A continuous and pulsatile flow circulation system for evaluation of cardiovascular devices

In a previous report we suggested that muscle fibers in distal myopathy with rimmed vacuoles (DMRV) were degraded by both lysosomal proteolysis (cathepsins) and Ca2+-dependent, nonlysosomal proteolysis (calpain). Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, we examined the role of the ATP-ubiquitin-dependent proteolytic pathway (proteasomes) in myofiber degradation in this myopathy. Immunohistochemically, proteasomes (26S) were located in the cytoplasm in normal human muscle, but the staining intensity was weak. Quantitative analysis showed more reactivity for proteasomes in DMRV muscles and, to a lesser extent, in muscles from muscular dystrophy, polymyositis, and amyotrophic lateral sclerosis patients. In DMRV, proteasomes often were located within or on the rim of rimmed vacuoles, and in the cytoplasm of atrophic fibers. Ubiquitin accumulation was marked within rimmed vacuoles and was seen less extensively in the cytoplasm of atrophic fibers. The latter proteins colocalized well. In other diseased muscles, proteasomes and ubiquitin showed a positive reaction in the atrophic or necrotic fibers. The results indicate increased proteasome and ubiquitin in these muscle fibers as well as in other diseased muscle fibers. We suggest that the ATP-ubiquitin-proteasome proteolytic pathway as well as the nonlysosomal calpain and the lysosomal proteolytic pathway may participate in the muscle fiber degradation in DMRV.     

The role of the ClpA chaperone in proteolysis by ClpAP

ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA-ClpP-substrate complexes assemble either by ClpA-substrate complexes interacting with ClpP or by ClpA-ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA-ClpP-substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.     

Proteolysis of Saccharomyces cerevisiae RNase H1 in E coli

Expression of S cerevisiae RNase H1 in E coli leads to the formation of a proteolytic product with a molecular mass of 30 kDa that is derived from the 39-kDa full length protein. The 30-kDa form retains RNase H1 activity, as determined by renaturation gel assay. The amount of proteolysis observed depends on the procedure used in preparing the cell extracts for protein analysis. The cleavage site on the amino acid sequence of the 39-kDa RNase H1 was determined by N-terminal sequence analysis of the 30-kDa proteolytic form. The cut occurs between two arginines located at the amino terminus region of the protein. The pattern of proteolysis was examined for both the wild-type RNase H1 and a mutant RNase H1 that was constructed in this work. In the mutant the second arginine of the cleavage site was changed to a lysine. Comparisons of the expression of the wild-type and altered protein in two different E coli strains demonstrate that the protease responsible for the degradation has a specificity very similar to that of the OmpT protease. However, the proteolysis observed in an OmpT background in extracts, prepared by boiling the cells in SDS containing buffer, indicates that the protease may, unlike OH108.     

Sequence elements that contribute to the degradation of yeast G alpha

BACKGROUND: Gpa1 is the alpha subunit of the yeast G-protein that regulates signal transduction during mating. The stability of Galpha/Gpa1 is influenced by the ubiquitin-dependent N-end rule pathway, suggesting that the regulation of G alpha levels may be important for effective mating response and recovery. RESULTS: The G alpha sequences that confer sensitivity to degradation by the N-end rule pathway were identified. The insertion of this degradation signal (G1-Deg) into the ordinarily stable Gpa2 protein conferred proteolytic targeting. We examined G alpha degradation under different conditions and found that it was efficiently degraded in haploid and diploid cells, but was stable if it was synthesized prior to expression of the N-end rule pathway. Interestingly, a specific mutation in G alpha that is believed to promote the GTP-bound form (N388K) caused accelerated degradation. CONCLUSION: A region encompassing a putative effector-binding domain (G1-Deg) is required for G alpha degradation via the N-end rule pathway. Our studies have shown that G alpha is susceptible to proteolysis soon after synthesis. These results are in agreement with the idea that G alpha is more unstable in the GTP-bound form, which is the predominant state of monomeric/free G alpha soon after synthesis. It is likely that the signal transduced by Gbetagamma can be regulated by adjusting the levels of G alpha through proteolysis.     

Determination of cardiac troponin I forms in the blood of patients with acute myocardial infarction and patients receiving crystalloid or cold blood cardioplegia

To determine the forms of cardiac troponin I (cTnI) circulating in the bloodstream of patients with acute myocardial infarction (AMI) and patients receiving a cardioplegia during heart surgery, we developed three immunoenzymatic sandwich assays. The first assay involves the combination of two monoclonal antibodies (mAbs) specific for human cTnI. The second assay involves the combination of a mAb specific for troponin C (TnC) and an anti-cTnI mAb. The third assay was a combination of a mAb specific for human cardiac troponin T (cTnT) and an anti-cTnI mAb. Fifteen serum samples from patients with AMI, 10 serum samples from patients receiving crystalloid cardioplegia during heart surgery, and 10 serum samples from patients receiving cold blood cardioplegia during heart surgery were assayed by the three two-site immunoassays. We confirmed that cTnI circulates not only in free form but also complexed with the other troponin components (TnC and cTnT). We showed that the predominant form in blood is the cTnI-TnC binary complex (IC). Free cTnI, the cTnI-cTnT binary complex, and the cTnT-cTnI-TnC ternary complex were seldom present, and when present, were in small quantities compared with the binary complex IC. Similar results were obtained in both patient populations studied. These observations are essential for the development of new immunoassays with improved clinical sensitivity and for the selection of an appropriate cTnI primary calibrator.     

CTL epitope generation is tightly linked to cellular proteolysis of a Listeria monocytogenes antigen

Listeria monocytogenes is a pathogenic intracellular bacterium that secretes proteins into the cytosol of host cells. A major secreted protein, p60, is processed by the host cell into the nonamer peptides p60 217-225 and p60 449-457, which are presented to CTL by H-2Kd MHC class I molecules. Herein, we use two membrane permeable peptide aldehyde protease inhibitors, LLnL and Z-LLF, to inhibit cytosolic proteolysis in L. monocytogenes-infected cells. These inhibitors, which have been shown to inhibit proteasomes, completely abrogate cytosolic p60 degradation. The effect of LLnL and Z-LLF on p60 epitope generation was determined by acid-eluting, HPLC-purifying, and quantifying p60 217-225 and p60 449-457 from infected cells. We show a direct linkage between p60 degradation and epitope generation. However, the two inhibitors have quantitatively different effects on the generation of the two epitopes. Our findings implicate proteasomes in the earliest stages of Ag degradation and suggest that different CTL epitopes can be generated by distinct proteolytic processes.     

Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P1-arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of alpha-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, alpha-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function as P. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components.     

Differential regulation of the pocket domains of the retinoblastoma family proteins by the HPV16 E7 oncoprotein

The human papillomavirus E7 oncoprotein binds to the retinoblastoma (Rb) tumor suppressor protein, and the binding to Rb correlates with the oncogenic potential of E7. Recent studies from several laboratories indicated that the half-life of the Rb protein is reduced in cells that are stably transformed with E7, suggesting that E7 could induce the proteolytic degradation of Rb. To investigate whether the Rb degradation is a primary effect of E7 or a result of altered cell phenotype, we sought to develop assays that can distinguish between the two possibilities. Using recombinant adenovirus expressing the human papillomavirus type 16 E7 protein, we show that the expression of E7 leads to an increased rate of decay of the Rb protein. Moreover, Rb degradation immediately follows the expression of E7 suggesting that it is an early and primary effect. Consistent with a previous study, we observed that the E7-induced degradation of Rb can be blocked by the inhibitors of the 26S proteasome. We have also developed a transient transfection assay for the E7-induced degradation of Rb. Using this assay, we show that the pocket domain of Rb is necessary and sufficient for the E7-induced degradation. However, the proteolysis is relatively specific for Rb because the level of p107 or p130 was not significantly altered by the expression of E7. Thus, although E7 binds to all three members of the Rb family of proteins, the proteolysis is much more efficient in the case of Rb. In the transient transfection assays, adenovirus E1A and SV40 large T antigen failed to induce degradation of Rb, suggesting that the Rb degradation is a unique property of the E7 oncoprotein.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) proteolysis in patients with colorectal cancer: possible association with the metastatic potential of the tumor

The limited proteolysis of insulin-like growth factor (IGF)-binding protein (IGFBP)-3 is a key event in the regulation of endocrine bioavailability of IGFs. Here, we investigated IGFBP-3 and IGFBP-3 proteolysis in serum from patients with colorectal cancer both before and at different times following surgery. In vivo IGFBP-3 proteolysis, estimated by immunoblot analysis of IGFBP-3 fragments in serum, and in vitro IGFBP-3 protease activity of serum, estimated by a 125I-IGFBP-3 degradation assay, allowed us to identify 2 groups of patients (IGF-M vs. IGF-NM) with respect to their status for mobilizing the IGF system. In IGF-M patients, in vivo and in vitro IGFBP-3 proteolysis were significantly elevated (156% and 181% of the age-matched control pool, respectively) and accompanied by a decrease in intact IGFBP-3 (38% of the control pool). The IGFBP-3 proteolytic processing was further increased in response to surgical ablation of the tumor (mean increase 45-55%), then gradually returned to levels comparable with controls. In contrast, IGF-NM patients exhibited a minimal alteration of in vitro IGFBP-3 protease activity and even an inhibition of in vivo IGFBP-3 proteolysis, whereas intact IGFBP-3 was unaltered when compared with controls. Moreover, this pattern was not further significantly altered in response to the surgical stress. None (0/6) of the IGF-M patients vs. 70% (5/7) of the IGF-NM patients developed a metastatic disease (median duration of follow-up 26 months). Neither elevated amounts of pro-IGF-II nor presence of detectable IGFBP-3 protease inhibitors in the circulation could explain the observed suppression of IGFBP-3 proteolytic processing in IGF-NM patients. These results indicate that inhibition of IGFBP-3 proteolysis and invasive properties of cancer cells are related in colorectal cancer patients.     

Proteolytic degradation of native hemoglobin and its constituent parts--isolated subunits and globin. I. Kinetic data and the character of the process of the breakdown of native forms

The rates of proteolytic breakdown for native human hemoglobin (Hb) in CNmet-and oxy-forms, for isolated native alpha- and beta-chains of human Hb with deprotected SH-groups and for apo-Hb--globin at constant temperature 20 degrees as well as for metHb and globin in the temperature range 4-25 degrees were studied. The proteolysis of oxy-forms of proteins was performed in the presence of CN- to prevent the appearance in solution of quickly splitted aqua and hydroxy met-forms. Pepsin (at pH 5.5), trypsin (at pH 7.0 and 8.5) and protease VI (pronase) (at pH 7.0 and 8.5) were used as proteases. The rate of proteolysis was registered simultaneously by proteolysate precipitation in concentrated salt solutions (to determine the content of the native form), by precipitation in aqueous solution of trichloroacetic or perchloric acid and by colouring the terminal NH2-groups by ninhidrin in the total proteolysate. For most cases the data of all the three independent methods fell on a single kinetic curve, each pair protein--protease being represented by their individual curves. Therefore the breakdown of all the protein studied possesses a burst-like ("one-by-one", "all-or-none") character. The protein resistance to the attack by proteolytic enzymes increases in the following order: globin less than oxy-alpha-chain less than metHb less than oxy-beta-chain less than HbO2 congruent to CNmetHb. The use of control repeated proteolysis has made it possible to prove that differences in the rate of proteolytic degradation are not the consequence of spontaneous denaturation of the least unstable forms of proteins in the course of proteolytic reaction but are predetermined by the conformational state of the native macromolecule.     

Identification and initial characterization of serum growth hormone binding protein in the turtle Chrysemys dorbigni

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential early during eye development to inhibit the determination of excess photoreceptors. Ubiquitin is a small polypeptide that tags proteins for degradation by a multisubunit proteolytic complex called the proteasome. The FAT FACETS protein is thought to be required to remove ubiquitin from a particular protein, thereby rescuing if from proteolysis. In order to identify the genes encoding the substrate of FAT FACETS and other components of the neural inhibition pathway, a mutagenesis screen for dominant enhancers of the fat facets mutant eye phenotype was performed. Several genes were identified, one of which is an excellent candidate for encoding a component of the pathway regulated by FAT FACETS. Three different eye phenotypes were observed when the fat facets mutants were dominantly enhanced by different mutations, suggesting that fat facets has other functions in addition to its critical role early in eye development.     

In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread.     

Dose- and wavelength-dependent oxidation of crystallins by UV light--selective recognition and degradation by the 20S proteasome

The lens of the human eye is a suitable model for age-related alterations at the molecular level. Age-related cataract formation is closely related to the accumulation of oxidatively altered proteins. In this study the influence of UV-A, UV-B, and UV-C irradiation on the proteolytic susceptibility of alpha-, betaL-, and betaH-crystallins by the isolated 20S proteasome was investigated. The proteins were irradiated with 280, 300, and 350 nm monochromatic light. Changes of the physical properties of the crystallins were characterized by absorbance measurements at 280 nm, fluorescence spectra, and SDS-PAGE-electrophoresis. The proteolytic susceptibility of crystallins was maximal after irradiation at 280 nm and three- to fivefold lower at 300 nm. Irradiation at 350 nm was not able to initiate proteolysis, probably due to protein-aggregate formation of higher molecular weight, as shown by SDS-PAGE. The damage of crystallins by UV-C light might be a signal for its proteolytic degradation by the 20S proteasome, whereas UV-B and UV-A do not increase the proteolytic susceptibility to the same extent but promote the formation of crosslinked proteins. Therefore, irradiation with UV, which is not followed by an increase in the proteolytic susceptibility, is accompanied by the formation of crosslinked proteins. It was concluded, that also long UV-B and UV-A may be involved in age-related alterations of the human lens and cataract formation.     

Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.     

Transfusion requirements are correlated with the degree of proteolysis of von Willebrand factor during orthotopic liver transplantation

During orthotopic liver transplantation (OLT) excessive bleeding is the main cause of death and graft failure. The acute bleeding tendency that accompanies OLT, particularly during the anhepatic period and after reperfusion of the graft, is due to the depletion or functional abnormalities of several hemostasis components caused by the enhanced activity of enzymes such as plasmin, trypsin and leukocyte proteases. We surmised that enhanced proteolysis might also cause abnormalities of von Willebrand factor (vWF), and that these abnormalities are implicated in the bleeding tendency that develops during OLT. Therefore, the pattern of vWF proteolysis was studied with 16 patients with chronic liver disease, in serial blood samples obtained before OLT, during the anhepatic stage, after graft reperfusion and at the end of the surgical procedure. vWF became markedly degraded during the anhepatic and reperfusion stages, as shown by the partial loss of high molecular weight multimers, the relative decrease of the intact 225 kD subunit and the increase of the native proteolytic fragments of 176 and 140 kD. Novel proteolytic fragments also became detectable. Using monoclonal antibody epitope mapping, it could be demonstrated that some of the proteolytic fragments corresponded in apparent molecular mass to those produced in vitro by incubating purified vWF with plasmin or elastase, but other fragments could not be attributed to these proteases. During the anhepatic and reperfusion stages there was a significant correlation between the degree of vWF degradation and the total amount of blood components transfused to replace blood losses. To evaluate whether or not vWF degradation could be controlled by the administration of a broad-spectrum protease inhibitor such as aprotinin, 5 patients were given a bolus dose of 500,000 U before surgery followed by 100,000 U/h during surgery, 5 were given a 2,000,000 U bolus followed by 500,000 U/h, and no aprotinin was given to the remaining 6 patients. There were no differences in the patterns or degrees of vWF degradation between patients treated with aprotinin or not. In conclusion, there is a marked degradation of a key hemostasis protein during OLT. These alterations may be of clinical significance, because they are correlated with the transfusion requirements.     

Immunopurification and characterization of a collagenase/gelatinase domain issued from basement membrane fibronectin

The proteolytic potential of cellular fibronectin fragments issued from a basement membrane hydrolysate was investigated. Three different gelatinase activities (47, 43 and 37 kDa), located by gelatin zymography, were isolated using successively heparin-agarose, gelatin-agarose and immunopurification with polyclonal antibodies directed against bovine plasma fibronectin. These fragments were also characterized using a monoclonal antibody directed against the extra-domain EDA of cellular fibronectin as a probe. A collagenase activity, reliably indicated by the gelatin zymography pattern, was also found using MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2, the intramolecularly quenched fluorogenic substrate of collagenases. From these results, cellular fibronectin was found to be able to exhibit a proteolytic function after limited proteolysis. This MMP-like function could be associated with tissue remodeling in both normal and pathological states, such as metastasis, angiogenesis and tissue repair.     

Retransplantation of patients with severe posttransplant hepatitis B in the first allograft

The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex. All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria. Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover. As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction. A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a. In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a. The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6. The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3. Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.