoprotease, which was induced by beta-naphthoflavone, was a dimer of 90 kDa and 40 kDa subunits, each containing Ser protease domains. The purified protease processed CYP1A1 in a sequence-specific manner, leading to its mitochondrial import. The glucocorticoid receptor, retinoid X receptor, and p53 underwent similar processing-coupled mitochondrial transport. The inducible 90 kDa subunit was a limiting factor in many cells and some tissues and, thus, regulates the mitochondrial levels of these proteins. A number of other mitochondria-associated proteins with noncanonical targeting signals may also be substrates of this endoprotease. Our results describe a new mechanism of mitochondrial protein import that requires an inducible cytoplasmic endoprotease for activation of cryptic mitochondrial targeting signals. 相似文献
2.
Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than half to serine proteases, with a minor role of metalloproteases. Treatment of isolated tepals with the purported serine protease inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] or DFP (diisopropyl-fluorophosphate) prevented the increase in endoprotease activity and considerably delayed or prevented the normal senescence symptoms. The specific cysteine protease-specific E-64d reduced maximum endoprotease activity by 30%, but had no effect on the time to visible senescence. Zinc chloride and aprotinin reduced maximum endoprotease activity by c. 50 and 40%, respectively, and slightly delayed visible senescence. A proteasome inhibitor (Z-leu-leu-Nva-H) slightly delayed tepal senescence, which indicates that protein degradation in the proteasome may play a role in induction of the visible senescence symptoms. It is concluded that visible senescence is preceded by large-scale protein degradation, which is apparently mainly due to cysteine- and serine protease activity, and that two (unspecific) inhibitors of serine proteases considerably delay the senescence symptoms. 相似文献
3.
The nuclear lamina protein, lamin A is produced by proteolytic cleavage of a 74 kDa precursor protein, prelamin A. The conversion of this precursor to mature lamin A is mediated by a specific endoprotease, prelamin A endoprotease. Subnuclear fractionation indicates that the prelamin A endoprotease is localized at the nuclear membrane. The enzyme appears to be an integral membrane protein, as it can only be removed from the nuclear envelope with detergent. It is effectively solubilized by the detergent n-octyl-beta-D-glucopyranoside and can be partially-purified (approximately 1200-fold) by size exclusion and cation exchange (Mono S) chromatography. Prelamin A endoprotease from HeLa cells was eluted from Mono S with 0.3 M sodium chloride as a single peak of activity. SDS-PAGE analysis of this prelamin A endoprotease preparation shows that it contains one major polypeptide at 65 kDa and smaller amounts of a second 68 kDa polypeptide. Inhibition of the enzyme activity in this preparation by specific serine protease inhibitors is consistent with the enzyme being a serine protease. 相似文献
4.
Ring cleavage of atrial natriuretic peptide (ANF) between Cys7 and Phe8 by endoprotease 3.4.24.11 yields X-ANF. Since endoprotease 3.4.24.11 may contribute to ANF metabolism in vivo, the present study determined if X-ANF exhibits reduced biological activity in comparison to the parent molecule. 相似文献
5.
A neutral metallo endoprotease involved in the processing of an F1-ATPase subunit precursor in mitochondria 总被引:31,自引:0,他引:31
A post-translational processing assay of the precursor to the yeast F1-ATPase subunit has been utilized to examine a mitochondrial endoprotease which cleaves this subunit precursor to the size of a mature subunit. The endoprotease is extracted from purified mitochondria as a soluble complex of Mr = 115,000 which is composed of subunits of lower molecular weight when examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibits a pH optimum of between pH 7 and 8 and is inactive at pH 6.5 and below. The mitochondrial endoprotease is insensitive to serine esterase inhibitors, but is inhibited by EDTA and o-phenanthroline. Restoration of precursor subunit processing activity in the presence of metal chelators is strictly dependent on excess Co2+ and Mn2+ over other heavy metals examined. These and additional data indicate that this soluble metallo endoprotease is involved in the processing of other cytoplasmically synthesized precursor subunits of the ATPase complex in addition to the subunit 2 precursor. The role of this processing enzyme in the assembly of mitochondrial inner membrane complexes is discussed in light of the current model of mitochondrial biogenesis. 相似文献
6.
Protease activity associated with HeLa cell ribosomes 总被引:1,自引:0,他引:1
B D Korant 《Biochemical and biophysical research communications》1977,74(3):926-933
HeLa cells contain endoprotease activity, detected by a sensitive, solid phase assay. The endoprotease has the ability to cleave a variety of protein substrates and is trypsin-like in its sensitivity to inhibitors. The activity is in part associated with cellular ribosomes and polysomes. A variety of biological and physical-chemical treatments which alter ribosomes or protein synthesis also directly affect the ribosomal protease activity. 相似文献
7.
An enriched preparation of neurosecretory granules from bovine pituitary neural lobes was used as a source of processing enzymes possibly involved in the cleavage of the proocytocin/neurophysin precursor. A synthetic eicosapeptide reproducing the entire (1-20) sequence of the NH2-terminal domain of the bovine ocytocin/neurophysin precursor was used as a substrate to monitor an endoprotease activity cleaving at the Lys11-Arg12 doublet. The 58-kDa endoprotease detected in the lysate of neurohypophyseal granules produced a single cleavage, after the doublet, at the Arg12-Ala13 peptide bond. This endoprotease with pHi 6.9 and 7.2 exhibits maximal activity at pH around neutrality (7.0) and was strongly inhibited by divalent cation chelating agents [ethylenediaminetetraacetic acid and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',-N'-tetraacetic acid] and to some extent by p-(chloromercuri)benzoate and p-(chloromercuri)benzenesulfonic acid, while phenylmethanesulfonyl fluoride and pepstatin were not active. This endoprotease action was sensitive to any modification of the substrate at either basic amino acid of the doublet since replacement of either L-Lys11 or L-Arg12 by D-Lys or D-Arg and by L-Nle abolished the cleavage reaction. In contrast, reversal of the polarity of the doublet in [Arg11,Lys12]proocytocin/neurophysin(1-20) had no effect on the mode of endoproteolytic cleavage as well as modifications of Gly10 (replaced by Ala10). It is concluded that the selectivity of this endoprotease, which may be involved in the primary event occurring in proocytocin/neurophysin processing, is strictly dependent upon the integrity of the basic doublet but that other parameters determined by the amino acid sequence around this doublet may play an important role. 相似文献
8.
Stepniak D Spaenij-Dekking L Mitea C Moester M de Ru A Baak-Pablo R van Veelen P Edens L Koning F 《American journal of physiology. Gastrointestinal and liver physiology》2006,291(4):G621-G629
Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients. 相似文献
9.
Multiple selenocysteine content of selenoprotein P in rats 总被引:2,自引:0,他引:2
Partially purified selenoprotein P from rat plasma was digested with either trypsin, endoprotease Lys-C, or endoprotease Arg-C and analyzed by high pressure liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several 75Se-labeled peptides were detected. The moles of selenium in selenoprotein P were estimated based on the 75Se content of the 75Se-labeled peptide fragments. Using this method, selenoprotein P was shown to contain approximately 9 moles of selenium. This is the first report of a selenoprotein containing more than one selenium per polypeptide. These findings support the proposed function of this protein in selenium transport. 相似文献
10.
Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis 总被引:19,自引:0,他引:19
Furin is a secretory pathway endoprotease that catalyses the maturation of a strikingly diverse group of proprotein substrates, ranging from growth factors and receptors to pathogen proteins, in multiple compartments within the trans-Golgi network (TGN)/endosomal system. This review focuses on recent developments in the biochemistry and cell biology of the endoprotease, including the mechanism of TGN localization, phosphorylation-dependent regulation of protein traffic, and novel insights into early embryogenesis, extracellular matrix formation and pathogen virulence. 相似文献
11.
Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. 总被引:13,自引:0,他引:13
We have recently shown that furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, is involved in precursor cleavage at sites marked by the Arg-X-Lys/Arg-Arg motif within the constitutive secretory pathway. In this study, we analyzed molecular and enzymatic properties of furin expressed in Chinese hamster ovary cells using gene transfer techniques. COOH-terminal truncation analyses indicate that the polypeptide region significantly conserved among the Kex2 family members is required for the endoprotease activity of furin, while the COOH-terminal unconserved region containing the Cys-rich domain and the transmembrane domain is dispensable. A mutant of furin truncated up to the transmembrane domain from the COOH-terminus was secreted into the culture medium as an active form. The sequence requirements for precursor cleavage of this truncated furin determined in vitro were similar to those of wild-type furin determined by expression studies in cultured cells. It had a strong resemblance to the Kex2 protease in the inhibitor profile and pH dependency. These observations support the notion that furin is the endogenous endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. 相似文献
12.
The selective processing activity which generates both the NH2- and COOH-terminal fragments of the octacosapeptide somatostatin-28 (S-28) was investigated. Separation into two distinct proteolytic activities was achieved by ion-exchange chromatography. An endoprotease cleaving either the substrate Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr-NH2, i.e. [Ala17,Tyr20]S-28-(10-20)-NH2 (peptide I), or the octacosapeptide somatostatin-28, on the NH2 side of the Arg-Lys doublet was separated from an aminopeptidase B-like activity. Whereas the endoprotease cleaves a single peptide bond, between Glu12 and Arg13 of S-28, the aminopeptidase B-like enzyme removes both Arg13 and Lys14 stepwise from the NH2 terminus of the corresponding COOH-terminal fragment. This endoprotease activity peaks around pH 8.5, whereas the optimal aminopeptidase B-like activity is in the pH range 6.2-8.5. Combination of both enzymes resulted in the recovery of the overall S-28 convertase activity with an optimal pH at 7. In addition, this endoprotease appears to be very sensitive to divalent cations since it is strongly inhibited by chelating agents. The use of selectively modified undecapeptides derived from the reference substrate peptide I by a single modification of the amino acids Glu12, Arg13, and Lys14 at the cleavage locus showed that both basic residues are critically important, whereas Glu12 is not. It is proposed that S-28 processing involves a divalent cation-sensitive endoprotease that is sensitive to thiol reagents, which cleaves before the Arg-Lys doublet, which is not trypsin-like, and whose action is coupled to an aminopeptidase B-like enzyme. 相似文献
13.
We developed a sensitive fluorometric assay to study in vitro fusion between early endosomes isolated from the human hepatoma, Hep G2. Biochemical characterization of this assay showed that fusion between endosomal vesicles was dependent on physiologic temperature, cytosol, and ATP. Fusion was inhibited by pretreatment of vesicles and cytosol with either 1 mM N-ethylmaleimide or 20 microM GTP gamma S. Neither 3 mM ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid nor 1 mM CaCl2 significantly affected fusion. In addition, ATP gamma S neither inhibited fusion at 50 microM nor supported fusion at 5 mM. To further our understanding of the factors regulating fusion, inhibitors of endoprotease activity and phosphotyrosine phosphatase activity were assayed for their effect on fusion. The dipeptide inhibitor of endoprotease activity, Cbz-gly-phe-amide, inhibited fusion 70% at 3 mM whereas a dipeptide analogue, Cbz-gly-gly-amide, was without effect. Furthermore, orthovanadate, an inhibitor of phosphotyrosine phosphatase activity, stimulated fusion twofold at 0.5 mM. These results suggest that both tyrosine dephosphorylation and endoprotease activity contribute to the regulation of endosome fusion. 相似文献
14.
Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation. 总被引:16,自引:2,他引:14
The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway. 相似文献
15.
An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease. 相似文献
16.
Endoprotease in Plasmodium yoelii nigeriensis 总被引:2,自引:0,他引:2
E Aissi P Charet S Bouquelet J Biguet 《Comparative biochemistry and physiology. B, Comparative biochemistry》1983,74(3):559-566
1. P. yoelli nigeriensis has an acid endoprotease (cathepsin D) and an endoarylamidase. 2. The acid endoprotease is specific towards haemogloblin. It is found in 2 molecular forms, of molecular weight 100,000 and 50,000. It is inhibited by hematin and pepsatin. 3. In mouse normal red blood cells we find an acid protease having physico-chemical properties similar to those of the enzyme present in P. yoelii nigeriensis extracts, except as regards the pHi. 4. In parasite extracts there exists an enzyme active on the synthesis substrate N-acetyl alanine 4 nitro anilide. The main properties of this enzyme have been determined. 5. This enzyme must be also involved in the mechanism of haemoglobin degradation. 相似文献
17.
Helmerhorst EJ Sun X Salih E Oppenheim FG 《The Journal of biological chemistry》2008,283(29):19957-19966
The nonsterile environment of the oral cavity facilitates substantial proteolytic processing, not only of resident salivary proteins but also of dietary proteins. To gain insight into whole saliva enzymatic processes, the in vivo generated peptides in this oral fluid were subjected to nano-flow liquid chromatography electrospray ionization tandem mass spectrometry. The 182 peptides identified were predominantly derived from acidic and basic proline-rich proteins, statherin, and histatins. The proteolytic cleavages in the basic proline-rich proteins occurred preferentially after a Gln residue with predominant specificity for the tripeptide Xaa-Pro-Gln, where Xaa in the P(3) position was mostly represented by Lys. Using the synthetic substrates Lys-Pro-Gln-pNA and Gly-Gly-Gln-pNA, the overall K(m) values were determined to be 97 +/- 7.7 and 611 +/- 28 microm, respectively, confirming glutamine endoprotease activity in whole saliva and the influence of the amino acids in positions P(2) and P(3) on protease recognition. The pH optimum of Lys-Pro-Gln-pNA hydrolysis was 7.0, and the activity was most effectively inhibited by antipain and 4-(2-aminoethyl) benzenesulfonyl fluoride, was metal ion-dependent, and not inhibited by cysteine protease inhibitors. A systematic evaluation of enzyme activities in various exocrine and nonexocrine contributors to whole saliva revealed that the glutamine endoprotease is derived from dental plaque and likely microbial in origin. The P(1) site being occupied by a Gln residue is a nonarchetype with respect to known proteases and indicates the presence of novel glutamine-specific endoprotease(s) in oral fluid. 相似文献
18.
Structure-function relationship studies were conducted on the proocytocin/neurophysin endoprotease previously characterized in both bovine neurohypophyseal and corpus luteum granules, using as a reference substrate a synthetic peptide reproducing the entire (1-20) NH2-terminal domain of the precursor. The [D-Arg12] derivative of proocytocin/neurophysin (1-20) was found to be a good competitive inhibitor of the enzyme (Ki = 30 microM), while the [D-Lys11] derivative was not. This allowed the complete purification of two isoforms of the endoprotease (Mr 58,000 and 52,000, respectively) by affinity chromatography using covalently immobilized [D-Arg12] proocytocin/neurophysin (1-20) as the affinity adsorbent. The use of selectively modified or truncated forms of the reference substrate or of the [D-Arg12] competitive inhibitor of the endoprotease established clearly that this basic pair specific convertase is sensitive to modification of the substrate structure either at the basic residues of the cleavage locus or at amino acids around this site (i.e., Pro7 and Gly9). It is concluded that longer distance interactions between amino acids situated on both the NH2 and COOH sides of the basic doublet Lys11Arg12 may contribute to the stabilization of a preferred substrate conformation allowing recognition by the enzyme subsites. 相似文献
19.
Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA. 总被引:16,自引:0,他引:16
Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s). 相似文献
20.
Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. 总被引:36,自引:0,他引:36
S S Molloy P A Bresnahan S H Leppla K R Klimpel G Thomas 《The Journal of biological chemistry》1992,267(23):16396-16402
Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA. 相似文献