Inhibition of a cathepsin L-like cysteine protease by a chimeric propeptide-derived inhibitor

Like other papain-related cathepsins, congopain from Trypanosoma congolense is synthesized as a zymogen. We have previously identified a proregion-derived peptide (Pcp27), acting as a weak and reversible inhibitor of congopain. Pcp27 contains a 5-mer YHNGA motif, which is essential for selectivity in the inhibition of its mature form [Lalmanach, G., Lecaille, F., Chagas, J. R., Authié, E., Scharfstein, J., Juliano, M. A., and Gauthier, F. (1998) J. Biol. Chem. 273, 25112-25116]. In the work presented here, a homology model of procongopain was generated and subsequently used to model a chimeric 50-mer peptide (called H3-Pcp27) corresponding to the covalent linkage of an unrelated peptide (H3 helix from Antennapedia) to Pcp27. Molecular simulations suggested that H3-Pcp27 (pI = 9.99) maintains an N-terminal helical conformation, and establishes more complementary electrostatic interactions (E(coul) = -25.77 kcal/mol) than 16N-Pcp27, the 34-mer Pcp27 sequence plus the 16 native residues upstream from the proregion (E(coul) = 0.20 kcal/mol), with the acid catalytic domain (pI = 5.2) of the mature enzyme. In silico results correlated with the significant improvement of congopain inhibition by H3-Pcp27 (K(i) = 24 nM), compared to 16N-Pcp27 (K(i) = 1 microM). In addition, virtual alanine scanning of H3 and 16N identified the residues contributing most to binding affinity. Both peptides did not inhibit human cathepsins B and L. In conclusion, these data support the notion that the positively charged H3 helix favors binding, without modifying the selectivity of Pcp27 for congopain.     

Inhibition of experimental autoimmune encephalomyelitis by a nonimmunogenic non-self peptide that binds to I-Au.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory neurologic disease initiated by myelin basic protein-reactive CD4+ T cells, which are restricted by a particular MHC class II molecule. Recent studies have utilized inhibitor peptides that bind to restricting MHC class II molecules in order to inhibit EAE, presumably by means of competing with encephalitogenic epitopes. However, these studies leave open the possibility of alternative explanations, such as Ag-specific nonresponsiveness and immunodominance. In order to demonstrate that competition for MHC binding alone can inhibit EAE, the inhibitor peptide should ideally be structurally unrelated and nonimmunogenic yet physically associate with the MHC class II molecule. In this study, we show that the OVA-323-339 peptide, which is unrelated to the disease-inducing peptide, binds to A alpha uA beta u. However, although OVA-323-339 is extremely immunogenic in A alpha dA beta d-expressing BALB/c mice, it is nonimmunogenic in (PL/J x SJL)F1 and PL/J mice expressing A alpha uA beta u. When administered as a coimmunogen with Ac1-11, OVA-323-339 inhibited induction of EAE in (PL/J x SJL)F1 mice. Myelin basic protein-89-101, which does not bind A alpha uA beta u, had no effect on the disease process. This study provides evidence that MHC class II binding alone can modulate the induction of EAE. The use of a nonimmunogenic non-self peptide to modulate an autoimmune disease minimizes the potential complications of immunodominance or alternative regulatory mechanisms associated with immunogenic peptide therapies and further confirms the MHC-blocking model of immunosuppression.     

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.     

Ceramide synthase 6 plays a critical role in the development of experimental autoimmune encephalomyelitis

Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C(16:0)-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C(16:0)-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ-activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ-mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C(16:0)-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C(16:0)-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C(16:0)-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C(16:0)-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.     

Inhibition by acidic phospholipids of protein degradation by ER-60 protease, a novel cysteine protease, of endoplasmic reticulum.

A protein (ER60) with sequence similarity to phosphoinositide-specific phospholipase C-alpha purified from rat liver endoplasmic reticulum (ER) degraded ER resident proteins and is really a protease [(1992) J. Biol. Chem. 265, 15152-15159]. Therefore, ER60 is called ER-60 protease. We now show that negatively charged phospholipids, phosphatidylinositol, phosphatidylinositol 4,5-bisphosphate and phosphatidylserine inhibit ER protein degradation by ER-60 protease. Phosphatidylcholine and phosphatidylethanolamine show no effect on the activity of ER-60 protease. With the use of protease inhibitors, ER-60 protease is shown to be a novel cysteine protease distinct from those of the cytosol and lysosomes.     

Fcgamma receptor-ligating complexes improve the course of experimental autoimmune encephalomyelitis by enhancing basal Th2 responses

IL-12p40 and macrophages are essential for the induction of disease in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. In this paper, we show that treatment of mice with opsonized erythrocytes, which have been shown to ligate Fcgamma receptors on macrophages and alter their cytokine profile, significantly delayed the onset of experimental autoimmune encephalomyelitis. This protection correlated to the induction of Th2 responses by autoreactive T cells, enhanced basal systemic responses and a significant downregulation of IL-12p40 and nitric oxide synthase-2, but not IFN-gamma expression. IL-4 was essential for the protection by opsonized erythrocytes as the effects of treatment were eliminated in IL-4-deficient mice. Together these studies suggest that the ligation of Fcgamma receptors can modify the development of autoimmune disease by altering macrophage activation and enhancing Th2 responses.     

Inhibition of natural type I IFN-producing and dendritic cell development by a small molecule receptor tyrosine kinase inhibitor with Flt3 affinity

In vivo steady-state type I natural IFN-producing and dendritic cell (DC) development is largely dependent on Flt3 signaling. Natural IFN-producing and DC progenitors and their respective downstream cell populations express the flt3 receptor, and Flt3 ligand (Flt3L)(-/-) mice have reduced while Flt3L-injected mice develop markedly increased numbers of both cell types. In the present study, we show that SU11657, a small multitargeted receptor tyrosine kinase inhibitor with Flt3 affinity, suppressed in vitro natural IFN-producing and DC development in Flt3L-supplemented mouse whole bone marrow cell cultures in a dose-dependant manner, while DC development in GM-CSF-supplemented cultures was not affected. In vivo SU11657 application led to a significant decrease of both natural IFN-producing and DCs, comparable to the reduction observed in Flt3L(-/-) mice. Conversely, Flt3L plasma levels increased massively in inhibitor-treated animals, likely via a regulatory feedback loop, without being able to compensate for pharmacological Flt3 inhibition. No obvious toxicity was observed, and hemopoietic progenitor cell and stem cell function remained intact as assessed by myeloid colony-forming unit activity and in vivo bone marrow repopulation assays. Furthermore, upon treatment discontinuation, IFN-producing and DCs recovered to normal levels, proving that treatment effects were transient. Given the importance of IFN-producing and DCs in regulation of immune responses, these findings might lead to new pharmacological strategies in prevention and treatment of autoimmune diseases and complications of organ or blood cell transplantation.     

Inhibition of Plasmodium falciparum cysteine protease falcipain-2 by a human cross-class inhibitor serpinB3: A mechanistic insight

Falcipain-2(FP2), a cysteine protease from Plasmodium falciparum, cleaves host erythrocyte hemoglobin and specific membrane skeleton components during the parasite life cycle. Therefore its inhibition has been considered as an attractive approach to combat the disease. SerpinB3 (SPB3) belongs to the ovalbumin-serpin family and is a potent cross-class inhibitor of cysteine cathepsins L, K, S and papain. This study explored the possibility of inhibition of FP2 by SPB3. It turned out that general proteolytic activities as well as specific hemoglobinolytic activity of FP2 have been inhibited by SPB3. Furthermore, studies have been designed to investigate and characterize the mechanism of inhibition in comparison with proteases Cathepsin L (CTSL) and papain. The Ki value of inhibition for FP2, measured against its specific substrate (VLK-pNA), is 338.11 nM and stoichiometry (I/E ratio) of inhibition is 1. These values are comparable to CTSL and papain. Analytical gel filtration profile and CD spectroscopy data confirm FP2-SPB3 complex formation. Our studies revealed that interaction of SPB3 with FP2 is non-covalent type like that of CTSL and papain but unlike other serine protease-inhibiting serpins. An in-silico docking and simulation study have been performed with FP2 as well as CTSL and results suggest different binding mode for FP2 and CTSL, though both the complexes are stable with significant contribution from electrostatic energy of interaction. We further showed a disease state mutant SPB3-Gly351Ala performed better anti-protease activity against FP2. This study, for the first time, has shown a serpin family inhibitor from human could efficiently inhibit activity of FP2.     

Methylacetylenic putrescine (MAP), an inhibitor of polyamine biosynthesis, prevents the development of collagen-induced arthritis

The objective of the present investigation was to examine the effects of an irreversible inhibitor of ornithine decarboxylase (2R,5R)-6-heptyne-2,5,diamine (methylacetylenic putrescine, MAP) on experimentally induced arthritis in mice. MAP (0.5-0.05%) was administered in drinking water to DBA/1 mice immunized with native chick type II collagen (CII). The development of arthritis was inhibited only in those mice receiving 0.5% MAP; lower doses were ineffective. Putrescine and spermidine levels were decreased and spermine levels were increased in spleen and lymph node cells from drug-treated mice compared to control arthritic mice. Furthermore, when control mice were developing arthritis, serum anti-CII antibody levels were lower in the MAP-treated group. MAP inhibited antibody production early in the immune response to CII; there was an association between inhibition of antibody production and inhibition of the development of arthritis. When MAP was discontinued, the nonarthritic, drug-treated mice did not develop the disease. Late administration of MAP (beginning 19 days after CII immunization) did not affect the incidence or the severity of the arthritis. Cyclophosphamide treatment begun at the same time significantly inhibited the development of the disease. In vitro T cell responses to denatured type II collagen (dCII) in untreated and MAP-treated mice were examined 14 days after immunization with CII. This is a time of peak T cell responsiveness in untreated animals. MAP treatment had no effect on the T cell response to dCII. These results indicate that MAP can prevent the development of CII-induced arthritis, possibly by inhibiting the autoantibody response. Therefore, inhibitors of polyamine biosynthesis deserve further investigation as potential immunosuppressive agents.     

Low dose CP-690,550 (tofacitinib), a pan-JAK inhibitor, accelerates the onset of experimental autoimmune encephalomyelitis by potentiating Th17 differentiation

Th17 cells, which have been implicated in autoimmune diseases, require STAT3 signaling activated by IL-6 or IL-23 for their development. Other Th1 and Th2 cytokines such as IL-2, IFN-γ and IL-4 strongly suppress Th17 development. Recently, CP-690,550 (tofacitinib), originally developed as a JAK3 inhibitor, has been shown to be effective in phase III clinical trials of rheumatoid arthritis and collagen-induced arthritis (CIA) models, but the precise mechanism of the effect, especially with respect to Th17 cells, is poorly understood. To our surprise, a low dose CP-690,550 was found to accelerate the onset of experimental autoimmune encephalomyelitis (EAE) at a concentration that suppressed CIA. At an early stage after immunization, more IL-17 production was observed in 15mg/kg body weight CP-690,550-treated mice than in untreated mice. In vitro, CP-690,550 inhibited both Th1 and Th2 development, while promoting Th17 differentiation at 10-50nM concentrations. Enhancement of Th17 by CP-690,550 is probably due to suppression of IL-2 signaling, because anti-IL-2 antibodies cancel the Th17-promoting effect of CP-690,550. CP-690,550 selectively inhibited IFN--induced STAT1, IL-4-induced STAT6 and IL-2-induced STAT5 at 3-30nM, while suppression of IL-6-induced STAT3 phosphorylation required a concentration greater than 100nM. In HEK293T cells, CP-690,550 less effectively suppressed JAK1-mediated STAT3 phosphorylation compared with JAK3. These results suggest that CP-690,550 has a different effects among JAKs and STATs, thereby affecting helper T cell differentiation, and murine autoimmune disease models.     

Inhibition of murine relapsing experimental autoimmune encephalomyelitis by immune tolerance to proteolipid protein and its encephalitogenic peptides.

Tolerization of SJL/J mice with splenocytes coupled with proteolipid protein (PLP), the major protein component of central nervous system myelin, resulted in dramatic inhibition of relapsing experimental autoimmune encephalomyelitis (R-EAE) induced by mouse spinal cord homogenate (MSCH). Mice tolerized with splenocytes coupled with MSCH (a complex mixture of neuroantigens) or with purified PLP, but not purified myelin basic protein, were resistant to the development of clinical and histologic R-EAE. In addition, mice rendered tolerant to an encephalitogenic peptide of PLP were significantly protected, whereas mice tolerized to a nonencephalitogenic peptide of PLP were highly susceptible, to the induction of MSCH-induced R-EAE. Thus, immune responses directed against encephalitogenic regions of PLP appear to play a major role in the development of R-EAE induced by MSCH in SJL/J mice. These results also indicate that determinant-specific immune tolerance is a feasible approach to the regulation of a disease that involves autoimmune responses to a variety of Ag.     

Lipids and caplain in guinea pig tissues in the process of development of experimental autoimmune encephalomyelitis

The character of some lipids level change--cholesterol and phospholipids--as basic lipid components of cell membranes in the guinea-pig brain and liver tissue, and in serum in conditions of development of experimental autoimmune encephalomyelitis (EAE) have been investigated on the 11th, 21st, 27th day after inoculation. It has been detected, that the level of the investigated lipids changes wavely and indifferent-direction in the brain tissue on the 21st day of EAE. Similar variability observed in the activity of proteolytic ferment calpain, which is authentically reduced in the brain tissue by the 11th hour and increases up to the test objective level in the subsequent periods of EAE development. In the liver the level of alpha-tocopherol is reduced, while the content of studied lipids does not change. The investigated parameters can be attributed to the factors, which play an essential role in structural stability of cell membranes and their variability in conditions of EAE development is related to the processes of nervous cells demyelinisation and, hence to occurrence of such pathology as multiple sclerosis in people.     

Inhibition of experimental autoimmune encephalomyelitis induction in SJL/J mice by using a peptide with high affinity for IAs molecules.

Blocking of the Ag presenting function of MHC by peptides capable of high affinity binding to this molecule has been proposed as a potential immunotherapeutic intervention in MHC-associated diseases. Recent studies have used this strategy to prevent the induction of experimental allergic encephalomyelitis (EAE) in mice. However, because of the close structural relationship between the inhibitor and encephalitogenic peptides, the results of these previous studies have been difficult to interpret with regard to whether MHC blockade was the mechanism by which the inhibitory peptides functioned. In our study, we have determined the capacity of unrelated peptides capable of binding with high affinity to IAs in inhibiting the induction of EAE in SJL/J mice after immunization with the autoantigenic peptide PLP 139-151. Prevention of the disease was accomplished by two methods: 1) when inhibitor was administered together with the encephalitogenic peptide at the time of immunization, as in previous studies, and 2) when inhibitor was administered at a separate site from the autoantigen 1 day before the immunization with that Ag. Inhibition was due to binding of the inhibitor to IAs, as evidenced by the fact that a control peptide incapable of binding to this MHC had no effect on the course of the disease. The finding that inhibitor could also be efficacious when administered at a separate site has implications for potential use of such a strategy to reverse ongoing autoimmune diseases. The inhibitor had to be present during the time of Ag stimulation, and had no long term inhibitory effects, in that a secondary immune response to the encephalitogenic peptide was not inhibited in animals given the inhibitory peptide before the induction of a primary response. This is compatible with the conclusion that MHC blockade was, in fact, the mechanism of the inhibition, rather than as a result of any long term suppressive effects on immunoreactive T cells. Finally, not only did administration of the inhibitory peptide lead to a prevention of the induction of EAE, but it could also be shown to decrease the T cell proliferative response in vitro to the autoantigen.     

Partial purification and characterization of a cysteine protease inhibitor from the plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.     

Effects of peptide fraction and counter ion on the development of clinical signs in experimental autoimmune encephalomyelitis

The most commonly used immunogen to induce experimental autoimmune encephalomyelitis is MOG_35‐55, a 21‐residue peptide derived from myelin oligodendrocyte glycoprotein (MOG). In most studies, mice exhibit a chronic disease; however, in some studies mice show a transient disease. One variable that is not often controlled for is the peptide fraction of the purified MOG material, which can vary from less than 50% to over 90%, with the remainder of mass primarily comprised of the counter ion used for peptide purification. We compared the development of clinical signs in female C57Bl6 mice immunized with two commercially available MOG_35‐55 peptides of similar purity but different peptide fraction (MOG‐A being 45%; MOG‐B being 72%). A single immunization with MOG‐A induced a chronic disease course with some recovery at later stages, whereas immunization with MOG‐B induced a similar course of disease but with significantly lower average clinical scores despite a higher peptide content. The addition of a booster immunization significantly increased clinical severity with both preparations, and significantly reduced the average day of onset using MOG‐A. To determine if the counter ion could influence disease, we compared MOG‐B‐containing trifluoroacetate with MOG‐B‐containing acetate. Although disease incidence and severity were similar, the average day of disease onset occurred approximately 5 days earlier with the use of MOG‐B‐containing trifluoroacetate. These results demonstrate that differences in peptide fraction influence the course of encephalomyelitis disease, which may be due in part to the levels of counter ions present in the purified material. These findings underscore the fact that a knowledge of peptide fraction is as critical as knowledge of peptide purity when using peptides from different sources.

     

Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase

Chemotherapy that is used to treat human immunodeficiency virus type-1 (HIV-1) infection focuses primarily on targeting virally encoded proteins. However, the combination of a short retroviral life cycle and high mutation rate leads to the selection of drug-resistant HIV-1 variants. One way to address this problem is to inhibit non-essential host cell proteins that are required for viral replication. Here we show that the activity of HIV-1 integrase stimulates an ataxia-telangiectasia-mutated (ATM)-dependent DNA damage response, and that a deficiency of this ATM kinase sensitizes cells to retrovirus-induced cell death. Consistent with these observations, we demonstrate that a novel and specific small molecule inhibitor of ATM kinase activity, KU-55933, is capable of suppressing the replication of both wild-type and drug-resistant HIV-1.     

Inhibition of gut proteases and development of dengue vector, Aedes aegypti by Allium sativum protease inhibitor

The paper describes the bio efficacy of a protease inhibitor; isolated from Allium sativum ‘garlic’ (ASPI); against Aedes aegypti mosquito, a well-known transmitter of dengue and Chikungunya. The purification of protease inhibitor from Allium sativum ‘garlic’ (ASPI) was carried out by ammonium sulfate precipitation followed by Fast Protein Liquid Chromatography using akta DEAE-Cellulose column. The protein fraction demonstrating trypsin inhibitory activity was further evaluated for its insecticidal activity using gut protease inhibition assay and larvicidal assay. ASPI is an inhibitor of porcine trypsin (IC₅₀ of 650.726?μg/mL) and has molecular weight of ~15?kDa determined by SDS PAGE similar to other inhibitors of the Kunitz-type family (14–26?kDa). ASPI demonstrated 50% reduced activity of Ae. aegypti midgut proteases and showed a dose-dependent acute toxicity on Ae. aegypti 3rd instars exhibiting LC₅₀ value of ~50.827?μg/mL. After ten days of larval exposure ASPI resulted in a 24-h delay of larval development and ~72% mortality at 61.5?μg/mL. These results suggest that ASPI may serve as potent insecticidal agent and hence opens a new gateway in the field of phyto-remediation.     

Inhibition of digestive proteinases of stored grain Coleoptera by oryzacystatin, a cysteine proteinase inhibitor from rice seed

Electrophoresis of midgut extracts from the rice weevil, Sitophilus oryzae, and the red flour beetle, Tribolium castaneum, in polyacrylamide gels containing sodium dodecyl sulfate and gelatin revealed there was one major proteinase (apparent molecular mass = 40,000) in the rice weevil and two major proteinases (apparent molecular masses = 20,000 and 17,000) in the red flour beetle. The pH optima using [3H]casein as substrate were about pH 6.8 for the rice weevil and pH 5.2 for the red flour beetle. Use of specific inhibitors, including L-trans-epoxysuccinyl-leucylamino-(4- guanidino)-butane (E-64), p-chloromercuriphenylsulfonic acid (PCMS), and oryzacystatin, indicated that nearly all of the proteinase activity against casein was contributed by cysteine proteinases. The estimated IC50 values for oryzacystatin were 2 x 10(-6) M and 4 x 10(-7) M when tested against midgut extracts from T. castaneum and S. oryzae, respectively.