Leupeptin-sensitive Proteases Research Articles

Protein phosphorylation maintains the normal function of cloned human Cav2.3 channels.

R-type currents mediated by native and recombinant Cav2.3 voltage-gated Ca2+ channels (VGCCs) exhibit facilitation (run-up) and subsequent decline (run-down) in whole-cell patch-clamp recordings. A better understanding of the two processes could provide insight into constitutive modulation of the channels in intact cells, but low expression levels and the need for pharmacological isolation have prevented investigations in native systems. Here, to circumvent these limitations, we use conventional and perforated-patch-clamp recordings in a recombinant expression system, which allows us to study the effects of cell dialysis in a reproducible manner. We show that the decline of currents carried by human Cav2.3+β3 channel subunits during run-down is related to adenosine triphosphate (ATP) depletion, which reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials. Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. Protein kinase inhibition also mimics the effects of run-down in intact cells, reduces the peak current density, and hyperpolarizes the voltage dependence of gating. Together, our findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. These data also distinguish the effects of ATP on Cav2.3 channels from those on other VGCCs because neither direct nucleotide binding nor PIP2 synthesis is required for protection from run-down. We conclude that protein phosphorylation is required for Cav2.3 channel function and could directly influence the normal features of current carried by these channels. Curiously, some of our findings also point to a role for leupeptin-sensitive proteases in run-up and possibly ATP protection from run-down. As such, the present study provides a reliable baseline for further studies on Cav2.3 channel regulation by protein kinases, phosphatases, and possibly proteases.

Dendroaspis natriuretic peptide and the designer natriuretic peptide, CD-NP, are resistant to proteolytic inactivation

Designer natriuretic peptides (NPs) represent an active area of drug development. In canine and human studies, the designer natriuretic peptide CD-NP demonstrated more desirable therapeutic potential than recombinant B-type NP (BNP), which is known as nesiritide and is approved for treatment of acute decompensated heart failure. However, why CD-NP is more effective than BNP is not known. We previously reported that CD-NP is a poorer activator of human guanylyl cyclase-A (GC-A) and a better activator of human guanylyl cyclase-B than BNP. Here, guanylyl cyclase bioassays were used to compare the susceptibility of CD-NP verses ANP, BNP, CNP and DNP to inactivation by human kidney membranes. The half time ( t 1/2) for CD-NP inactivation was increased by factors of 13, 3 and 4 compared to ANP, BNP and CNP, respectively, when measured in the same assay. Surprisingly, DNP failed to undergo complete inactivation and was the most degradation resistant of the peptides tested. The neutral endopeptidase (NEP) inhibitor, phosphoramidon, blocked inactivation of CNP and CD-NP, but not BNP or DNP. In contrast, the general serine and cysteine protease inhibitor, leupeptin, completely blocked the degradation of BNP and CD-NP, but did not block CNP inactivation unless phosphoramidon was included in the assay. Thus, NPs with shorter carboxyl tails (ANP and CNP) are degraded by phosphoramidon-sensitive proteases and NPs with extended carboxyl tails (BNP, DNP and CD-NP) are resistant to NEP degradation and degraded by leupeptin-sensitive proteases. We conclude that DNP and CD-NP are highly resistant to proteolysis and that proteolytic resistance contributes to the beneficial cardiovascular properties of CD-NP. We suggest that this property may be exploited to increase the half-life of NP-based drugs.

Plasma membrane delivery, endocytosis and turnover of transcobalamin receptor in polarized human intestinal epithelial cells

Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B(12)) and they obtain it from the circulation bound to transcobalamin (TC) via the transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labelling, domain-specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life (T(1/2)) of 8 h following its delivery to the basolateral plasma membrane (BLM). The T(1/2) of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed (T(1/2), 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2 cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM.

Leupeptin‐sensitive proteases involved in cell survival after X‐ray irradiation in human RSa cells

Proteases have received attention as important cellular components responsible for stress response in human cells. However, little is known about the role of proteases in the early steps of cell response after X-ray irradiation. In the present study, we first searched for proteases whose activity levels are changed soon after X-ray irradiation in human RSa cells with a high sensitivity to X-ray cell-killing. RSa cells showed an increased level of fibrinolytic protease activity within 10 min after irradiation with X-ray (up to 3 Gy). The induced protease activity was proved to be inhibited by leupeptin. We next examined whether this protease inducibility is related to the X-ray susceptibility of cells. Treatment of RSa cells with leupeptin prior to X-ray irradiation resulted in lowered colony survival and an increased ratio of G(2)/M-arrested cells and apoptotic cells. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human RSa cells to X-ray cell-killing.

Increase in epidermal growth factor receptor protein induced in osteoblastic cells after exposure to flow of culture media.

To investigate how bone cells respond to mechanical stimuli, we subjected osteoblastic cells to fluid flow. We and others already reported that in a culture system of osteoblast-like cells, ERK1/2, Shc, and other proteins were tyrosine-phosphorylated by medium flow and the early response gene, egr-1 or c-fos mRNA, increased. These are the same as events found after stimulation by various growth factors. Moreover, because there were also reports suggesting that growth factor signaling is involved in the responses to mechanical stimuli, we examined the change in epidermal growth factor (EGF) receptor in the cells exposed to medium flow. The results demonstrated that EGF receptor protein increased after exposure to medium flow. This increase did not occur without serum in media, and the addition of EGF restored it. Furthermore, leupeptin blocked this increase. These results suggest that degradation of EGF-occupied EGF receptor by leupeptin-sensitive protease(s) in endosomes decreased with exposure to medium flow. This was presumed to participate, at least in part, in signaling of fluid flow.

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes.

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 M) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.

Evolution of the Ca 2+ current during dialysis of isolated bovine chromaffin cells: effect of internal calcium

We have examined the internal Ca 2+-dependence of the long-term evolution of whole cell high voltage activated Ca current in chromaffin cells. The evolution of the peak Ca current was characterized by 2 distinct phases: after an initial facilitation, there followed a rundown, which represented a reduction by 70% within some 10 min. The rundown process was shown not to depend on Ca 2+ entry nor on membrane depolarization. It resulted from cell dialysis with a saline solution and, once initiated, it proceeded at a rate of 0.28 min −1 at 4 different Ca 2+ concentrations (pCa 5–9). The facilitation is also initiated by cell dialysis but this process developed faster at higher internal Ca 2+ concentrations. Thus, globally, high-voltage activated Ca 2+ current runs down faster when using a recording pipette solution with a higher internal Ca 2+ concentration (pCa 5 or 6). Some leupeptin-sensitive proteases may be involved in the initiation of facilitation and rundown processes.

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.

Protein breakdown in submandibular glands rendered hypertrophic by amputation of lower incisor teeth in rats.

Protein breakdown in submandibular glands rendered hypertrophic by amputation of the lower incisor teeth in rats was investigated. Reduced protein breakdown was observed in the hypertrophic gland tissues, and was found to be inhibited by 20 mM epsilon-amino-n-caproic acid, an inhibitor of serine protease, and 50 microM leupeptin, an inhibitor of trypsin, plasmin, papain and cathepsin B, but not by 2 mM PMSF (phenylmethylsulfonyl fluoride), an inhibitor of serine protease, 10 microM pepstatin, an inhibitor of cathepsin D and 20 microM antipain, an inhibitor of cathepsin A and B. These results suggest that some serine proteases and leupeptin-sensitive proteases (presumably cathepsin B) participate in protein breakdown in hypertrophic gland tissues, and that hypertrophy of the submandibular glands is closely related to the reduced protein breakdown in these tissues.

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Cel and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited >95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.

Analysis of the disruptive action of an epididymal protease and the stabilizing influence of molybdate on nondenatured and denatured glucocorticoid receptor.

The nucleomyofibrillar fraction of epididymides from sexually mature rabbits contains a novel leupeptin-sensitive protease that disrupts the oligomeric conformation of cytosolic estrogen and progesterone receptors, and molybdate inhibits this process. In this report we used the AtT-20 cell glucocorticoid receptor as substrate and performed analyses under nondenaturing vs. denaturing conditions to further investigate the effects of the epididymal protease and molybdate on steroid receptor structure. Analysis on low salt sucrose gradients indicated that the protease partially converted the oligomeric (9-10S) glucocorticoid receptor to several more slowly sedimenting forms (3-7S), and this effect was not observed in the presence of molybdate. Paradoxically, gradient analysis under high salt conditions revealed that the protease induced a discrete, quantitative and molybdate-insensitive conversion of the 4-5S steroid-binding subunit to a 3S form. Further studies were done using denaturing polyacrylamide gel electrophoretic analysis of receptor that had been labeled covalently with [3H]dexamethasone 21-mesylate and partially purified by DNA/cellulose chromatography. At 0-4 C, the protease cleaved the steroid-binding subunit (mol wt, 96,900) of the receptor to a single steroid-labeled fragment (mol wt, 42,600). Under these conditions, digestion was complete within 30-60 min and was inhibited by leupeptin, but was unaffected by thiol-reactive reagents or molybdate. The epididymal protease and alpha-chymotrypsin produced steroid-labeled receptor fragments that were indistinguishable in size, shared an epitope recognized by our BuGR-2 monoclonal antibody, and retained DNA-binding activity. Despite the apparent similarity of these two enzymes, they are distinct, since the chymotrypsin-dependent cleavage event was not inhibited by leupeptin. These studies show that the epididymal protease attacks a site on the steroid-binding subunit of glucocorticoid receptors as well as estrogen and progestin receptors. It also appears that the cleavage site is situated close to that most readily attacked by alpha-chymotrypsin. Finally, our data provide independent confirmation of a recent report indicating that molybdate ions interact directly with the cytosolic steroid receptor to stabilize its oligomeric structure even after proteolysis within the steroid-binding subunit.

Mode of action of bestatin and leupeptin to induce the accumulation of acid soluble peptides in rat liver in vivo and the properties of the accumulated peptides. The important role of bestatin- and leupeptin-sensitive proteases in the protein degradation pathway in vivo

1. 1. The chemical properties of acid soluble peptides accumulated in liver or excreted into urine after administration of bestatin or leupeptin to rats were investigated extensively. 2. 2. At the same time, the effects of glucagon on the bestatin-induced accumulation of acid soluble peptides were studied. 3. 3. The results show the important role of bestatin- and leupeptin-sensitive proteases in the degradation pathway of intracellular proteins in vivo.

A protease acting on the estrogen receptor may modify its action in the adult rabbit epididymis

We have previously shown that the cytosolic estrogen receptor in adult rabbit epididymides sediments as an ∼- 3S species on sucrose gradients containing 0.01 M KC1 while that from immature rabbit epididymides sediments at ∼- 9 S. This age-dependent decrease in sedimentation coefficient is attributable to the appearance of a leupeptin-sensitive protease as the animals mature. We now show that if adult epididymides are homogenized in buffer containing leupeptin, the 9 S receptor can be demonstrated, indicating inhibition of receptor degradation. In vitro nuclear uptake studies conducted in the absence of leupeptin indicated that the proteolyzed receptor was not an efficient nuclear binder. When leupeptin was present, nuclear uptake increased 6-fold and it was accompanied by depletion of receptor from the cytosol. Binding of the receptor to nuclei was specific since it could be inhibited by unlabeled estrogens but not by unlabeled 5α-dihydrotestosterone or progesterone. In vitro mixing experiments indicated that the proteolytic activity was associated with the crude nuclear fraction since, in the absence of leupeptin, they had reduced ability to bind estrogen receptor present in immature epididymal cytosol. Specific in vivo binding of [ 3H]estradiol by adult and immature rabbit epididymides could be demonstrated. The time course of in vivo binding of [ 3H]estradiol by adult rabbit epididymal nuclei indicated maximum binding (70 fmol/g tissue) at 30 min following injection. By 60 min, the amount of binding had decreased to about 25 fmol. The accessory sex organs, which do not contain the protease, also exhibited maximum binding (150 fmol/g tissue) at 30 min. However, at the 60 min period binding was still about 140 fmol. Processing the tissues in buffers containing leupeptin had no effect on the results obtained. These results are intrepreted to indicate that the presence of the protease decreases nuclear binding of the estrogen receptor and shortens nuclear occupancy. This combination of factors may be responsible for the decrease in estrogen action in the adult rabbit epididymis.

Further characterization of a steroid receptor-active protease from the mature rabbit epididymis

The nucleomyfibrillar fraction of mature rabbit epididymides contains a salt-extractable and leupeptin-sensitive protease that alters the sedimentation coefficient of cytosolic steroid receptors. We refer to this modification as receptor conversion. The substrate used in these studies was cytosolic estrogen receptor obtained from frozen rabbit uteri. The unactivated form of the receptor exists as an oligomer under hypotonie (0.01 M KCl) conditions ( s 20,w ∼- 9.6. Stokes radius (R s) ∼- 7.4nm, M r ∼- 320,000) and dissociates under hypertonic (0.4 M KCl) conditions to yield the steroid-binding monomer ( s 20,w ∼- 4.7, R s ∼- 5.1 nm, M r ∼- 104,000). According to analysis under hypotonie conditions, the epididymal protease disrupts the oligomeric architecture of the receptor and reduces the size of the steroid-binding monomer ( s 20,w ∼- 3.2, R s ∼- 3.0 nm, M r ∼- 42,000). The epididymal protease had no detectable effect on the structure of the proteins used as standards for the ultracentrifugal or gel filtration analyses. Although inhibited by leupeptin, the epididymal enzyme is not a typical thiol protease since it was unaffected by thiol-blocking agents (iodoacetamide and N-ethylmaleimide), and was partially inhibited by thiol-reducing agents (monothioglycerol and dithiothreitol). Calcium and magnesium ions alone, or in combination with ATP, had no effect on the activity of the protease. However, both cations selectively suppressed recovery of the oligomeric receptor form. These results, in conjunction with those from previous studies, serve to distinguish the epididymal protease from receptor-active proteases described in extracts of other animal tissues. Molybdate, at a concentration of 50 mM, blocked receptor conversion. The ability of the receptor to be stabilized by molybdate was lost following conversion. Finally, the epididymal protease appears to remove a portion of the estrogen receptor that is necessary for nucleotide-binding.

A new protease in hog thyroid lysosomes

The presence of protease activity at pH 7.5 and at acidic pH was demonstrated in the soluble fraction of lysosomes which were prepared from hog thyroid homogenates by subcellular fractionation. The ratios of specific activity measured at pH 7.5 to that at pH 3.5 were 0.07 to 0.08 for casein (at pH 7.5) or haemoglobin (at pH 3.5) as substrate and 0.13 for iodoamino acid-releasing activity from thyroglobulin as substrate. The protease activity measured at pH 7.5 was inhibited by inhibitors for neutral type protease, such as leupeptin, and by sulfhydryl inhibitors, such as iodoacetamide, indicating that the protease was a leupeptin-sensitive protease and contained a sulfhydryl group for its active site. Insensitiveness of the protease activity to di-isopropyl fluorophosphate excluded the involvement of a serine group at the active site. At pH 5.5 the proteolytic and iodoamino acid-releasing activities using casein and thyroglobulin, respectively, were also investigated. Several lines of evidence suggested that both activities measured at pH 5.5 were due to a mixture of acidic and leupeptin-sensitive protease activities.

Effects of leupeptin and pepstatin on protein turnover in adult rat hepatocytes in primary culture

Addition of pepstatin, an inhibitor of acid protease, to 2-day cultures of rat hepatocytes rapidly inhibited the activity to hydrolyze hemoglobin (Hb), but did not affect the activity to hydrolyze α- N-benzoyl- dl-arginine-β-naphthylamide (BANA). On the other hand, addition of leupeptin, an inhibitor of thiol protease, inhibited the activity of BANA hydrolase and caused a sixfold increase in the activity of Hb hydrolase within 1 day. Neither protease inhibitor affected the rate of protein synthesis. Release of amino acids from hepatocytes into Hanks' salt solution was measured by the ninhydrin method. Pepstatin inhibited the release only 15% within 2 days, but leupeptin inhibited it 65% within 10 h. These two inhibitors had additive inhibitory effects on the release, suggesting that they inhibit the degradations of different groups of proteins. The inhibitory effect of leupeptin gradually decreased after 10 h, which is consistent with the observed induction of a protease activity mentioned above. A preferential involvement of leupeptin-sensitive protease in the degradation of proteins with longer half-lives was suggested from studies on [ 14C]leucine release from hepatocytes prelabeled for 30 h. On the other hand, the two inhibitors had similar effects on the release of [ 14C]leucine from hepatocytes labeled for only 1 h. Their inhibitory effects were again additive, but there was no reduction in the inhibition by leupeptin on prolonged incubation, suggesting that proteins with short half-lives were not substrates for the induced protease. These results suggest that in hepatocytes, proteins with longer half-lives are degraded more by cathepsin B than by cathepsin D, while those with short half-lives are degraded equally by these two proteases.