Conformational preferences of substrates for human prolyl 4-hydroxylase

Prolyl 4-hydroxylase (P4H) catalyzes the posttranslational hydroxylation of (2 S)-proline (Pro) residues in procollagen strands. The resulting (2 S,4 R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. Even though its product (Hyp) differs from its substrate (Pro) by only a single oxygen atom, no product inhibition has been observed for P4H. Here, we examine the basis for the binding and turnover of substrates by human P4H. Synthetic peptides containing (2 S,4 R)-4-fluoroproline (Flp), (2 S,4 S)-4-fluoroproline (flp), (2 S)-4-ketoproline (Kep), (2 S)-4-thiaproline (Thp), and 3,5-methanoproline (Mtp) were evaluated as substrates for P4H. Peptides containing Pro, flp, and Thp were found to be excellent substrates for P4H, forming Hyp, Kep, and (2 S,4 R)-thiaoxoproline, respectively. Thus, P4H is tolerant to some substitutions on C-4 of the pyrrolidine ring. In contrast, peptides containing Flp, Kep, or Mtp did not even bind to the active site of P4H. Each proline analogue that does bind to P4H is also a substrate, indicating that discrimination occurs at the level of binding rather than turnover. As the iron(IV)-oxo species that forms in the active site of P4H is highly reactive, P4H has an imperative for forming a snug complex with its substrate and appears to do so. Most notably, those proline analogues with a greater preference for a C (gamma)- endo pucker and cis peptide bond were the ones recognized by P4H. As Hyp has a strong preference for C (gamma)- exo pucker and trans peptide bond, P4H appears to discriminate against the conformation of proline residues in a manner that diminishes product inhibition during collagen biosynthesis.     

4-chloroprolines: synthesis, conformational analysis, and effect on the collagen triple helix

Collagen is an abundant, triple-helical protein comprising three strands of the repeating sequence: Xaa-Yaa-Gly. (2S)-Proline and (2S,4R)-4-hydroxyproline (Hyp) are common in the primary structure of collagen. Here, we use nonnatural proline derivatives to reveal determinants of collagen stability. Specifically, we report high-yielding syntheses of (2S,4S)-4-chloroproline (clp) and (2S,4R)-4-chloroproline (Clp). We find that the molecular structure of Ac-Clp-OMe in the solid state is virtually identical to that of Ac-Hyp-OMe. In contrast, the conformational properties of Ac-clp-OMe are similar to those of Ac-Pro-OMe. Ac-Clp-OMe has a stronger preference for a trans amide bond than does Ac-Pro-OMe, whereas Ac-clp-OMe has a weaker preference. (Pro-Clp-Gly)(10) forms triple helices that are significantly more stable than those of (Pro-Pro-Gly)(10). Triple helices of (clp-Pro-Gly)(10) have stability similar to those of (Pro-Pro-Gly)(10). Unlike (Pro-Clp-Gly)(10) and (clp-Pro-Gly)(10), (clp-Clp-Gly)(10) does not form a stable triple helix, presumably due to a deleterious steric interaction between proximal chlorines on different strands. These data, which are consistent with previous work on 4-fluoroprolines and 4-methylprolines, support the importance of stereoelectronic and steric effects in the stability of the collagen triple helix and provide another means to modulate that stability. (     

Cyclin-dependent kinase inhibitors block leukocyte adhesion and migration

Leukocyte trafficking is a tightly regulated process essential for an appropriate inflammatory response. We now report a new adhesion pathway that allows unstimulated leukocytes to adhere to and migrate through exposed endothelial matrix or high-density ligand, a process we have termed ligand-induced adhesion. This ligand-induced adhesion is integrin mediated, but in contrast to phorbol ester-stimulated adhesion, it is not dependent on the small GTPase Rap-1 activity. Instead, we show a critical role for cyclin-dependent kinase (Cdk) 4 in ligand-induced adhesion by three independent lines of evidence: inhibition by pharmacological inhibitors of Cdk, inhibition by dominant-negative construct of Cdk4, and inhibition by Cdk4 small interfering RNA. The major substrate of Cdk4, Rb, is not required for ligand-induced adhesion, suggesting the involvement of a novel Cdk4 substrate. We also demonstrate that Cdk4(-/-) mice have impaired recruitment of lymphocytes to the lung following injury. The finding that Cdk inhibitors can block leukocyte adhesion and migration may expand the clinical indications for this emerging class of therapeutics.     

MMP-9 Sheds the ��2 Integrin Subunit (CD18) from Macrophages

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, β₂ integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of β₂ integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala⁷⁰⁵ and Ile⁷⁰⁶ of the β₂ integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that β₂ integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of β₂ integrin.Matrix metalloproteinases (MMPs),¹ a subfamily of metazincins, are a structurally related group of zinc-dependent proteases (1). They are synthesized in latent form as pro-MMPs, and their prodomain must be removed or modified before they are proteolytically active. Some MMPs are secreted, whereas others are anchored to the cell surface, but their proteolytic activity is thought to be confined locally within the secretory pathway at the cell surface and nearby extracellular space (1–3). Individual MMPs have distinct substrate specificities and act on diverse extracellular and membrane proteins, such as chemokines, cell surface adhesion proteins, and extracellular matrix components. Proteolysis by MMPs plays an important role in a wide variety of normal and pathological processes, such as host defense, inflammation, and tumor progression (1–9).High levels of MMP-9 (gelatinase B) are expressed by activated macrophages (10), which are key effector cells of both innate and acquired immunity. In addition to having homeostatic functions, MMP-9 secreted by macrophages has been implicated in aneurysm formation, tumor progression, and disruption of atherosclerotic plaques (8, 9, 11, 12). Although the pathogenesis of those processes is generally thought to involve inappropriate degradation of extracellular matrix proteins, it has become increasingly clear that MMPs cleave a number of diverse substrates to mediate their varied functions (3, 13). Because MMP-9 can accumulate on the cell surface (14), it is likely to act on membrane proteins.To understand the specific roles of individual MMPs in inflammatory and immune responses, it is critical to identify their physiological substrates (3, 15–17). Most studies have focused on identifying substrates by their ability to be cleaved in defined in vitro reactions (18, 19), but this approach is biased in two ways. First, the candidate substrate must be selected a priori. Second, in vitro reactions fail to account for the complexity of the pericellular environment. Another method is to identify sequences in synthetic peptides that MMPs can cleave (20, 21). However, individual MMPs cleave different proteins at a variety of sites rather than at a consensus site. Moreover MMPs often interact with substrates through domains remote from the active site (exosites) (22), and exosites of MMP-2 have been used in a yeast two-hybrid system to trap candidate substrates (23). However, some substrates may bind weakly or not at all to exosites, limiting the utility of this approach for global substrate screening.An emerging strategy for finding MMP substrates is to conduct an unbiased, global search by coupling gel electrophoresis or liquid chromatography with MS-based protein identification. For example, two-dimensional (2D) gel electrophoresis (24) and derivatization of cysteine-containing peptides with an isotope affinity tag (25) have identified candidate substrates for membrane type-1 MMP (MT1-MMP) in plasma and cultured cells. Quantitative approaches using 2D difference gel electrophoresis have identified potential substrates of MMP-2 and MMP-9 in bronchoalveolar lavage fluid (26) and of MMP-9 and the related metalloproteinases ADAM-10 and ADAM-17 in cancer cells (27, 28). Lectin affinity chromatography detected glycosylated proteins that were selectively enriched in medium from a monocyte cell line expressing ADAM-17 and in phorbol ester-stimulated monocytes (16). Recently iTRAQ (isobaric tags for relative and absolute quantitation) labeling was used to identify substrates of MMP-2 (29). It is important to note, however, that proteases can affect protein abundance by pathways not involving proteolysis. Thus, an important limitation of many of these studies is that they fail to provide evidence that proteins with altered abundance in cells expressing a protease are direct substrates for proteolytic cleavage.In the current studies, we used subtractive proteomics to identify proteins enriched in the medium of a macrophage cell line. Subtractive proteomics compares two or more proteomes to identify proteins that are specifically enriched or depleted under certain conditions (30, 31). Our biochemical studies confirmed that two integral membrane proteins, amyloid precursor protein (APP) and the β₂ integrin subunit (CD18), were shed by macrophages expressing autoactivating MMP-9. We next used a peptide substrate mapping strategy to identify potential MMP-9 cleavage sites in β₂ integrin subunit. Targeted MS/MS analysis demonstrated that β₂ integrin subunit peptides with the same cleavage site were detected only in the medium of macrophages expressing autoactivating MMP-9, providing strong evidence that β₂ integrin is a direct substrate for proteolysis. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a robust, high throughput technique for identifying cellular substrates that are proteolytically shed from macrophages.     

TRPM channels are required for rhythmicity in the ultradian defecation rhythm of <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

Ultradian rhythms, rhythms with a period of less than 24 hours, are a widespread and fundamental aspect of life. The mechanisms underlying the control of such rhythms remain only partially understood. Defecation in C. elegans is a very tightly controlled rhythmic process. Underlying the defecation motor programme is an oscillator which functions in the intestinal cells of the animal. This mechanism includes periodic calcium release and subsequent intercellular calcium waves which in turn regulate the muscle contractions that make up the defecation motor programme. Here we investigate the role of TRPM cation channels in this process.     

Effects of exogenous interleukin-1β on primary sporocysts of Schistosoma mansoni (Trematoda) incubated with plasma and hemocytes from schistosome-susceptible and resistant Biomphalaria glabrata (Gastropoda)

Abstract. The cytokine interleukin-1β (IL-1β) mediates interactions of immune and inflammatory cells in mammals. Previous reports also have linked plasma (cell-free hemolymph) levels of IL-1β in the snail Biomphalaria glabrata to resistance against Schistosoma mansoni . In the present study, fluorescent probes were used to study larval schistosome and snail hemocyte viability during in vitro encounters. Hemolymph (plasma and hemocytes) from schistosome-susceptible (M-line) and resistant (13–16-R1) B. glabrata was added to sporocysts of S. mansoni and the viability of hemocytes and parasites was assessed. Next, IL-1β was added to sporocyst-hemolymph samples, the viability of sporocysts and hemocytes determined and then compared to control assays. The number of live sporocysts present after incubation for 1 h with hemolymph from M-line snails was significantly greater than the number seen when hemolymph from 13–16-R1 snails was tested. Nearly all sporocysts survived the 1 h incubation with M-line hemolymph, and most of the hemocytes attached to sporocysts were dead. In contrast, nearly all sporocysts were dead when hemolymph from 13–16-R1 snails was tested, and most attached hemocytes were alive. Addition of IL-1β to M-line hemolymph resulted in a dramatic increase in sporocyst death. Addition of IL-1β to 13–16-R1 hemolymph produced a small but significant increase in the rate of sporocyst death. These results indicate that the concentration of IL-1β present in hemolymph from B. glabrata is directly related to the ability of this snail to kill S. mansoni sporocysts in vitro.     

Comparison of Worm Control Strategies in Grazing Sheep in Denmark

Control of nematode parasites with reduced reliance on the use of anthelmintics was studied in 16 ewes with suckling twin lambs on contaminated pasture in Denmark. Ewes and lambs were treated with albendazole at turn-out 3 May. Ewes were removed from the groups on 26 July, and lambs were slaughtered on 11 October. The animals were allocated to 4 groups of 8 lambs and their 4 ewes. Group TS was treated with albendazole at weeks 3, 6 and 8 after turnout and set-stocked; group TM was similarly treated but moved to clean pasture in conjunction with the last drenching; group US was untreated and set-stocked, and group UM was left untreated but moved to clean pasture week 8 after turn-out. Supplementary feed was offered in June and August due to scarcity of pasture. Strategic treatments of ewes and lambs weeks 3, 6 and 8 after turn-out, with or without a move to clean pasture, were highly effective in controlling nematode infections for most of the season. This was reflected in better weight gains and carcass characteristics in the treated compared to untreated lambs, resulting in an average increase in the value of the product by 36%. The effect of moving without treatment (UM) on faecal egg counts was limited but peak pasture infectivity was reduced to less than 10% compared to the set-stocked group and weight gains of lambs were significantly better despite poor feed availability in late season. The study showed that under set-stocked conditions repeated anthelmintic treatments of both ewes and lambs in early season may ensure sufficient nematode control whereas moving animals to clean pasture without dosing was less efficient. The latter may, however, still be a viable option in organic and other production systems where routine use of anthelmintics is banned, particularly if weaning and moving are combined or a second move is performed.     

<i>In vitro</i> evaluation of antifungal activity of Agave (<i>Agave scabra</i>, Salm Dyck) extracts against post-harvest mushrooms

The agricultural sector, and particularly the horticultural production, has a singular importance in agriculture, considering that it ranks second on agricultural products, nationally and worldwide. Fungal diseases are one of the major causes of vegetable loss during storage, reducing their nutritional value, quality and sale price. Vegetables are usually exposed to diverse treatments with chemical products before storage; as a result, fungal populations develop an increased resistance over time becoming more difficult to control. Because of this, research efforts toward finding more suitable chemicals to control fungal diseases are needed. Natural extracts may be an alternative solve this problem. In the present investigation the fungicidal activity of aqueous and ethanol extracts of Agave scabra was evaluated on the growth of Botrytis cinerea, Mucor sp., Aspergillus niger, Fusarium sp. and Penicillium sp., whose strains were isolated from potato and tomato. To assess their effects, the agar-dilution and agar-well techniques were performed. The ethanol extract was more effective against Botrytis cinerea and Mucor sp. when the agar-well method was used. However, when using the agar-dilution method the ethanol extract of Agave scabra inhibited the growth of Botrytis cinerea, Mucor sp. and Penicillium sp.     

KFERQ sequence in ribonuclease A-mediated cytotoxicity.

Onconase(ONC) is an amphibian ribonuclease that is in clinical trials as a cancer chemotherapeutic agent. ONC is a homolog of ribonuclease A (RNase A). RNase A can be made toxic to cancer cells by replacing Gly(88) with an arginine residue, thereby enabling the enzyme to evade the endogenous cytosolic ribonuclease inhibitor protein (RI). Unlike ONC, RNase A contains a KFERQ sequence (residues 7-11), which signals for lysosomal degradation. Here, substitution of Arg(10) of the KFERQ sequence has no effect on either the cytotoxicity of G88R RNase A or its affinity for RI. In contrast, K7A/G88R RNase A is nearly 10-fold more cytotoxic than G88R RNase A and has more than 10-fold less affinity for RI. Up-regulation of the KFERQ-mediated lysosomal degradation pathway has no effect on the cytotoxicity of these ribonucleases. Thus, KFERQ-mediated degradation does not limit the cytotoxicity of RNase A variants. Moreover, only two amino acid substitutions (K7A and G88R) are shown to endow RNase A with cytotoxic activity that is nearly equal to that of ONC.     

The mycoflora and toxicity of feedstuffs from a production plant in córdoba, Argentina

Feedstuffs used for poultry nutrition in Argentina were analyzed for fungal flora and natural incidence of mycotoxins. Survey of 120 samples of poultry feeds, taken from May 1998 to April 1999, showed the presence of 15 genera of filamentous fungi. The predominant genera wereFusarium spp. andPenicillium ssp., isolated in 67.5 % of the samples, followed byAspergillus spp. (57.5 %). Yeast, were significantly isolated from most of the samples. Species identification was carried down for the toxigenic genera. Fungal total counts of poultry feeds ranged from 2.0 × 10³ to 3.0 × 10⁵ CFU g^-1 The fungal total counts during two months of sampling, were slightly over the limit value of 1 × 105 CFU g^-1, which ensure the hygienic quality of the feed. Potentially toxicogenic species presented moderate mean colony counts. Many of the fungi isolated from poultry feeds are mycotoxin producers. Fumonisins had the highest incidence, and were found in 97 % of the analyzed samples followed by aflatoxin B₁ (46 %), zearalenone (18 %) and deoxynivalenol (6 %). On the co-occurrence of both carcinogenic mycotoxins, all of the FBs contaminated feed samples were co-contaminated with AFB₁. The results show the relevance of the samples screening for viable fungi propagules and the surveillance of their associated mycotoxins in poultry feeds.