Selective killing of human M1 macrophages by Smac mimetics alone and M2 macrophages by Smac mimetics and caspase inhibition.

The inflammatory and anti-inflammatory Mϕs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting Mϕ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory Mϕ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated Mϕs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human Mϕ subsets are resistant to the cytotoxic effects of SMs, we show that M1 Mϕs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 Mϕs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 Mϕs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human Mϕs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.

Optimisation of the antifungal potency of the amidated peptide H-Orn-Orn-Trp-Trp-NH2 against food contaminants.

The design of novel efficient antimicrobial peptides (AMPs) faces several issues, such as cost of synthesis, proteolytic stability or cytotoxicity. The identification of key determinants involved in the activity of AMPs, such as cationicity and amphipathicity, allowed the synthesis of short peptides with optimized properties. An ultrashort peptide made of the sequence H-Orn-Orn-Trp-Trp-NH2 (O3TR) showed antifungal activity against several contaminants from food products. This peptide inhibited the growth of the filamentous fungi Fusarium culmorum, Penicillium expansum and Aspergillus niger within a range of concentration of 12.5-50µg/ml. In addition, O3TR inhibited the growth of the yeast Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii and Kluyveromyces lactis within the range 12.5-50µg/ml. A derivative peptide, called C12O3TR, made by the addition of lauric acid at the N-terminus of O3TR was 2- to 8-fold more active than O3TR against every species. In addition to the inhibition of conidial germination, O3TR and C12O3TR killed F. culmorum hyphae at 100 and 50µg/ml respectively. The MIC of the two peptides against F. culmorum and Z. bailii after heat treatment at 100°C for 60 min and within the pH range 3-10, were not changed. However, the activity of O3TR against F.culmorum and Z. bailii was strongly reduced in salt solutions, whereas the lauric acid peptide kept its antifungal activity and resistance to proteolytic digestion. The conjugation with lauric acid reduced the random coiled structure and increased the α-helical content of O3TR. After conjugation with the dye tetramethylrhodamine (TMR), both peptides entered F. culmorum spores. They also both induced permeabilization of F. culmorum hyphae but only C12O3TR permeabilized Z. bailii membrane. In contrast to the lipopeptide, O3TR did not show haemolytic or cytotoxic activity when applied at the concentrations that exhibited antifungal potency. The two peptides were challenged against a yeast cocktail of S. cerevisiae and Z. bailii, and A. niger in different commercial beverages. After 7 days, O3TR was able to inhibit the yeast cocktail in a commercial lager and carbonated drink. Due to its antifungal potency, high stability and low cytotoxicity, the tetrapeptide could represent a promising starting point of a novel food preservative.

Identification of mycobacterial membrane proteins and their types using over-represented tripeptide compositions.

Mycobacterium can cause many serious diseases, such as tuberculosis and leprosy. Its membrane proteins play a critical role for multidrug-resistance and its tenacious survival ability. Knowing the types of membrane proteins will provide novel insights into understanding their functions and facilitate drug target discovery. In this study, a novel method was developed for predicting mycobacterial membrane protein and their types by using over-represented tripeptides. A total of 295 non-membrane proteins and 274 membrane proteins were collected to evaluate the performance of proposed method. The results of jackknife cross-validation test show that our method achieves an overall accuracy of 93.0% in discriminating between mycobacterial membrane proteins and mycobacterial non-membrane proteins and an overall accuracy of 93.1% in classifying mycobacterial membrane protein types. By comparing with other methods, the proposed method showed excellent predictive performance. Based on the proposed method, we built a predictor, called MycoMemSVM, which is freely available at http://lin.uestc.edu.cn/server/MycoMemSVM. It is anticipated that MycoMemSVM will become a useful tool for the annotation of mycobacterial membrane proteins and the development of anti-mycobacterium drug design.

Unique structural features of the peptidoglycan of Mycobacterium leprae.

The peptidoglycan structure of Mycobacterium spp. has been investigated primarily with the readily cultivable Mycobacterium smegmatis and Mycobacterium tuberculosis and has been shown to contain unusual features, including the occurrence of N-glycolylated, in addition to N-acetylated, muramic acid residues and direct cross-linkage between meso-diaminopimelic acid residues. Based on results from earlier studies, peptidoglycan from in vivo-derived noncultivable Mycobacterium leprae was assumed to possess the basic structural features of peptidoglycans from other mycobacteria, other than the reported replacement of l-alanine by glycine in the peptide side chains. In the present study, we have analyzed the structure of M. leprae peptidoglycan in detail by combined liquid chromatography and mass spectrometry. In contrast to earlier reports, and to the peptidoglycans in M. tuberculosis and M. smegmatis, the muramic acid residues of M. leprae peptidoglycan are exclusively N acetylated. The un-cross-linked peptide side chains of M. leprae consist of tetra- and tripeptides, some of which contain additional glycine residues. Based on these findings and genome comparisons, it can be concluded that the massive genome decay in M. leprae does not markedly affect the peptidoglycan biosynthesis pathway, with the exception of the nonfunctional namH gene responsible for N-glycolylmuramic acid biosynthesis.

Immunomodulatory potential of hydrophobic analogs of Rigin and their role in providing protection against Plasmodium berghei infection in mice.

Here, we report the immunomodulating potential of N-palmitoyl-amino-ethyl-rigin amide (PR) and N-cholestanyl-amino-ethyl-rigin amide (CR), the two new structural analogs of rigin (an IgG-derived tetrapeptide). Their activity profiles are compared with native tuftsin (NT) and/or N-palmitoyl-amino-ethyl-tuftsin amide (PT) taken as positive control. To explore the possibility of their use as targeting molecules, they are incorporated into the liposome bilayer and, subsequently, interacted with macrophages in an in vitro study. The new analogs of rigin with the hydrophobicity introduced at the C-terminus are found to considerably improve both the cell-mediated and the humoral immune responses in mice. However, unlike tuftsin and its analog, which mainly activate polymorphonuclear leukocytes and macrophages, the rigin analogs appear to manifest their response more through lymphocytes. When administered prophylactically to a group of mice, at the dose of 100 micrograms/0.5 ml/mouse/day for 2 days (i.v.), followed by a challenge presented with 1 x 10(6) rbcs parasitised with Plasmodium berghei on day 0, substantial reduction in parasitaemia and rate of mortality is observed. This led to increase the median survival time (MST) of the treated group in comparison to the control group. The response is found to be more prominent in CR-treated mice possibly because of the presence of steroid moiety, which is likely to have more productive interaction with cell membranes. Incorporation of these peptides into the bilayer of liposomes does not alter the permeability behavior of vesicles and, in fact, enhances their uptake by the macrophages in an in vitro study. The effect, however, is dependent on both, the concentration of peptide liposomes and the time of incubation. Present study, thus, establishes the possible use of these analogs not only as adjuvant in chemotherapy, but also as a prophylactic supplement to boost the natural immune status. The activity response of rigin analogs is manifested through lymphocytes, they can also find use in the chemotherapy of diseases, like leishmaniasis, tuberculosis and leprosy, where macrophage activity is either tamed or impaired by pathogens.

Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site.

Plasminogen activation by the urokinase-type plasminogen activator (uPA) is facilitated in the presence of cells expressing the glycolipid-anchored high-affinity receptor for uPA (denoted uPAR). Structures involved in the interaction between human uPAR and a decamer peptide antagonist of uPA binding (SLNFSQYLWS) were previously tagged by specific site-directed photoaffinity labeling [Ploug, M., Ostergaard, S., Hansen, L. B. L., Holm, A., and Dano, K. (1998) Biochemistry 37, 3612-3622]. Replacement of the key functional residues Phe4 and Trp9 with either benzophenone or (trifluoromethyl)aryldiazirine rendered this peptide antagonist photoactivatable, and as a consequence, it incorporated covalently upon photolysis into either uPAR domain I or domain III depending on the actual position of the photophore in the sequence. The residues of uPAR specifically targeted by photoaffinity labeling were identified by matrix-assisted laser desorption mass spectrometry, NH2-terminal sequence analysis, and amino acid composition analysis after enzymatic fragmentation and HPLC purification. According to these data, the formation of the receptor-ligand complex positions Phe4 of the peptide antagonist very close to Arg53 and Leu66 in uPAR domain I and Trp9 of the antagonist in the vicinity of His251 in uPAR domain III. The gross molecular arrangement of the deduced receptor-ligand interface provides a rational structural basis for the observed requirement for the intact multidomain state of uPAR for achieving high-affinity ligand binding, since according to this model ligand binding must rely on a close spatial proximity of uPAR domains I and III. In addition, these data suggest that the assembly of the composite ligand binding site in uPAR may resemble the homophilic interdomain dimerization of kappa-bungarotoxin, a structural homologue of the Ly-6/uPAR domain family.

Biosynthesis of mycobacterial lipoarabinomannan.

The mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), are potent immunomodulators in tuberculosis and leprosy. Little is known of their biosynthesis, other than being based on phosphatidylinositol (PI), and they probably originate in the phosphatidylinositol mannosides (PIMs; PIMans). A novel form of cell-free incubation involving in vitro and in situ labeling with GDP-[14C]Man of the polyprenyl-P-mannoses (C35/C50-P-Man) and the simpler PIMs of mycobacterial membranes, reisolation of the [14C]Man-labeled membranes, and in situ chase demonstrated the synthesis of a novel alpha(1-->6)-linked linear form of LM at the expense of the C35/C50-P-Man. There was little or no synthesis under these conditions of PIMan5 with its terminal alpha(1-->2)Man unit or the mature LM or LAM with copious alpha(1-->2)Man branching. Synthesis of the linear LM, but not of the simpler PIMan2, was susceptible to amphomycin, a lipopeptide antibiotic that specifically inhibits polyprenyl-P-requiring translocases. A mixture of P[3H]I and P[3H]IMan2 was incorporated into the linear LM, supporting other evidence that, like the PIMs, LM and LAM, it is a lipid-linked mannooligosaccharide and a new member of the mycobacterial glycosylphosphatidylinositol lipoglycan/glycolipid class. Hence, the simpler PIMs originate in PI and GDP-Man, but further growth of the linear backbone emanates from C35-/C50-P-Man and is amphomycin-sensitive. The origin of the alpha(1-->2)Man branches of mature PIMan5, LM, and LAM is not known at this time but is probably GDP-Man.

Characterization of the fibronectin-attachment protein of Mycobacterium avium reveals a fibronectin-binding motif conserved among mycobacteria.

Mycobacterium avium is an intracellular pathogen and a major opportunistic infectious agent observed in patients with acquired immune deficiency syndrome (AIDS). Evidence suggests that the initial portal of infection by M. avium is often the gastrointestinal tract. However, the mechanism by which the M. avium crosses the epithelial barrier is unclear. A possible mechanism is suggested by the ability of M. avium to bind fibronectin, an extracellular matrix protein that is a virulence factor for several extracellular pathogenic bacteria which bind to mucosal surfaces. To further characterize fibronectin binding by M. avium, we have cloned the M. avium fibronectin-attachment protein (FAP). The M. avium FAP (FAP-A) has an unusually large number of Pro and Ala residues (40% overall) and is 50% identical to FAP of both Mycobacterium leprae and Mycobacterium tuberculosis. Using recombinant FAP-A and FAP-A peptides, we show that two non-continuous regions in FAP-A bind fibronectin. Peptides from these regions and homologous sequences from M. leprae FAP inhibit fibronectin binding by both M. avium and Mycobacterium bovis Bacillus Calmette-Guerin (BCG). These regions have no homology to eukaryotic fibronectin-binding proteins and are only distantly related to fibronectin-binding peptides of Gram-positive bacteria. Nevertheless, these fibronectin-binding regions are highly conserved among the mycobacterial FAPs, suggesting an essential function for this interaction in mycobacteria infection of their metazoan hosts.

Isolation and primary structure of a novel adipokinetic peptide from the pyrgomorphid grasshopper, Phymateus leprosus.

Using a heterologous bioassay, monitoring lipid increase in the haemolymph of migratory locusts, two peptides have been purified from methanolic extracts of corpora cardiaca of the pyrgomorphid grasshopper, Phymateus leprosus. The structures of both peptides were elucidated by a combination of Edman degradation, after deblocking the N-terminal pyroglutamic acid residue, and mass spectrometric techniques. One peptide is an octapeptide (pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH2) which also occurs in desert locusts; the second peptide is a novel decapeptide member of the AKH-family (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Ser-NH2).

Sera of leprosy patients with type 2 reactions recognize selective sequences in Mycobacterium leprae recombinant LSR protein.

Type 2 reactions (erythema nodosum leprosum [ENL]) are episodic, reactional states causing significant morbidity in lepromatous leprosy patients. With a view to defining the immunological differences between the stable and reactional forms of lepromatous leprosy, we determined antibody responses to LSR, a recombinant protein of Mycobacterium leprae previously described by us (S. Laal, Y.D. Sharma, H.K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. S. Mishra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991), as well as to 10- to 15-mer overlapping peptides synthesized on the basis of the LSR amino acid sequence. We report here the selective recognition of B cell epitopes by sera from patients with ENL as compared with a control group with nonreactional lepromatous leprosy. Peptides 2 and 3, with the sequences GVTYEIDLTNKNAA and IDLTNKNAAKLRGD, respectively, were recognized by > 95% of sera from patients with active ENL. Peptide 3 in addition showed reactivity with sera taken from 91.6% of lepromatous leprosy patients who were apparently stable but who were recorded to have had ENL several weeks before or after the sample collection. The core sequence IDLTNKNAA common to both these peptides may be a major target of humoral responses in ENL. In addition, the RGD motif at the C terminus appeared to influence the antigenicity of peptide 3 in enzyme-linked immunosorbent assay. It would appear that humoral responses during ENL are directed to selective antigenic determinants of the leprosy bacillus. The use of such serological markers to identify lepromatous leprosy patients with a high risk for developing ENL would be of clinical and predictive value.

Roles of soluble fibronectin and beta 1 integrin receptors in the binding of Mycobacterium leprae to nasal epithelial cells.

The mechanisms by which Mycobacterium leprae invades the human host are presently unknown. We investigated the ability of M. leprae to bind to human RPMI 2650 cells, a human nasal septal epithelial cell line, using both microscopic observation and an ELISA technique. The results demonstrated that M. leprae adheres to nasal cells after binding to soluble fibronectin. Furthermore, it was observed that M. leprae could bind to the beta 1 chain of the integrins in the absence of serum or mucus. These results demonstrated that M. leprae uses fibronectin and fibronectin receptors on the surface of epithelial cells to bind and possibly invade the nasal epithelial cells.

Purification and characterization of a paired basic residue-specific yeast aspartic protease encoded by the YAP3 gene. Similarity to the mammalian pro-opiomelanocortin-converting enzyme.

Yeast cells express an alternate enzyme encoded by the YAP3 gene which can process pro-alpha-mating factor when this pheromone is overexpressed in KEX2-deficient mutants. The YAP3 gene product is an aspartic protease (YAP3) that cleaves at paired basic residues (Egel-Mittani, M., Flygenring, H.P., and Hansen, M. T. (1990) Yeast 6, 127-137). In this study, the YAP3 gene was overexpressed in the BJ 3501 strain of Saccharomyces cerevisiae. YAP3 was purified to apparent homogeneity using concanavalin A and pepstatin A affinity chromatography. The enzyme was characterized as an M(r) 68,000 glycoprotein with a pH optimum of 4.0-4.5. It was inhibited by pepstatin A and activated by 5 mM Ca2+. YAP3 cleaved at paired basic residues of mouse pro-opiomelanocortin (POMC) to yield adrenocorticotropin (ACTH) and beta-lipotropin (LPH); human beta-LPH to yield beta-endorphin-(1-31), beta-endorphin-(1-29), beta-endorphin-(1-28), gamma-LPH, and beta-melanocyte-stimulating hormone; and bovine N-POMC1-77 to yield gamma 3-melanocyte-stimulating hormone. It also cleaved the tetrabasic residues of ACTH1-39 to yield primarily ACTH1-15 and Lys-Arg-corticotropin-like intermediate lobe peptide. The physical properties, pH optimum, and specificity of YAP3 indicate that it is a homologue of the mammalian POMC-converting enzyme (EC 3.4.23.17), a paired basic residue-specific aspartic protease from bovine pituitary intermediate lobe secretory granules (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205).

Angiotensin-converting enzyme: clinical applications and laboratory investigations on serum and other biological fluids.

Angiotensin I-converting enzyme (ACE) is a peptidyldipeptide hydrolase that is located mainly on the luminal surface of vascular endothelial cells but also in cells derived from the monocyte-macrophage system. Physiologically, ACE is a key enzyme in the renin-angiotensin system, converting angiotensin I into the potent vasopressor angiotensin II and also inactivating the vasodilator bradykinin. Increased serum ACE activity (SACE) has been reported in pathologies involving a stimulation of the monocytic cell line, primarily granulomatous diseases. Sarcoidosis is the most frequent and the better studied of these diseases; high SACE is not only a well-established marker for the diagnosis but is also a useful tool for following its course and evaluating the effect of therapy. SACE can also be increased in nonsarcoidotic pulmonary granulomatous diseases such as silicosis and asbestosis, in extrathoracic granulomatous pathologies such as Gauchers disease and leprosis, and, to a lesser extent, in nongranulomatous disorders such as hyperthyroidism or cholestasis. On the other hand, monitoring sarcoidosis obviates the measurement of ACE activity in other biological fluids, e.g., broncho-alveolar and cerebrospinal fluids, in the search of a locoregional dissemination or dis-simulation of the disease. Decreased SACE has been reported in vascular pathologies involving an endothelial abnormality, e.g., deep vein thrombosis, and in endothelium dysfunctions related to the toxicity of chemo- and radiotherapy used in cancers, leukemias, and hematopoietic or organ transplantations. SACE is also of interest for monitoring arterial hypertension treated with specific synthetic ACE inhibitors. These various reasons for determining ACE activity have led to the development of numerous methods. The most widely used is the spectrophotometric assay using hippuryl-histidyl-leucine as substrate. Fluorimetric and radiochemical assays using both classic and novel substrates have been proposed, but they are time consuming, require special apparatus, and are not suited to automation. Kinetic spectrophotometry of furylacryloyl-phenylalanyl-glycyl-glycine hydrolysis is now used extensively because it is easy to automatize. Efforts are now required to standardize one or more of these assays. Indeed, "normal" plasma values differ not only according to the substrate, but also to the method of determination and to sex and age.

Use of flanking sequences to study secondary structure-activity correlations of a Mycobacterium leprae T cell epitope.

The 65-kDa protein of the intracellular pathogen M. leprae is prominent in the immune response to this mycobacterium, and individual T cell epitopes from this protein sequence have been defined. We have tested the stimulatory activity of extended analogs of the minimal peptide representing one such epitope, LQAAPALDKL, with a variety of tetrapeptide extensions added to enhance or destabilize alpha helix formation. The conformational potential of the peptides was measured by circular dichroism using aqueous trifluoroethanol as a secondary structure inducer. Although analogs with high helical potential activated T cells at low concentrations, a less helical variant was similarly potent. Activity also did not correlate with predicted overall alpha helical amphipathicity. One analog was found which stimulated T cell proliferation in the 50 pM range. The effect of tetrapeptide extensions on epitope activity is not consistent with the importance in activity of only a single stable secondary structure such as an alpha helix.