Degradation of the immunogenic peptide gp100(280-288) by the monocyte-like U937 cell line

The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.     

Positive and negative modulation of peptidases by pro-inflammatory cytokines

The capacity of pro-inflammatory cytokines to modulate proteolysis was analyzed by liquid chromatography using human fibroblasts as cell model and enzyme source, and the immunodominant epitope gp100(280-288) (YLEPGPVTA) as substrate. The measurements made after fibroblast pre-incubation with either IL-1, TNF, or IL-6 plus its soluble receptors have been compared with those made with un-stimulated fibroblasts. The results obtained suggest an uneven association of cytokine treatment with substrate degradation, and with a prevailingly positive - but also negative - association with release of smaller peptides and free amino acids. Data obtained by separately measuring these two groups of by-products indicate that, after IL-1 cell pre-treatment, the velocity of formation of both groups of by-products increased, resulting in a net increase of substrate degradation. After TNF and IL-6 pre-treatment, the increase of one group was compensated by a decrease of the other group; specifically, the compensation was only partial for TNF, and overall substrate hydrolysis increased. In the case of IL-6, the increase of free amino acids was almost exactly compensated by a reduction of peptidic by-products, resulting in a negligible increase of substrate hydrolysis. In addition, the existence of reaction time-related modifications in the apparent velocity of substrate degradation and formation of by-products, allows hypothesizing different effects of cytokines on the enzymes degrading the substrate with different time constants. Taken together, these data can be interpreted as indicating different, positive and negative, effects of the three cytokines on the individual enzymes expressed by fibroblasts and capable of degrading peptidic substrates.     

Effect of IL-1 on the hydrolysis of the tumor antigen epitope gp100(280-288) by fibroblast-expressed enzymes

The role of proinflammatory cytokines in increasing the activity of specific proteases suggests the hypothesis that, by altering the expression of these mediators, adjuvants may modulate the effectiveness of peptides used as vaccines. The possible effect of IL-1 on fibroblast-expressed, peptidases was, thus, investigated by analyzing the degradation of a tumor antigen epitope (gp100(280-288), YLEPGPVTA) in the presence of cultured human fibroblasts. The data obtained indicate an increase of substrate hydrolysis after IL-1 treatment as compared with non-treated controls. Hydrolysis increase was accompanied by defined changes in the population of the by-products formed: specifically, the amount of peptidic by-products increased more than the amount of single amino acids, and the amount of the C-terminal by-products increased more than the amount of their N-terminal counterpart. These data appear to indicate that the positive effect of IL-1 on the activity of substrate-active enzymes is function of modified expression of a number of these enzymes by fibroblasts. From these data it can be inferred that the use of IL-1-inducing adjuvants, increasing the activity of peptidases expressed by bystander cells, may reduce the bio-availability of peptides used for immunization.     

Soluble enkephalin-degrading enzymes released by lymphomic and erythroleukaemic cell lines

The possible existence of soluble proteolytic enzymes released by cells of lymphomic (U937 and 1301) and erythroleukaemic (K562) lines was studied measuring the hydrolysis of³H-leucine enkephalin in the presence of cell-free supernatants obtained from these lines. Results indicate that leu-enkephalin is rapidly degraded in the presence of these supernatants, and that enkephalin disappearance is paralleled by the formation of peptides that can be interpreted as its hydrolysis fragments. To characterize the factors involved in leu-enkephalin degradation, cell supernatants were analyzed by ion exchange and by steric exclusion chromatography. Data obtained indicate the presence of three groups of proteins active in leu-enkephalin degradation: aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In all three lines, these enzymes are represented by a considerable number of distinct activities. The sizable number of soluble enzymes identified and the signficant total activity observed suggest a possible role in the regulatory degradation of informational peptides, as proposed by several groups for the membrane-bound proteolytic enzymes of immunocompetent cells.     

Expansion of tumor-T cell pairs from fine needle aspirates of melanoma metastases

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.     

Neutral endopeptidase from nuchal ligament of fetal calves

The nuchal ligament of unborn calves contains a neutral endopeptidase that is biochemically and immunologically similar to the neutral endopeptidase (NEP), or enkephalinase, from human kidney. Enzymatic activity was inhibited more than 90% by phosphoramidon (1 microM). The specific activity in membrane fractions, as determined by hydrolysis of the dansylated substrate, DAPGN, was similar in tissue from fetuses of gestational ages ranging from 100 to 280 days. NEP activity in adult ligament tissue, however, was less than 10% of that in fetal tissue. Fibroblasts dissociated from ligament tissue by collagenase displayed less NEP activity than did preparations of intact ligament, and activity was even lower in cultured cells. By contrast, fibroblasts cultured from fetal calf lungs had NEP activity comparable to that in the ligament tissue. When ligament fibroblasts were cultured on subcellular matrices derived from fetal lung fibroblasts the NEP activity increased relative to those cultured on plastic alone. These studies confirm the presence of neutral endopeptidase (NEP) in the nuchal ligament of the fetal calf. The consistent activity through a range of gestational ages and the influence of the subcellular matrix suggest that this enzyme might be involved in growth of the ligament during fetal life.     

Effect of Differentiation on the Leucine Enkephalin-Degrading Soluble Enzymes Released by the K562(S) Cell Line

Leu-enkephalin hydrolysis kinetics were measured in the presence of soluble supernatants obtained from cultures of the K562(S) leukaemic cell line. Under these conditions, the substrate is degraded with formation of two distinct patterns of the hydrolysis by-products: in one pattern, similar amounts of Tyr and Tyr-Gly are formed; in the other, only Tyr-Gly can be measured. Kinetic data suggest that soluble proteolyses are released by these cells, and that either dipeptidylaminopeptidases alone, or both aminopeptidases and dipeptidylaminopeptidases are involved in substrate hydrolysis. This alternation of hydrolysis patterns appears consistent with existing data on the heterogeneity of K562 cells. In contrast with these results, chromatographic separation of the soluble enzymes indicates the release of all three classes of proteolyses known to hydrolyze enkephalins: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. In cells induced to differentiate by treatment with butyric acid, substrate hydrolysis is increased, and the pattern of the enzymes released is modified. In these cells, variations in both total proteolytic activity, and ratio between the three enzyme classes mentioned above are only minor, while the ratio between the different enzyme species within each class is greatly modified. Data obtained suggest that the expression of soluble enzymes is modified by differentiation. These data may also be interpreted as stressing the role of competition in controlling substrate hydrolysis by the multiple enzymes co-released by K562(S) cells.     

Proteolysis of Microtubule-Associated Protein 2 and Tubulin by Cathepsin D

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and tubulin by the lysosomal aspartyl endopeptidase cathepsin D was studied. MAP-2 was very sensitive to cathepsin D-induced hydrolysis in a relatively broad, acidic pH range (3.0-5.0). However, at a pH value of 5.5, cathepsin D-mediated hydrolysis of MAP-2 was significantly reduced and at pH 6.0 only a small amount of MAP-2 was degraded at 60 min. Interestingly, the two electrophoretic forms of MAP-2 showed different sensitivities to cathepsin D-induced degradation, with MAP-2b being significantly more resistant to hydrolysis than MAP-2a. To our knowledge, this is the first clear demonstration that MAP-2 is a substrate in vitro for cathepsin D. In contrast to MAP-2, tubulin was relatively resistant to cathepsin D-induced hydrolysis. At pH 3.5 and an enzyme-to-substrate ratio of 1: 20, only 35% of the tubulin was degraded by cathepsin D at 60 min. The cathepsin D-mediated hydrolysis of tubulin was optimal only at pH 4.5. These results demonstrate that MAP-2 and tubulin are unequally susceptible to degradation by cathepsin D. These data also imply a potential for rapid degradation of MAP-2 in vivo by cathepsin D either in lysosomes or perhaps autophagic vacuoles of the neuron.     

The recBC enzyme of Escherichia coli K12: premature cessation of catalytic activities in vitro and reactivation by potassium ions.

It is shown that in vitro the degradation of native and single-stranded DNA as well as the hydrolysis of ATP by purified recBC enzyme ceases 2-3 min after the start of the reaction. The presence of potassium ions (60-100 mM), bovine serum albumin (1 mg/ml) or protein from cell-free Escherichia coli extract (10 microgram/ml) prevents the cessation of the activity. Once the cessation has occurred, the activity of the enzyme can be completely restored by the addition of potassium ions, but not by bovine serum albumin. Sedimentation studies revealed that, in contrast to the active recBC enzyme, the 'silent' enzyme is no longer associated with substrate DNA of high molecular weight. On the basis of these results and other observations it is hypothesized that during the degradation of DNA in the absence of potassium ions or bovine serum albumin the recBC enzyme is subject to an alteration of its molecular conformation which results in an inactive form.     

Pattern of protein degradation during germination of Streptomyces antibioticus spores

The pattern of protein degradation during germination of Streptomyces antibioticus spores was studied by the pulse and chase technique. Two different protein fractions were found. First, a fraction of the proteins synthesized during the darkening process (20-30%) was quickly degraded in the 30 min following the labelling period. This rapid protein degradation was partially inhibited by protease inhibitors: p-chloromercuribenzoic acid, phenylmethylsulphonylfluoride, and o-phenanthroline. Second, the remaining 70-80% and the entire protein population formed during spore swelling and germ tube emergence were degraded with a lower and constant rate (3.3-6.0% /h). A stable mRNA fraction of the dormant spores was translated upon incubation of the spores in a minimal synthetic medium (MSM) or in distilled water. However, the degradation of these proteins did not occur unless the spores were then incubated in the MSM. A strong correlation between the degradation pattern of these proteins and that of those quickly degraded at the beginning of germination was observed. Protease activity in cell-free extracts of dormant spores was detected. Inhibition studies suggest the presence of serine, thiol, and metalloproteases. The protease activity, using casein as substrate, remained constant during the darkening process and started to increase progressively from the beginning of spore swelling.     

A melanoma multiepitope polypeptide induces specific CD8+ T-cell response

Strategies using epitope-based vaccination are being considered for melanoma immunotherapy, in an attempt to overcome failure of other modalities. In the present study, we designed and produced a multiepitope polypeptide for melanoma (MEP-mel), which contains three repeats of four antigenic epitopes (gp100: 209-217 (210M); gp100: 280-288 (288V); Mart1: 26-35 (27L); tyrosinase: 368-376 (370D). The peptides were attached to each other by linkers containing sequences recognized by the proteasome, to improve protein cleavage and antigen presentation. The results show that peptide-specific T cells produced IFN-gamma when stimulated with MEP-mel-transfected dendritic cells. The presentation of peptides by MEP-mel-transfected dendritic cells was proteasome-dependent and was more long-lasting than the presentation of exogenously delivered native peptides. When dendritic cells were loaded with MEP-mel protein, weak cross presentation was induced. The production of multiepitope molecules based on several peptides linked by sequences sensitive to proteasomal cleavage represents a promising new tool for the improvement of cancer immunotherapy.     

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.     

Human interferon-gamma is internalized and degraded by cultured fibroblasts

Human interferon-gamma (IFN-gamma) binds specifically and with high affinity to receptors on the surface of cultured fibroblasts (GM-258). At 37 degrees C about 50% of the receptor-bound IFN-gamma was rapidly internalized (t 1/2 = 4-5 min) by these cells. Following an initial lag of 15-30 min, internalized IFN-gamma was continuously degraded over a period of at least 8 h. The total uptake of IFN-gamma over this time period was found to exceed by 5 times the number of occupied IFN receptors present on the surface of these cells, suggesting that either there is a large intracellular pool of IFN-gamma receptors, or that receptors are recycled during the course of incubation. Cycloheximide (100 micrograms/ml) inhibited uptake only after the first 2 h of incubation and then only moderately. It is therefore unlikely that de novo receptor synthesis plays a major role in the observed uptake process. Both sodium azide (15 mM) and methylamine (20 mM) inhibited both the uptake and degradation of IFN-gamma at all times up to 6 h. While uptake was only slightly reduced in the presence of chloroquine (25 microM), degradation was markedly inhibited, suggesting that degradation occurs intracellularly, probably within lysosomes.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.     

Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.     

ClpA and ClpP remain associated during multiple rounds of ATP-dependent protein degradation by ClpAP protease.

The Escherichia coli ClpA and ClpP proteins form a complex, ClpAP, that catalyzes ATP-dependent degradation of proteins. Formation of stable ClpA hexamers and stable ClpAP complexes requires binding of ATP or nonhydrolyzable ATP analogues to ClpA. To understand the order of events during substrate binding, unfolding, and degradation by ClpAP, it is essential to know the oligomeric state of the enzyme during multiple catalytic cycles. Using inactive forms of ClpA or ClpP as traps for dissociated species, we measured the rates of dissociation of ClpA hexamers or ClpAP complexes. When ATP was saturating, the rate constant for dissociation of ClpA hexamers was 0.032 min(-1) (t(1/2) of 22 min) at 37 degrees C, and dissociation of ClpP from the ClpAP complexes occurred with a rate constant of 0. 092 min(-1) (t(1/2) of 7.5 min). Because the k(cat) for casein degradation is approximately 10 min(-1), these results indicate that tens of molecules of casein can be turned over by the ClpAP complex before significant dissociation occurs. Mutations in the N-terminal ATP binding site led to faster rates of ClpA and ClpAP dissociation, whereas mutations in the C-terminal ATP binding site, which cause significant decreases in ATPase activity, led to lower rates of dissociation of ClpA and ClpAP complexes. Dissociation rates for wild-type and first domain mutants of ClpA were faster at low nucleotide concentrations. The t(1/2) for dissociation of ClpAP complexes in the presence of nonhydrolyzable analogues was >/=30 min. Thus, ATP binding stabilizes the oligomeric state of ClpA, and cycles of ATP hydrolysis affect the dynamics of oligomer interaction. However, since the k(cat) for ATP hydrolysis is approximately 140 min(-1), ClpA and the ClpAP complex remain associated during hundreds of rounds of ATP hydrolysis. Our results indicate that the ClpAP complex is the functional form of the protease and as such engages in multiple rounds of interaction with substrate proteins, degradation, and release of peptide products without dissociation.     

Ethanol potentiates the stimulatory effects of phorbol ester, sphingosine and 4-hydroxynonenal on the hydrolysis of phosphatidylethanolamine in NIH 3T3 cells

Ethanol and other alcohols have been shown to specifically stimulate phospholipase-D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here, we further examined the possible mechanism of this ethanol action. Ethanol (10-300 mM) and the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol 13-acetate (TPA) had synergistic stimulatory effects on the degradation of preformed [14C]PtdEtn when added in combination to [14C]ethanolamine-labelled suspended NIH 3T3 cells 30 min after collection of cells by scraping. Scraping caused a transient increase, lasting for less than 30 min, in the cellular content of 1,2-diacylglycerol, another PKC activator. Initially (0-50 min incubation), the main water-soluble product of [14C]PtdEtn degradation in ethanol plus TPA-treated cells was [14C]ethanolamine, while later (90 min) the main product of [14C]PtdEtn hydrolysis was [14C]ethanolamine phosphate in the presence of these agents. Ethanol also potentiated the specific stimulatory effects of sphingosine (through phospholipase D) and 4-hydroxynonenal (not involving phospholipase D) on PtdEtn hydrolysis. The effects of these latter agents were unrelated to PKC activation. These data indicate that the observed potentiating effects of ethanol on PtdEtn hydrolysis do not involve direct regulation of PKC or phospholipase D activities.     

Degradation of the precursor of mitochondrial aspartate aminotransferase in chicken embryo fibroblasts

The precursor of mitochondrial aspartate aminotransferase accumulates in the cytosol of cultured chicken embryo fibroblasts if its import into mitochondria is inhibited by an uncoupling agent. However, its accumulation is limited by degradation with a half-life of only approximately 5 min (Jaussi, R., Sonderegger, P., Flückiger, J., and Christen, P. (1982) J. Biol. Chem. 257, 13334-13340). The aim of the present study was the characterization of the proteolytic system(s) responsible for this very rapid intracellular degradation. On depleting chicken embryo fibroblasts of ATP, the rate of degradation of the precursor was lowered by approximately 70%. Chicken embryo fibroblasts depleted of divalent metal ions showed a degradative activity of 10% of the initial value. Reconstitution of these cells with Mg2+ and Ca2+ increased the degradative activity from 10 to 107 and 24%, respectively. Thiol reagents almost completely prevented the degradation, whereas specific peptide inhibitors of cysteine proteases or inhibitors of intralysosomal proteolysis decreased the rate of degradation by only approximately 30%. Inhibitors of serine proteases had little effect. No rapid degradation of the precursor was observed in crude extracts of chicken embryo fibroblasts. The data indicate that the bulk of the precursor accumulated under conditions of import block is degraded by one or several cytosolic proteases dependent on ATP, Mg2+, and thiol groups of unknown localization, conceivably by proteolytic enzymes identical with or similar to one of the high molecular weight cytosolic proteases (Waxman, L., Fagan, J.M., Tanaka, K., and Goldberg, A. L. (1985) J. Biol. Chem. 260, 11994-12000). The rest of the precursor appears to be degraded by lysosomes.     

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.     

Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: Cross-talk between E3s in the endoplasmic reticulum

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.