Search

Prostate cancer has the highest incidence among men in advanced countries, as well as a high mortality rate. Despite the efforts of numerous researchers to identify a gene-based therapeutic target as an effective treatment of prostate cancer, there is still a need for further research. The cathepsin gene family is known to have a close correlation with various cancer types and is highly expressed across these cancer types. This study aimed at investigating the correlation between the cathepsin A (CTSA) gene and prostate cancer. Our findings indicated a significantly elevated level of CTSA gene expression in the tissues of patients with prostate cancer when compared with normal prostate tissues. Furthermore, the knockdown of the CTSA gene in the representative prostate cancer cell lines PC3 and DU145 led to reduced proliferation and a marked reduction in anchorage-independent colony formation, which was shown to be caused by cell cycle arrest in the S phase. In addition, CTSA gene-knockdown prostate cancer cell lines showed a substantial decrease in migration and invasion, as well as a decrease in the marker genes that promote epithelial mesenchymal transition (EMT). Such phenotypic changes in prostate cancer cell lines through CTSA gene suppression were found to be mainly caused by reduced p38 MAPK protein phosphorylation; i.e. the inactivation of the p38 MAPK cell signaling pathway. Tumorigenesis was also found to be inhibited in CTSA gene-knockdown prostate cancer cell lines when a xenograft assay was carried out using Balb/c nude mice, and the p38 MAPK phosphorylation was inhibited in tumor tissues. Thus, the CTSA gene is presumed to play a key role in human prostate cancer tissues through high-level expression, and the suppression of the CTSA gene leads to the inhibition of prostate cancer cell proliferation, colony formation, and metastasis. The mechanism, by which these effects occur, was demonstrated to be the inactivation of the p38 MAPK signaling pathway., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background & Aims: Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. However, the cellular defense mechanisms underlying ALD are not well understood. Recent studies highlighted the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Sorting nexin (SNX)-10 has a regulatory function in endolysosomal trafficking and stabilisation. Here, we investigated the roles of SNX10 in CMA activation and in the pathogenesis of alcohol-induced liver injury and steatosis., Methods: Snx10 knockout (Snx10 KO) mice and their wild-type (WT) littermates fed either the Lieber-DeCarli liquid alcohol diet or a control liquid diet, and primary cultured WT and Snx10 KO hepatocytes stimulated with ethanol, were used as in vivo and in vitro ALD models, respectively. Activation of CMA, liver injury parameters, inflammatory cytokines, oxidative stress and lipid metabolism were measured., Results: Compared with WT littermates, Snx10 KO mice exhibited a significant amelioration in ethanol-induced liver injury and hepatic steatosis. Both in vivo and in vitro studies showed that SNX10 deficiency upregulated lysosome-associated membrane protein type 2A (LAMP-2A) expression and CMA activation, which could be reversed by SNX10 overexpression in vitro. LAMP-2A interference confirmed that the upregulation of Nrf2 and AMPK signalling pathways induced by SNX10 deficiency relied on CMA activation. Pull-down assays revealed an interaction between SNX10 and cathepsin A (CTSA), a key enzyme involved in LAMP-2A degradation. Deficiency in SNX10 inhibited CTSA maturation and increased the stability of LAMP-2A, resulting in an increase in CMA activity., Conclusions: SNX10 controls CMA activity by mediating CTSA maturation, and, thus, has an essential role in alcohol-induced liver injury and steatosis. Our results provide evidence for SNX10 as a potential promising therapeutic target for preventing or ameliorating liver injury in ALD., Lay Summary: Alcoholic liver disease is a major cause of morbidity and mortality worldwide. Recent studies highlight the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Our study reveals that deficiency of sorting nexin (SNX) 10 increases the stability of LAMP-2A by inhibiting cathepsin A maturation, resulting in the increase of CMA activity and, thus, alleviates alcohol-induced liver injury and steatosis., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Accessory sex gland (ASG) secretory proteins of the Chinese mitten crab (Eriocheir sinensis) can effectively digest the spermatophore wall. In order to identify which proteins participate in spermatophore wall digestion, a 50-kDa protein secreted from the ASG was purified to homogeneity by a series of isolation steps, including ammonium sulfate fractionation, Sephadex G-25 S gel-filtration, ion exchange chromatography on a DEAE-Sephacel column and Sephacryl S-200 gel-filtration. The purified protein was effective in spermatophore wall rupture, and the subsequent HPLC-ESI-MS/MS shotgun analysis showed the digestive protein to be cathepsin A (cathA). This finding was also confirmed by Western blot analysis and a cathA inhibitor digestion experiment. ELISA analysis showed that cathA enzymatic activity from ASG secretions increased during its purification process. Furthermore, enzymatic activity was significantly higher in the mating period of E. sinensis parallel to the latest developmental stage of the gland. Moreover, analysis from a cathA inhibitor that inhibits spermatophore wall digestion showed that cathA is the main enzyme involved. Hence, we first report the characterization of cathA from the ASG, which might play a key role in digesting the spermatophore wall of E. sinensis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.

Selective proteolysis is an important regulatory mechanism in all cells. In eukaryotes, this process gains specificity by tagging proteins with the small protein ubiquitin. K48 linked polyubiquitin chains of four and more ubiquitin moieties target proteins for hydrolysis by the proteasome. Prior to degradation the polyubiquitin chain is removed from the protein, cleaved into single units, and recycled. The deubiquitinating enzyme Ubp14 is an important catalyst of this process. Mutants of Ubp14 had been shown to accumulate non-cleaved oligo- and polyubiquitin chains, which resulted in inhibition of overall ubiquitin-proteasome linked proteolysis as well as in inhibition of degradation of some known substrates. Here we show that accumulation of ubiquitin chains due to defective Ubp14 does not uniformly lead to inhibition of ubiquitin-proteasome linked protein degradation. Instead, inhibition of degradation depends on the substrate tested. The results indicate the existence of different paths through which proteins enter the proteasome.

The gene product of the Saccharomyces cerevisiae open reading frame YDR229w (named IVY1 for: Interacting with Vps33p and Ypt7p) was found to interact with both the GTPase Ypt7p and the Sec1-related Vps33 protein. While deletion of IVY1 does not lead to any recognized change in phenotype, overexpression of Ivy1p leads to fragmentation of the vacuole, missorting of the vacuolar enzyme carboxypeptidase Y (CPY) to the exterior of the cell, and an accumulation of multivesicular bodies inside the cell. All effects caused by the overexpression of Ivy1p can be reset by simultaneously raising the amount of Vps33p. This suppression activity of Vps33p suggests that Ivy1p and Vps33p at least partially counteract the action of each other in the cell. The intracellular level of Ivy1p increases in cells approaching stationary growth phase at which part of the protein is located at the rim of the vacuole. In addition to its specific interactions with members of two regulatory protein families, Ivy1p in vitro shows a marked propensity for binding phospholipids with high affinity.

The ER-associated degradation (ERAD) pathway directs ubiquitin-mediated degradation of a variety of ER-associated misfolded and normal proteins. Recent studies have delineated the molecular machinery responsible for protein ubiquitination and highlighted mechanistic questions surrounding the recognition, extraction and proteasomal destruction of the diverse array of ERAD substrates. Consideration of separate lines of work on this versatile pathway now indicate that despite its central role as an avenue of cellular quality control, ERAD is also harnessed for feedback regulation of sterol synthesis, and most likely numerous other cellular processes. These studies give ERAD a larger role in cellular function, and imply that cellular quality-control pathways could be widely employed in both natural and pharmaceutical control of individual proteins.

Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disorder galactosialidosis (GS). PPCA(-/-) mice display a disease course similar to that of severe human GS, resulting in nephropathy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overexpressing the corrective enzyme in either erythroid cells or monocytes/macrophages has proven effective for the improvement of the phenotype, and encouraged the use of genetically modified BM cells for ex vivo gene therapy of GS. Here, we established stable donor hematopoiesis in PPCA(-/-) mice that received hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based, bicistronic retroviral vector overexpressing PPCA and the green fluorescent protein (GFP) marker. We observed complete correction of the disease phenotype in the systemic organs up to 10 months after transplantation. PPCA(+) BM-derived cells were detected in all tissues, with the highest expression in liver, spleen, BM, thymus, and lung. In addition, a lysosomal immunostaining was seen in nonhematopoietic cells, indicating efficient uptake of the corrective protein by these cells and cross-correction. Expression in the brain occurred throughout the parenchyma but was mainly localized on perivascular areas. However, PPCA expression in the central nervous system was apparently sufficient to delay the onset of Purkinje cell degeneration and to correct the ataxia. The long-term expression and internalization of the PPCA by cells of systemic organs and the clear improvement of the neurologic phenotype support the use of this approach for the treatment of GS in humans. (Blood. 2002;99:3169-3178)

The influence of tannic acid in the rate of growth (BWG), feed intake, protein efficiency ratio, and liver proteolytic activities (cathepsin A and D) were measured in growing male chickens. These birds were fed ad libitum over a 15-d experiment on 20% protein standard diets containing heated soybean (control, C) as the main source of protein. Tannic acid (TA; 25 g/kg diet) was added to all diets, except the control. It has been found that in comparison to control-fed birds, TA-fed birds showed a significant reduction (P < 0.01) in BWG, protein efficiency ratio, and relative weight of liver, together with a significant increase (P < 0.01) in the activities of cathepsin A and D in liver. Addition of TA to the control diet had no significant effect on feed intake. The possible nature of these results is discussed.

Tamm-Horsfall protein (THP), also known as uromodulin, is a major glycoprotein synthesized in the kidney. THP is expressed on the luminal surface of the membrane with the glycosyl phosphatidylinositol (GPI) anchor and excreted in urine at a rate of 50-100 mg per day. Although THP is the most abundant urinary protein, the function of THP remains unclear. In addition, little is known about the mechanism by which large amounts of THP are actively released into the urinary fluid. In this study, we examined the C-terminal structure of highly purified THP derived from human urine. Carboxypeptidase Y efficiently degraded urinary THP, indicating that the C-terminal structure of the protein contains an amino acid residue with a free carboxyl moiety. These results are consistent with our previous finding that urinary THP does not bind anti-CRD antibody. We obtained peptides from the complete digestion of urinary THP with lysylendopeptidase. We purified the most C-terminal peptide with p-phenylene diisothiocyanate-controlled pore glass (DITC-CPG) beads. N-terminal sequence analysis indicated the peptide begins with Tyr 520 and ends between E539 and E576. Direct C-terminal amino acid sequencing of highly purified urinary THP gave a sequence of -X-(Q)-G-(R)-F, corresponding to amino acids 544-548, -S-Q-G-R-F. We therefore conclude that urinary THP is generated by a proteolytic cleavage between F548 and S549, 66 amino acids upstream of a possible GPI-anchor attachment site. Because the sequence of THP, including the cleavage site, is highly homologous to that of GP2, a GPI-anchored protein within the pancreas, and both THP and GP2 are abundantly found as soluble forms in the excreted fluids, a common mechanism may exist governing the proteolytic release of GPI-anchored membrane proteins.

The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast., (Copyright 2001 Academic Press.)

We describe a new method for the site-specific incorporation of an extrinsic cysteine to the C-termini of immunoglobulin G (IgG) using carboxypeptidase Y (CPase Y) catalyzed transpeptidation. The transpeptidase activity of CPase Y was employed to attach cysteine esters to the C-termini of the IgG molecule (cysteinylation) at alkaline pH. No CPase Y catalyzed transpeptidation products were found when native IgG was used as the substrate or when cysteine was used as the nucleophile. However, C-terminal labeling occurred when cysteine ethyl ester (CysOEt) or cysteine isobutyl ester (CysOiBu) was used as the nucleophile and IgG methyl ester as the substrate. When CysOiBu was used as the nucleophile, the maximal labeling yield obtained with IgG methyl ester as substrate was 25%, assuming all four C-termini in the IgG molecule were labeled equally. The C-terminal labeling pattern of cysteinylated IgG was determined by autoradiography followed by the integration of radiodensity. It revealed that both the C-termini of the heavy and light chains of IgG methyl ester were labeled with CysOiBu., (Copyright 2000 Academic Press.)

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight "tongue-and-groove" fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens., (Copyright 2000 Academic Press.)

A rapid and sensitive assay for the determination of cathepsin A activity is reported. This method is based on fluorimetric detection of a dansylated peptide, 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe-L-Leu, after separation by high-performance liquid chromatography using a C18 reversed-phase column and isocratic elution. This method is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-L-Phe at concentrations as low as 300 fmol, yields highly reproducible results and requires less than 7.0 min per sample for separation and quantitation. The optimum pH for cathepsin A activity was 4.5-5.0. The Km and Vmax values were respectively 14.9 microM and 27.91 pmol/microg/h with the use of enzyme extract obtained from mouse kidney. Cathepsin A activity was strongly inhibited by Ag+, Hg2+, diisopropylfluorophosphate and p-chloromercuriphenylsulphonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in kidney. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (yPDI) has been analysed by exchanging the active-site cysteine residues for serine residues. The activity of the mutant forms of yPDI was determined quantitatively by following the refolding of bovine pancreatic trypsin inhibitor in vitro. In this assay the activity of the wild-type yPDI is quite similar to that of human PDI, both in rearrangement and oxidation reactions. However, while the a domain active site of the human enzyme is more active than the a'-site, the reverse is the case for yPDI. This prompted us to set up an assay to investigate whether the situation would be different with a native yeast substrate, procarboxypeptidase Y. In this assay, however, the a' domain active site also appeared to be much more potent than the a-site. These results were unexpected, not only because of the difference with human PDI, but also because analysis of folding of procarboxypeptidase Y in vivo had shown the a-site to be most important. We furthermore show that the apparent difference between in vivo and in vitro activities is not due to catalytic contributions from the other PDI homologues found in yeast., (Copyright 1999 Academic Press.)

The 32/20-kDa two-chain form of protective protein/cathepsin A (CathA) secreted by human platelets was thermostable in the aggregation supernatant at acidic pH, but was denatured at neutral pH. Leupeptin partly protected the CathA against denaturation, which was not observed in the supernatant after depletion of the cosecreted lysosomal acid beta-galactosidase (beta-Gal) by affinity separation with p-aminophenylthiogalactose (PATG)-agarose beads even at pH 4.8. The purified recombinant human beta-Gal proteins, the 84-kDa precursor and 64-kDa mature-like enzyme (the tryptic product of the 84-kDa precursor), also protected the CathA against denaturation at neutral pH in part. Biospecific interaction analysis revealed that the CathA secreted by platelets dose dependently bound to the immobilized recombinant beta-Gal proteins. The association rate constant of CathA with the 64-kDa mature-like beta-Gal was 4.0 x 10(6) (M-1 s-1) at acidic pH, which was three times larger than that with the 84-kDa beta-Gal precursor. The calculated affinity constants for the enzyme molecules at acidic pH were approximately 1 x 10(9) (M-1), and those at neutral pH were two orders lower. These results first demonstrated that beta-Gal stabilizes the catalytic activity of CathA through direct binding in vitro. The affinity was shown to increase with removal of the carboxy-terminal domain of the beta-Gal precursor., (Copyright 1998 Academic Press.)

Galactosialidosis is a human autosomal recessive lysosomal storage disease caused by a genetic defect of protective protein/cathepsin A (PPCA). The patients in a Japanese family with the severe early-infantile form of galactosialidosis were revealed to be homozygous for the A1184-G transition in the PPCA gene in both alleles, which leads to the Y395C substitution. The acid carboxypeptidase (cathepsin A) and lysosomal neuraminidase activities were markedly decreased in cultured fibroblasts and chorionic villus cells derived from the patients, although the decrease in beta-galactosidase activity was less. Immunoblot and immunocytochemical analyses showed that neither the precursor nor the mature form of the PPCA gene product was present in the cultured cells. The Y395C mutation was revealed to cause the loss of the translated product, that determines the severity of the clinical phenotype.

The vps (vacuolar protein sorting) mutants have been used to dissect and characterize the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. The vps mutants were isolated through their loss of ability to correctly sort the vacuolar hydrolase CPY, which travels from Golgi membranes to the vacuole through a prevacuolar compartment. Over 50 VPS genes have been divided into 6 classes according to vacuolar morphology. Mutations in any one of the class E VPS genes, such as VPS27, lead to an exaggerated form of the prevacuolar compartment. This class E compartment contains endocytosed proteins as well as proteins en route to the vacuole, and is thus taken to represent an intersection point between the endocytic and biosynthetic pathways. Mutations in the class D gene VPS45 can be used to define a second transport intermediate along the vacuolar biogenesis pathway, Golgi-derived transport vesicles carrying vacuolar membrane proteins on their way to the vacuole. Here we demonstrate that the Sec1p-like protein Vps45p is required for the fusion of Golgi-derived vesicles with the prevacuolar compartment indicating that VPS45 functions before VPS27 in the vacuolar biogenesis pathway. In addition, we show that VPS45 function is not required for the delivery of endocytosed proteins to the prevacuolar compartment from the plasma membrane suggesting that the function of Vps45p is restricted to a single vesicular pathway.

In the present study we describe a novel method for obtaining highly pure carboxypeptidase Y, or derivatives thereof, in a single-step purification procedure. The method is based on affinity chromatography and the results demonstrate that an efficient method is obtained only when the affinity gel is fully saturated with enzyme. Thus, pilot experiments are required to determine the binding capacity of the resin with respect to a given enzyme. To avoid this additional experimental effort, we have developed a method utilizing reversed-flow affinity elution. The method has been successfully employed to purify hundreds of carboxypeptidase Y mutant enzymes.

Sec7 protein (Sec7p) is required for membrane traffic in the yeast secretory pathway. Because Sec7p regulates more than one stage in the pathway, it has been difficult to assign the most proximal requirement for Sec7p action. We have engineered a novel mutant whose Sec7p levels are regulated by growth conditions and by selective protein destabilization according to the N-end rule. Sec7p depletion causes cell growth arrest and accumulation of transport proteins with post-translational modifications indicative of Sec7p dependence for ER-to-Golgi traffic, in addition to the already characterized Golgi requirements. Immuno-EM of sec7 revealed exaggeration of ER and Golgi membranes with protein accumulation in these exaggerated structures, suggesting that these regions may represent staging areas for cargo sorting and vesicle assembly.

To identify new genes whose products act on the endocytic and vacuolar protein sorting pathways, we screened for mutants that display synthetic growth defects with delta ypt51, a mutant impaired in membrane traffic at a point where these pathways intersect. Seven mutants that fell into six different complementation groups were found to fit this criterium. Two of the mutants (ysl1 and ysl2) are new, two others are defective in the VAN1 gene. Mutants in VAN1 were previously identified by their resistance to the drug orthovanadate. The others represent known endocytosis (rvs167 and sac6) and vacuolar protein sorting (vps41) mutants. As suggested by their genetic interactions with delta ypt51, the newly identified mutants are impaired in endocytosis, vacuolar protein sorting and vacuole biogenesis. In addition, van1 and ysl2 display actin cytoskeletal defects.

The fetal diagnosis of galactosialidosis is performed by measuring carboxypeptidase (cathepsin A) activity in cultured villous cells and by immunofluorescence analysis with an antibody against an oligopeptide corresponding to the N-terminal domain of the human mature protective protein. Neither carboxypeptidase activity nor immunofluorescence was detected in cultured villous cells derived from an at-risk fetus or in cultured fibroblasts derived from the sister with galactosialidosis. Neuraminidase and beta-galactosidase activities were also confirmed to be deficient or low. A direct assay system for protective protein/cathepsin A is useful for the accurate prenatal diagnosis of galactosialidosis.

The third multicopy suppressor gene of the PDI1 deletion from Saccharomyces cerevisiae, MPD2, was isolated and characterized. The MPD2 gene encodes a protein with a putative signal sequence, ER retention signal, and a disulfide isomerase active site like sequence. The amino acid sequence around the active site like sequence is similar to the thioredoxin-like domains of PDI and PDI related proteins, although the similarity is comparatively low. A delta-pdi1 strain over-producing Mpd2p showed slow growth and was sensitive to 1 mM dithiothreitol. Mpd2p can be detected in wild type cells and is a glycoprotein. Although the MPD2 gene was not essential for growth, overexpression of the gene partially restored the maturation defect of carboxypeptidase Y caused by the PDI1 deletion. Mutagenesis analysis revealed that Mpd2p can compensate for the loss of PDI with its CXXC sequence.

Lactacystin, the most specific inhibitor of the proteasome, strongly inhibited at pH 5.5 the activity of human platelet lysosomal cathepsin A-like enzyme. At a concentration as low as 1-5 microM it almost completely decreased the hydrolysis rate of cathepsin A specific substrates: Cbz-Phe-Ala and FA-Phe-Phe. This inhibition was probably due to the lactacystin intermediate beta-lactone formed during 10 min hydrolysis at pH 8.0 since nonhydrolyzed inhibitor did not affect cathepsin A activity. Basing on similarities in the inhibitor sensitivity, pH optimum, and substrate preferences it is suggested that the cathepsin A-like activity may be involved in chymotrypsin-like activity of the proteasome.

The ability to carry out specific C-terminal modification or labeling of peptides and proteins has a broad range of applications. It is well established that this may be achieved by protease-catalyzed transacylation reactions and that carboxypeptidase Y (CPD-Y) is suitable for this due to its broad specificity and stability in the presence of denaturants. Furthermore, CPD-Y is characterized by a S'1 binding site that is open to solvent and, thus, capable of catalyzing a transpeptidation reaction with nucleophiles that extend beyond the perimeter of the active site. However, one major drawback with CPD-Y is that the yield of the reaction is highly dependent on the nature of the leaving group; e.g., with large apolar leaving groups the yield of the reaction does not exceed 15%. In the present publication it is demonstrated that mutants of CPD-Y, designed for low leaving group dependence, efficiently incorporate biocytin amide as well as a new fluorescent nucleophile, N'-Abz-Lysine amide (ablysin amide), into peptides and proteins.

Aquaporin-1 (AQP1) is an abundant protein in human erythrocyte membranes which functions as a specific and constitutively active water conducting pore. Solubilized and isolated as tetramer, it forms well-ordered two-dimensional (2D) crystals when reconstituted in the presence of lipids. Several high resolution projection maps of AQP1 have been determined, but information on its three-dimensional (3D) mass distribution is sparse. Here, we present surface reliefs at 0.9 nm resolution that were calculated from freeze-dried unidirectionally metal-shadowed AQP1 crystals as well as surface topographs recorded with the atomic force microscope of native crystals in buffer solution. Our results confirm the 3D map of negatively stained AQP1 crystals, which exhibited tetramers with four major protrusions on one side and a large central cavity on the other side of the membrane. Digestion of AQP1 crystals with carboxypeptidase Y, which cleaves off a 5 kDa intracellular C-terminal fragment, led to a reduction of the major protrusions, suggesting that the central cavity of the tetramer faces the outside of the cell. To interpret the results, sequence based structure predictions served as a guide.

Part of the vacuole in the mother cell of Saccharomyces cerevisiae is segregated early in the cell cycle to establish a new vacuole in the bud. Investigation of the molecular mechanism of vacuolar segregation has previously been limited by the lack of an efficient screen for mutants defective in this process. We developed a new screening procedure based on a cascade for activation of vacuolar proteases. Carboxypeptidase Y (CPY) is activated by proteinase A (PrA). However, upon PrA depletion, CPY continues to be activated, supposedly by self-sustaining proteinase B (PrB) activity that is transferred from one generation to the next generation through vacuolar segregation. In this study fourteen mutants were isolated that failed to sustain CPY activation upon PrA depletion. While these mutants had altered vacuolar protease-activity levels, two mutants showed specific vacuolar segregation defects. They formed large-budded cells that contained no vacuole or extremely small vacuoles in the bud. These mutants represented two complementation groups, named VAC6 and VAC7. The data indicate that constitutive vacuolar segregation mutants are viable, but that they are unable to transfer proteolytic activities from mother vacuole to the bud. Surprisingly, despite the apparent lack of quantitative vacuolar inheritance, all daughter cells of vac6 and vac7 had obtained a vacuole before cell division.

The entire human protective protein gene has been cloned, and structural analysis revealed that the gene spans 7.5kb and comprises 15 exons. Furthermore, it partially overlaps on the opposite strand with the gene encoding phospholipid transfer protein. This region of DNA on chromosome 20 appears to encode two distinct mRNAs expressing defined functional products, and the mRNAs overlap by 58 nucleotides at their 3'-untranslated ends.

A rapid and sensitive assay method for the determination of deamidase activity is reported. This method is based on fluorometric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val (N-Dns-D-Tyr-Val), enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr-Val-NH2 (N-Dns-D-Tyr-Val-NH2), after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure N-Dns-D-Tyr-Val at concentrations as low as 100 fmol, yields highly reproducible results and requires less than 8.5 min per sample for separation and quantitation. The optimum pH for deamidase activity was 4.0-4.5. Greater than 5 mM of reduced glutathione was needed for maximal enzyme activity. The Km and Vmax values were respectively 125 microM and 14.12 pmol/micrograms/h with the use of enzyme extract obtained from mouse spleen. Deamidase activity was strongly inhibited by Ag+, Cu2+, diisopropylfluorophosphate, and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in spleen. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this enzyme.

We describe a method to determine the substrate specificity of human lysosomal carboxypeptidase, cathepsin A/protective protein, using furylacryloyl (FA)-Phe-X dipeptides as substrates. These dipeptides contain a chromophore which allows continuous spectrophotometric assay at wavelengths above 324 nm with little interference from protein absorbance. The results obtained with cathepsin A purified from human placenta demonstrate that the enzyme has the highest affinity for substrates with large hydrophobic (Phe, Leu) or positively charged (Arg) amino acid residues in P1' position. The three substrates (FA-Phe-Phe, FA-Phe-Leu, and FA-Phe-Ala) which demonstrated the highest specificity (kcat/Km) for the purified enzyme were then used to assay cathepsin A activity in cultured skin fibroblasts from patients affected with galactosialidosis, an inherited lysosomal storage disease caused by the genetic deficiency of cathepsin A. Residual cathepsin A activity in galactosialidosis fibroblasts was lower than 6% of controls, indicating the high specificity of the assay method.

The C-terminal region of the chaperone-like pro-sequence (py) of yeast carboxypeptidase Y (CPY) is suggested to be crucial for the folding of mature CPY. In order to study the influence of hydrophobic residues in this domain, a set of mutations have been introduced in py. Unexpectedly, only small amounts of CPY precursors are expressed when Leu108, at the C-terminal end of py, is substituted for polar residues Lys, Arg or Asp. In contrast, substitution with hydrophobic residues Val, Ile or Ala permit normal expression. Interestingly, the poorly expressed molecules are coreglycosylated, implying that they have failed to leave the endoplasmic reticulum (ER). The ER-retained molecules caused an induction in the levels of BiP, signifying that polar substitutions at position 108 of pre-py-CPY induce misfolding. Quite surprisingly, a reporter gene, linked to concatamerized unfolded-protein-response elements, reveals that py-mediated misfolding of CPY is not really identical in all mutants. This shows that a simple transcriptional assay can assess the subtleties of pro-sequence mediated protein folding in an eukaryotic cell.

The hydrolytic cleavage of a cyanine (Cy3)-labeled angiotensin, catalyzed by various proteases, was studied by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The end-labeled peptides and the Cy3 diacid internal standard were separated on a 20-microns x 27-cm capillary with LIF detection (emission, 580 nm) using a frequency-doubled solid-state diode laser emitting at 532 nm or a He-Ne laser emitting at 543 nm. Hydrolysis of the Cy3-labeled angiotensin I, catalyzed by proteinase K, is a sequential process beginning from the C-terminal of the peptide, instead of from random cleavages. Trypsin catalyzes a specific cleavage of Cy3-angiotensin I to Cy3-Asp-Arg as anticipated. Using a combination of endopeptidase and carboxypeptidases, the remnant of the labeled species was characterized by CE-LIF. The method provides a general tool for studying the mechanism of protease-catalyzed hydrolysis of peptide.

We raised two polyclonal antibodies against synthetic oligopeptides comprising amino acid sequences in the human lysosomal protective protein. The first antibody recognized the 54-kDa precursor and the N-terminal sequence of the 32-kDa mature protein subunit, and the second one recognized the precursor and the C-terminal sequence of the 20-kDa subunit. In normal fibroblasts, mature protective protein was detected on immunoblotting with these antibodies. Considerable amounts of mature protective protein also were detected in kidney, lung, liver, and spleen, but not in brain from a patient with Gaucher disease. Neither the precursor nor the mature protective protein was detected in cultured fibroblasts, liver or cerebrum from a galactosialidosis patient with protective protein deficiency.

Cathepsin A (also named "protective protein" and carboxypeptidase L) stabilizes beta-galactosidase and activates neuraminidase by forming with them a high-molecular-weight lysosomal complex. We determined the main forms of the supramolecular organization of human placental cathepsin A and the quantitative relationship between them, using an affinity chromatography on agarose-Phe-Leu for direct purification of cathepsin A. We found that cathepsin A in human placenta exists as the following three forms: a 1270-kDa complex with beta-galactosidase and neuraminidase (about 1% of total cathepsin A), a 680-kDa complex with beta-galactosidase (30-40% of total), and a free 98-kDa cathepsin A dimer (60-70% of total). All forms are in dynamic equilibrium with each other, but almost all placental beta-galactosidase is associated with cathepsin A in the 680-kDa complex. The main properties of free cathepsin A (including the capacity to associate with beta-galactosidase) were found to be identical to those of cathepsin A obtained by dissociation of the 680-kDa complex. The presence of a free cathepsin A pool in the lysosome is connected with its sixfold overproduction in the cell compared to beta-galactosidase and may be necessary to ensure cathepsin A proteolytic function in addition to its protective role for beta-galactosidase and neuraminidase in the lysosomal multienzymatic complex. Such a dual function of cathepsin A is also confirmed by our finding that it is the only carboxypeptidase of placenta extract able to catalyze the hydrolysis of both carbobenzoxy (CBZ)-Glu-Tyr and CBZ-Phe-Leu dipeptide substrates.

We propose a method to study multienzyme complex formation in vitro based on nondenaturing agarose gel electrophoresis. The enzymes with different isoelectric points (pI) were loaded at the opposite ends of the same lane of agarose gel and electrophoresis was performed at a pH value intermediate between their pI's. In cases where a complex of the enzymes was formed, an additional protein band of low electrophoretic mobility was found corresponding to the point where they crossed on the gel. This band contained both enzyme activities. The method was used to demonstrate association between two enzymes of the mitochondrial citric acid cycle, malate dehydrogenase and citrate synthase, and between the lysosomal hydrolases, beta-galactosidase and cathepsin A. Relative proportions of free and bound enzymes after electrophoresis suggest that interaction between the mitochondrial enzymes is relatively weak compared to that of lysosomal hydrolases. Microdensitometric scanning of countermigration electrophoresis gels was used to determine the stoichiometry of components in the complex.

Cathepsin A (EC 3.4.16.1), a lysosomal carboxypeptidase, has been purified 1374-fold from pig kidney. Purification steps included concanavalin A-Sepharose and phenyl-Sepharose chromatography and chromatofocusing. The specific activity (16.9 U/mg) of the purified enzyme was significantly higher than previously reported values. The enzyme preparation appeared homogeneous when analyzed by non-denaturing polyacrylamide gel electrophoresis and was free of detectable protease contamination. The molecular mass (M(r) = 97,000), isoelectric point (5.0), and sensitivity to inhibitors were consistent with reported properties of cathepsin A. However, the previously reported three-peptide chain structure was not observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol demonstrated that the enzyme is composed of two M(r) 47,000 subunits, each of which dissociate in the presence of 2-mercaptoethanol into two polypeptide chains of 19,000 and 31,000.

A simple, rapid method of epitope mapping has been developed that avoids the often cumbersome requirement of obtaining amino acid sequence information. The protein antigen is digested with various concentrations of carboxypeptidase into a nearly continuous series of polypeptides of different molecular weights, all containing a common N-terminus. The peptides are separated by polyacrylamide gel electrophoresis and then transferred to nitrocellulose paper. After developing the blot with the antibody to be mapped, a nearly continuous stain is observed extending from the position of the intact antigen to the molecular weight of the smallest N-terminal fragment still containing the antibody's epitope. By noting the molecular weight where the stain terminates, the position of the epitope relative to the N-terminus can be determined. Using this methodology, and taking special precautions to inhibit all endoproteinases in the carboxypeptidase preparation, the previously mapped epitopes of six nonoverlapping antibodies to the erythrocyte anion transporter were confirmed.