Thiol Protease Inhibitor Research Articles

A Novel Endoprotease Responsible for the Specific Cleavage of Transducin .gamma. Subunit

Isoprenylated/methylated heterotrimeric G proteins play important roles in a large number of signal transduction processes. While the enzymology of isoprenylated/methylated protein biosynthesis is well understood, nothing is known about how these proteins are degraded. In this article, a novel endoproteolytic activity has been identified from bovine retina and is shown specifically to remove the glycylfarnesylcysteine moiety from the carboxyl terminus of T gamma. When tested in a GTP binding assay, freshly prepared proteolyzed T beta gamma was unable to catalyze the binding of guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S) to T alpha in the presence of detergent solubilized rhodopsin. The optimum pH for this proteolytic activity is approximately 6, and the pH profile corresponds to an enzyme having pKa's of 4.4 +/- 0.1 and 7.7 +/- 0.1 for its active site residues. After analyzing a series of protease inhibitors, we found E-64, a specific thiol protease inhibitor, to be the most effective irreversible inhibitor of this enzyme, suggesting that the endoprotease might be a thiol protease. Affinity labeling studies using biotinylated affinity labeling probes have identified a 35 kDa protein as a candidate for the endoprotease.

Inhibition of neutrophil chemotaxis by protease inhibitors. Differential effect of inhibitors of serine and thiol proteases

Previous studies have demonstrated that neutrophils possess an active serine protease(s) which may be involved in the process of chemotaxis but the precise identity of this enzyme(s) remains to be determined. In this study fourteen different protease inhibitors were tested over a wide concentration range for their ability to inhibit unstimulated neutrophil movement and chemotaxis to C5a, fMLP and IL-8. Pretreatment of neutrophils with aspartyl or metallo-protease inhibitors had no effect on either chemotaxis or random cell movement. The thiol protease inhibitors E-64 and cystatin, as well as the thiol/serine inhibitors antipain and leupeptin, diminished only C5a-induced chemotaxis. Pretreatment of neutrophils with the serine protease inhibitors PMSF or 3,4-DCI significantly reduced chemotaxis to C5a, fMLP and IL-8. The inhibitor of trypsin-like serine proteases, TLCK, and the neutrophil elastase inhibitor MeO-Suc-AAPV-CMK had no inhibitory effect on cell movement. However, two different inhibitors of chymotrypsin-like serine proteases, TPCK and chymostatin, significantly inhibited movement to any chemoattractant. These results suggest that an active chymotrypsin-like serine protease is essential for neutrophils to respond to chemotactic stimuli.

Insulin Resistance Is Mediated by a Proteolytic Fragment of the Insulin Receptor

Insulin resistance is a common clinical feature of obesity and non-insulin-dependent diabetes mellitus, and is characterized by elevated serum levels of glucose, insulin, and lipids. The mechanism by which insulin resistance is acquired is unknown. We have previously demonstrated that upon chronic treatment of fibroblasts with insulin, conditions that mimic the hyperinsulinemia associated with insulin resistance, the membrane-associated insulin receptor beta subunit is proteolytically cleaved, resulting in the generation of a cytosolic fragment of the beta subunit, beta', and that the generation of beta' is inhibited by the thiol protease inhibitor E64 (Knutson, V. P. (1991) J. Biol. Chem. 266, 15656-15662). In this report, we demonstrate that in 3T3-L1 adipocytes: 1) cytosolic beta' is generated by chronic insulin administration to the cells, and that E64 inhibits the production of beta'; 2) chronic administration of insulin to the adipocytes leads to an insulin-resistant state, as measured by lipogenesis and glycogen synthesis, and E64 totally prevents the generation of this insulin-induced cellular insulin resistance; 3) E64 has no effect on the insulin-induced down-regulation of insulin receptor substrate-1, and therefore insulin resistance is not mediated by the down-regulation of insulin receptor substrate-1; 4) under in vitro conditions, partially purified beta' stoichiometrically inhibits the insulin-induced autophosphorylation of the insulin receptor beta subunit; and 5) administration of E64 to obese Zucker fatty rats improves the insulin resistance of the rats compared to saline-treated animals. These data indicate that beta' is a mediator of insulin resistance, and the mechanism of action of beta' is the inhibition of the insulin-induced autophosphorylation of the beta subunit of the insulin receptor.

A study of a synaptosomal thyrotropin releasing hormone-inactivating pyroglutamate aminopeptidase from bovine brain

Pyroglutamate aminopeptidase type II is a highly specific membrane-bound neuropeptidase that has the ability to remove N-terminal pyroglutamate (Glp) from Thyrotropin Releasing Hormone (Glp-His-Pro-NH 2) or very closely related tripeptides or tripeptide amides. In this paper we report on the purification and characterisation of a pyroglutamate aminopeptidase activity from the synaptosomal membranes of bovine brain. The Triton X-100 solubilised enzyme was purified nearly 600-fold by a combination of conventional column chromatography steps with a recovery/yield of 17.0%. Phase-partitioning experiments with Triton X-114 showed the activity to be an integral membrane protein. This detergent-solubilised pyroglutamate aminopeptidase activity was found to have a relative molecular mass of 240 kDa on a calibrated S-200 column. HPLC analysis on a C18 reverse-phase column showed that the purified activity displayed a very narrow substrate specificity cleaving only Thyrotropin Releasing Hormone (TRH) or the very closely related acid-TRH, LHRH (1–3) and the TRH-analogue (methyl-His)-TRH and had a K m of 100 μM for the fluorimetric substrate Glp-His-Pro-methyl-coumarin. The enzyme was inactivated by the metalchelator 1,10-ortho-phenanthroline but showed less sensitivity to EDTA. It also showed some inhibition by thiol protease inhibitors such as iodoacetate and n-ethylmaleimide. In summary, we have purified a pyroglutamate aminopeptidase from the synaptosomal membrane of bovine brain. This enzyme displays characteristics consistent with it being classified as a PAP type II neuropeptidase with only minor differences from other proteases in this group.

Rat hepatic lipocytes synthesize and secrete transin (Stromelysin) in early primary culture

Background & Aims : Hepatic lipocyte proliferation and activation are pivotal in liver fibrosis. Disruption of normal lipocyte-matrix interactions may contribute to this process. The synthesis of transin, which degrades normal liver matrix, by culture-activated hepatic lipocytes was investigated. Methods : Lipocytes were isolated by pronase/collagenase perfusion, density gradient centrifugation, and centrifugal elutriation. Transin messenger RNA in lipocytes was analyzed by Northern blotting. Transin activity was analyzed by zymography, Western blotting, immunocytochemistry, and quantitative [ 14C]β-casein degradation assay. Results : Transin messenger RNA was detected in early primary culture (3–5 days) but not in freshly isolated lipocytes or late primary culture. Zymography of lipocyte medium showed caseinolytic activity (relative molecular weight, 57 kilodaltons and 60 kilodaltons) inhibited by ethylenediaminetetraacetic acid but not thiol or serine protease inhibitors. Immunoblotting and immunocytochemistry confirmed the presence of transin in media and cells. Quantitative transin activity decreased progressively with increasing duration of primary lipocyte culture and myofibroblastic transformation. Conclusions : Rat hepatic lipocytes express the transin gene and secrete its product during the early phase of lipocyte activation in primary culture. Because this enzyme degrades a wide spectrum of normal basement membrane proteins and activates progelatinase B and interstitial collagenase, it may have an important role in liver injury and fibrosis.

Kupffer cell-derived 95-kd type IV collagenase/gelatinase B: Characterization and expression in cultured cells

Release of 92-kd type IV collagenase/gelatinase, also known as gelatinase B, by inflammatory and tumor cells is increasingly recognized and is believed to facilitate cellular migration across basement membranes. It has been implicated in the pathogenesis of many diseases, but little is known of its cellular origin(s) and function in liver. In this study we have demonstrated synthesis and release of gelatinase B by human and rat Kupffer cells in primary culture. Northern analysis of RNA extracted from Kupffer cells stimulated with phorbol ester demonstrated a 2.8 kb transcript for gelatinase B. Immunoblotting and zymography of serum-free Kupffer cell-conditioned media demonstrated extracellular release of immunoreactive enzyme and gelatinase activity, Mr 92,000 (95,000 from rat cells). The organomercurial 4-aminophenyl mercuric acetate (APMA) activated the enzyme in vitro, indicating secretion primarily as a proenzyme. Stimulation of Kupffer cells by phorbol ester markedly induced gelatinase B release, which was inhibited by cycloheximide. In contrast, cycloheximide had no effect on constitutive secretion in culture, suggesting that there is some intracellular storage. Kupffer cell-derived gelatinase B was also partially purified and characterized. After separation by gelatin sepharose and gel filtration chromatogrpahy, gelatin-degrading activities of 95, 88, 75, and 65 kd were detected, the three lower-molecular-weight species probably representing activated forms. Enzyme activity was inhibited by ethyl-enediaminetetra-acetic acid (EDTA), but not by serine- and thiol-protease inhibitors, and was restored by zinc. Activity was also inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1) and alpha-2 macroglobulin. The partially purified enzyme rapidly degraded denatured collagens (gelatin) as well as native types III, IV, and V collagens, but had no activity against casein, types I and VI collagens.

Kupffer cell-derived 95-kd type iv collagenase/gelatinase b: characterization and expression in cultured cells

Release of 92-kd type IV collagenase/gelatinase, also known as gelatinase B, by inflammatory and tumor cells is increasingly recognized and is believed to facilitate cellular migration across basement membranes. It has been implicated in the pathogenesis of many diseases, but little is known of its cellular origin(s) and function in liver. In this study we have demonstrated synthesis and release of gelatinase B by human and rat Kupffer cells in primary culture. Northern analysis of RNA extracted from Kupffer cells stimulated with phorbol ester demonstrated a 2.8 kb transcript for gelatinase B. Immunoblotting and zymography of serum-free Kupffer cell-conditioned media demonstrated extracellular release of immunoreactive enzyme and gelatinase activity, Mr 92,000 (95,000 from rat cells). The organomercurial 4-aminophenyl mercuric acetate (APMA) activated the enzyme in vitro, indicating secretion primarily as a proenzyme. Stimulation of Kupffer cells by phorbol ester markedly induced gelatinase B release, which was inhibited by cycloheximide. In contrast, cycloheximide had no effect on constitutive secretion in culture, suggesting that there is some intracellular storage. Kupffer cell-derived gelatinase B was also partially purified and characterized. After separation by gelatin sepharose and gel filtration chromatography, gelatin-degrading activities of 95, 88, 75, and 65 kd were detected, the three lower-molecular-weight species probably representing activated forms. Enzyme activity was inhibited by ethyl-enediaminetetra-acetic acid (EDTA), but not by serine-and thiol-protease inhibitors, and was restored by zinc. Activity was also inhibited by tissue inhibitor of metallo-proteinase-1 (TIMP-1) and α-2 macroglobulin. The partially purified enzyme rapidly degraded denatured collagens (gelatin) as well as native types III, IV, and V collagens, but had no activity against casein, types I and VI collagens.

Protease inhibitors diminish lymphocyte stimulation in vitro

Lymphocyte stimulation initiates activation of signal transduction pathways presumably enzymatic in nature. The next step includes gene activation and specific protein molecule production. Proteases of different specificity are crucial in enzyme activation: protein cleavage and biologically active molecule production. To elucidate the role of proteases in peripheral blood mononuclear cell (PBMC) activation, the broad specificity inhibitors of thiol (PHMB) and serine (TLCK) proteases have been used in vitro. Both inhibitors diminished the PHA-induced lymphocyte stimulation when applied at the beginning of culture; inhibitory effect was abrogated when inhibitors were introduced to the culture system 4 h later. Exogenous IL-2 abolished inhibition. PHMB activity was reversible whereas TLCK was irreversible. The inhibition of T lymphocyte-enriched proliferation is more distinctive as compared to that of PBMC. It can be concluded that proteases are involved in early events of lymphocyte proliferation and IL-2 production which is responsible for different states of cell growth.

Thiol-Protease Inhibitors Selectively Inhibit Presentation of Antigen Incorporated through B Cell Antigen Receptors

B cells efficiently uptake exogenous antigens through cell surface IgM molecules and present them to specific T cells. Although it is implicated that antigens taken up by surface IgM associate with newly synthesized class II molecules, the precise pathway of the presentation is still unclear. Here we report that thiol-protease inhibitors inhibit the presentation of trinitrophenol-conjugated ovalbumin by a leukemic B cell line, A20.HL, which constitutively expresses trinitrophenol-specific surface IgM although they had only a minimum effect on the presentation of unconjugated ovalbumin. In addition, thiol-protease inhibitors slightly reduced the surface expression of class II molecules. Our results suggest that exogenous antigen taken up through surface IgM is presented by a pathway which involves proteolysis by thiol-proteases and distinct from that for the antigens taken up through non-specific endocytosis.

Host Plasma Low Density Lipoprotein Particles as an Essential Source of Lipids for the Bloodstream Forms of Trypanosoma brucei

In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.

Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus

The foot-and-mouth disease virus (FMDV) Lb gene was cloned into bacterial expression vectors under the control of a T7 RNA polymerase promoter. The Lb protein was expressed in both an in vitro transcription-translation system and in Escherichia coli. In vitro expression of a construct containing the Lb gene fused to a portion of the VP4 and 3D genes demonstrated cis cleavage activity that could be blocked by the thiol protease inhibitor E-64. Lb expressed in E. coli was purified from the soluble fraction by metal chelation chromatography. Purified Lb had trans cleavage activity at the L/P1 junction and cleaved the p220 component of the cap-binding protein complex.

Changes in cathepsin B and lipofuscin during development and aging in rat brain and heart.

While results with inhibitors of thiol proteases have led to the suggestion that the progressive increase with age of lipofuscin in post-mitotic and some stable cells may be due to an age-related decline in the activity of these enzymes (Ivy et al., 1989), no direct evidence has been yet presented to support this hypothesis. In this study Wistar female rats were killed at age of 5, 14, and 24 months and the amounts of lipofuscin were histologically quantitated in neurons of the left cerebral parietal cortex and in cardiac myocytes of left ventricle. The sites of cathepsin B activity histochemically detected were quantitated in sections from left cerebral parietal cortex and left ventricle, and the activity of this enzyme was also measured biochemically in brain and heart homogenates. In line with previous findings, the amounts of lipofuscin in neurons and cardiac myocytes increased linearly during development and aging (from 5 to 14 and from 14 to 24 mo.). The sites of cathepsin B activity histochemically detected in sections from cerebral cortex significantly increased from 5 to 14 mo., but remained unchanged from 14 to 24 mo, while in sections from the left cardiac ventricle these sites of activity remained unchanged during development, and significantly increased during aging. On the other hand the biochemically determined activities of cathepsin B in brain and heart homogenates remained unchanged from 5 to 14 mo., but significantly decreased from 14 to 24 mo. These results suggest that the increase in lipofuscin with age may not be due to an age-wise decline in cathepsin B activity.

Mouse macrophage metalloelastase expressed in bacteria absolutely requires zinc for activity.

Mouse macrophage metalloelastase was expressed in Escherichia coli. This recombinant enzyme (rMME) was present in the inclusion bodies that were solubilized in 7 M guanidine HCl. After removal of guanidine HCl, rMME was purified with a Q-Sepharose column. Degradation of [3H]elastin by rMME absolutely required Ca2+; the optimal Ca2+ concentration was 5 mM. NaCl stimulated the enzyme activity; maximal stimulation was obtained at 400 mM. The rMME activity was inhibited by metalloprotease inhibitors, but not by serine, aspartyl, or thiol protease inhibitors. Among the divalent cations tested, only Ba2+ and Sr2+ exhibited marginal stimulation of rMME activity in the absence of Ca2+. Cu2+, Zn2+, or Cd2+ strongly inhibited rMME activity with IC50 values between 68 and 180 microM, while Mg2+, Ba2+, Mn2+, Co2+, and Sr2+ had no effect. The requirement of Zn2+ for rMME activity was determined. Significant enzyme activity was present in rMME treated with EDTA followed by Q-Sepharose column chromatography. Only when the inclusion bodies were solubilized in the presence of 20 mM EDTA, did an enzyme preparation which was absolutely dependent on exogenous Zn2+ for activity result. The optimal Zn2+ concentration for rMME activation was 100 microM. These results indicate that Zn2+ is tightly bound to rMME.

Isolation and molecular cloning of a novel bone phosphoprotein related in sequence to the cystatin family of thiol protease inhibitors.

We describe here the isolation of a novel non-collagenous protein from the acid demineralization extract of bovine cortical bone. This 24-kDa protein is multiply phosphorylated at serine residues in Ser-X-Glu/Ser(P) sequences, a recognition motif for phosphorylation by the secretory pathway protein kinase, and we have termed this protein secreted phosphoprotein 24 (spp24). The cDNA structure of spp24 was determined by sequencing cDNA fragments obtained by reverse transcription-polymerase chain reaction, 3'-rapid amplification of cDNA ends, and screening a lambda gt11 cDNA library. This cDNA sequence predicts a 200-residue initial translation product which consists of a 20-residue signal sequence and the 180-residue mature spp24. Northern blot analysis using the spp24 cDNA showed that spp24 mRNA is in liver and bone but not in heart, lung, kidney, or spleen. A search of existing protein sequences revealed that the N-terminal 107 residues of mature spp24 are related in sequence to the cystatin family of thiol protease inhibitors, which suggests that spp24 could function to modulate the thiol protease activities that are known to be involved in bone turnover. Several of the proteins in the cystatin family that are most closely related to spp24 are not only thiol protease inhibitors but are also precursors to peptides with potent biological activity, peptides such as bradykinin and the neutrophil antibiotic peptides. It is therefore possible that the intact form of spp24 found in bone could also be a precursor to a biologically active peptide, a peptide which could coordinate an aspect of bone turnover.

Identification and localisation of a synaptosomal membrane prolyl endopeptidase from bovine brain.

Prolyl endopeptidase, which has long been recognised for its importance in the degradation of several neuropeptides such as thyroliberin, luteinising hormone releasing hormone, angiotensin, substance P and neurotensin, has been widely characterised as a cytosolic enzyme. However, in this paper, we report the presence of a prolyl endopeptidase activity in the particulate fractions of bovine brain, which is distinct from that in the cytoplasm. This previously uncharacterised activity was found to reside in the synaptosomal membranes, a location which is highly significant for the inactivation of neuropeptides in brain. Following vigorous salt washing and osmotic shock, the prolyl endopeptidase activity was released from the membranes by treatment with the detergent Triton X-100, and was partially purified by gel filtration on a Sephacryl S-200HR column. This prolyl endopeptidase activity was shown to have a molecular mass (87 kDa) higher than the cytosolic prolyl endopeptidase but, from initial investigation, appears to demonstrate a similarly broad substrate specificity towards proline-containing neuropeptides. The partially purified enzyme was inhibited by certain thiol-protease inhibitors and was also found to be sensitive to the metal chelator 1,10-phenanthroline.

Aggrecan degradation in osteoarthritis and rheumatoid arthritis

Aggrecan turnover is critically important to maintain extracellular matrix homeostasis in articular cartilage. Cartilage aggrecan metabolism has been studied in chondrocyte cell cultures, cartilage explant cultures, and in whole animal models. In many of these studies, matrix metalloproteinases (MMPs) are proposed to degrade cartilage aggrecan. MMP expression appears elevated in joint tissues from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) (Dean et al. 1989, Wolfe et al. 1993) and inhibitors of both MMPs and thiol proteinases have been shown to block aggrecan (Buttle et al. 1992) and type II collagen (Mort et al. 1993) degradation in cartilage explant cultures. Using antibodies and cDNA probes, elevations in expression and concentration of many of these enzymes in animal models and in OA and RA have been described. Human cartilage aggrecan has now been cloned and sequenced (Doege et al. 1991). This information, in combination with the ability to sequence aggrecan and aggrecan fragment...

Peptides related to thyrotrophin-releasing hormone are degraded in seminal plasma by an enzyme similar to prolyl endopeptidase.

A TRH-like peptide, fertilization-promoting peptide (FPP), is present in high concentrations in mammalian prostate and semen and enhances the fertilization potential of spermatozoa. In this study, we have examined the properties of the enzyme that degrades TRH and FPP in rabbit seminal plasma. The enzyme responsible had a pH optimum of approximately 7.0, was inhibited by serine (di-isopropyl flurophosphate) and thiol (N-ethylmaleimide) protease inhibitors, bacitracin and concentrations of Zn2+ naturally present in seminal plasma: these functional reagents are all known to be potent inhibitors of prolyl endopeptidase. The major product after incubation of [3H]TRH in seminal plasma for 100 min was acid TRH (deamidated TRH) which is also the product after incubation of TRH with prolyl endopeptidase. Our results are consistent with the enzyme responsible for degradation of TRH and FPP in seminal plasma being similar to prolyl endopeptidase. The enzyme identified in this study is secreted and is therefore likely to be different from prolyl endopeptidase characterized from porcine brain, because the latter enzyme is known to be located in the cytosolic compartment of the cell.

Amino Acids and Peptides. XXXIX. Synthesis of iNoc-Gln-Val-Val-Ala-Ala-pNA and Its Action on Thiol Proteinases.

Based on the results of X-ray analysis of the complex between Suc-Gln-Val-Val-Ala-Ala-pNA, a fairly potent thiol proteinase inhibitor, and papain, iNoc-Gln-Val-Val-Ala-Ala-pNA was designed and prepared and its inhibitory activity against thiol proteinases was examined. iNoc-Gln-Val-Val-Ala-Ala-pNA inhibited cathepsin L fairly specifically, although its potency is not high.

Mast Cell Procarboxypeptidase A: MOLECULAR MODELING AND BIOCHEMICAL CHARACTERIZATION OF ITS PROCESSING WITHIN SECRETORY GRANULES

Previously, we characterized murine mast cell procarboxypeptidase A (MC-proCPA) as an inactive zymogen. To investigate the mechanisms for this lack of enzymatic activity and the processing of the zymogen to the active form, we now have performed molecular modeling of the tertiary structure of murine MC-proCPA based on the x-ray crystallographic structures of porcine pancreatic procarboxypeptidases A and B. Our model predicts that MC-proCPA retains a high degree of structural similarity to its pancreatic homologues. The globular propeptide physically blocks access to the fully formed active site of the catalytic domain and contains a salt bridge to the substrate-binding region that precludes docking of even small substrates. Based on consideration of the predicted tertiary structure and charge field characteristics of the model, the activation site (between GluA94 and Ile1) appears to be highly exposed even after MC-proCPA binds to secretory granule proteoglycans. Based on the steady-state levels of MC-proCPA versus MC-CPA, cycloheximide inhibition of protein synthesis, and brefeldin A blockage of protein sorting, we show that MC-proCPA is processed rapidly in murine mast cell line KiSV-MC14 with a half-life of 26 +/- 5 min (mean +/- S.D., n = 3), and the processing occurs within the secretory granules. The enzyme responsible for this processing may be a thiol protease since treatment of the KiSV-MC14 with 200 microM E-64d, a selective thiol-protease inhibitor, increases MC-proCPA by 2.7 +/- 0.2-fold (mean +/- S.D., n = 3) within 6 h of application.

Detection of the substance immunologically cross-reactive with insulin in insulin ria is an artifact caused by insulin tracer degradation: involvement of the insulin-degrading enzyme

In previous literature, the existence of a new insulin-like substance found in tumor tissues, termed substance immunologically crossreactive with insulin (SICRI), has been proposed. In these studies, insulin-specific radioimmunoassay (RIA) was the only detection method for SICRI. The mouse melanoma B16BL6 cell line was found to be a rich source of SICRI. In this paper, we show that SICRI is not expressed in B16BL6 cells. Previous RIA measurements were wrongly ascribed to SICRI. What was really measured was a positive artifact caused by insulin tracer degradation in RIA. Several lines of evidence indicate that protease responsible for insulin degradation in B16BL6 cells is insulin-degrading enzyme (IDE; EC 3.4.22.11). First, SICRI activity of B16BL6 cytosol measured by insulin RIA was inhibited by thiol protease inhibitor N-ethylmaleimide (NEM). Thiol active agents as well as metal chelators, both potent IDE blockers, inhibited also the insulin-degrading activity of the same sample. Second, cross-linking to 125I-labeled insulin of partially purified sample with highest insulin RIA activity specifically labeled only a single protein with molecular mass similar to IDE (110 kDa). Labeling was blocked by ‘cold’ insulin in excess. Third, kinetic studies of insulin degradation by RIA active Chromatographic fractions revealed an apparent K d of 90 nM which is very similar to the reported affinity of insulin for IDE ( K d = 100 nM). Additionally, in B16BL6 as well as in mouse myeloid leukemia cells, IDE gene is actively transcribed and this expression was found to be much stronger than in normal mouse tissues. In conclusion, our results strongly question the real existence of SICRI. They showed that IDE is responsible for false positive measurements of SICRI in RIA. These artifacts were circumvented by IDE inhibitor NEM. Finally, increased expression of IDE gene in mouse tumor tissues suggests a possible important role for IDE in the process of malignant transformation.