Novel natural peptide substrates for endopeptidase 24.15, neurolysin,and angiotensin-converting enzyme

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.     

Intracellullar peptides as putative natural regulators of protein interactions

Extralysosomal proteolysis by multicatalytic complexes such as the 26S proteasome produces large amounts of peptides in the cytosol, mitochondria and nuclei of eukaryotic cells, and there is increasing evidence that the resulting free intracellular peptides can modulate specific protein interactions. The demonstration that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions suggests new putative roles for these molecules in gene regulation, metabolism, cell signaling and protein targeting. Such interactions frequently involve specific consensus amino acid sequences that can be predicted based on similarities in domain composition. We have recently developed a new strategy for identifying novel natural peptides, the sequences of which correspond to fragments of intracellular proteins and contain putative post-translational modification sites. In this review, we examine the evidence that intracellular peptides released by proteasomes may be involved in regulating protein interactions. In particular, the role of endopeptidase 24.15 (thimet oligopeptidase; EC 3.4.24.15) is discussed in detail as this enzyme has been implicated in intracellular peptide metabolism in vivo in concert with the 26S proteasome.     

Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry

In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.     

Identification of the major phosphorylation site of the hepatitis C virus H strain NS5A protein as serine 2321.

The hepatitis C virus (HCV) NS5A protein is phosphorylated by a cellular, serine/threonine kinase. To identify the major site(s) of NS5A phosphorylation, radiolabeled HCV-H NS5A phosphopeptides were purified and subjected to phosphoamino acid analysis and Edman degradation. These data identified the major intracellular phosphorylation site in the HCV-H NS5A protein as Ser(2321), a result verified by two additional, independent methods: (i) substitution of Ala for Ser(2321) and the concomitant disappearance of the major in vivo phosphorylated peptides and corresponding in vitro phosphorylated peptides; and (ii) comigration of the digestion products of a synthetic peptide phosphorylated on Ser(2321) with the major in vivo phosphorylated NS5A peptides. Site-directed mutagenesis of Ser(2321) suggested that phosphorylation of NS5A is dispensable for previously described interactions with NS4A and PKR, a cellular, antiviral kinase that does not appear to catalyze NS5A phosphorylation. The proline-rich nature of the amino acid sequence flanking Ser(2321) (PLPPPRS(2321) PPVPPPR) suggests that a proline-directed kinase is responsible for the majority of HCV NS5A phosphorylation, consistent with previous kinase inhibitor studies.     

Purification and characterization of enzymes involved in the degradation of chemotactic N-formyl peptides

N-Formyl peptides are derived from proteolytic degradation/processing of bacterial and mitochondrial proteins and serve as potent chemoattractants for mammalian phagocytic leukocytes. A response to the chemotactic N-formyl peptides released by commensal bacteria in the gut region could be detrimental, leading to unwanted inflammation. Here, two enzymes that act sequentially to degrade N-formyl peptides were purified from the rat intestinal mucosal layer and biochemically characterized. The first enzyme cleaves chemotactic peptide f-MLF to release N-formylmethionine (f-Met) and dipeptide leucylphenylalanine, with a k(cat) value of 14 s(-)(1), a K(M) value of 0.60 mM, and a k(cat)/K(M) value of 22 500 M(-)(1) s(-)(1). In-gel tryptic digestion followed by mass spectral fingerprinting identified the protein as the alpha-N-acylpeptide hydrolase (or acylamino acid-releasing enzyme, EC 3.4.19.1). The second enzyme hydrolyzes N-formylmethionine into formate and methionine with a k(cat) value of 7.9 s(-)(1), a K(M) value of 3.1 mM, and a k(cat)/K(M) value of 2550 M(-)(1) s(-)(1). This protein was identified as the N-acylase IA (or N(alpha)-acyl-l-amino acid amidohydrolase, EC 3.5.1.14). Together, these two enzymes play a protective role in degrading bacterial and mitochondrial N-formylated peptides.     

A dipalmitoyl peptide that binds SH3 domain,disturbs intracellular signal transduction,and inhibits tumor growth in vivo

The Src homology 3 (SH3) domain plays a crucial role in protein-protein interactions during intracellular signal transduction. Blocking the SH3-mediated protein binding may inhibit the corresponding signal transduction, and thus, block the cellular functions. In this study, a peptide that specifically binds to SH3 domain could be introduced into the intracellular region when the peptides were conjugated with dipalmitic acid and appeared to disturb intracellular signaling. The dipalmitoyl peptide appeared to inhibit the phosphorylation of ZAP-70, Lck, and T-cell antigen receptor zeta in Jurkat. Mobilization of the intracellular free calcium induced by anti-CD3 antibody was reduced after treatment with the dipalmitoyl peptide. It was also observed that the dipalmitoyl peptide inhibited cancer cell growth both in vitro and in vivo. These results suggest that the dipalmitoyl peptide that presumably disturbs SH3-mediated signal transduction may have a potent anti-proliferative activity, which would be useful as a potential anti-tumor agent.     

Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.     

Effect of carbon and nitrogen growth limitation upon nutrient uptake and metabolism in batch cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle, Catharanthus roseus (L). G. Don were grown as batch cultures in two different types of media; in one medium the limiting nutrient was inorganic nitrogen, and in the other it was carbon. The response of the cells to these growth-limiting conditions was monitored by measuring cellular fresh weight, dry weight and protein accumulation, cell viability, medium sugar and nitrate levels, and the activities of certain intracellular enzymes throughout growth in batch culture. The enzymes investigated were glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), hexokinase (EC 2.7.1.40), phosphofructokinase (EC 2.7.1.11), nitrate reductase (EC 1.6.6.1), glutamate dehydrogenase (EC 1.4.1.2) and glutamine synthetase (EC 6.3.1.2). The effect of culturing the cells under different nutritional regimes was apparent in all aspects of growth; only some enzyme activities were unaffected. Cell viability remained at a high level for several days after growth limitation in both types of culture. The possibility that protein degradation in nitrogen-limited batch cultures is under very stringent control is discussed.     

Subcellular localization and levels of aminopeptidases and dipeptidase in Saccharomyces cerevisiae.

Three aminopeptidases (L-aminoacyl L-peptide hydrolases, EC 3.4.11) and a single dipeptidase (L-aminoacyl L-amino acid hydrolase, EC 3.4.13) are present in homogenates of Saccharomyces cerevisiae. Bassed on differences in substrate specificity and the sensitivity to Zn2+ activation, methods were developed that allow the selective assay of these enzymes in crude cell extracts. Experiments with isolated vacuoles showed that aminopeptidase I is the only yeast peptidase located in the vacuolar compartment. Aminopeptidase II (the other major aminopeptidase of yeast) seems to be an external enzyme, located mainly outside the plasmalemma. The synthesis of aminopeptidase I is repressed in media containing more than 1% glucose. In the presence of ammonia as the sole nitrogen source its activity is enhanced 3--10-fold when compared to that in cells grown on peptone. In contrast, the levels of aminopeptidase II and dipeptidase are less markedly dependent on growth medium composition. It is concluded that aminopeptidase II facilitates amino acid uptake by degrading peptides extracellularly, whereas aminopeptidase I is involved in intracellular protein degradation.     

The Dual Effects of Ca2+ on Binding of the Glycolytic Enzymes,Phosphofructokinase and Aldolase,to Muscle Cytoskeleton

We show here that in rat diaphragm muscle, a short time of incubation with the Ca²⁺-ionophore A23187 induced an increase in cytoskeleton-bound phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13), whereas a longer period of incubation, which causes a pathological rise in intracellular Ca²⁺, induced a decrease in bound enzymes. Lactate concentration correlated with both phases of Ca²⁺ action on the binding of the enzymes. The increase in cytoskeleton-bound enzymes could be prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a novel, potent, and selective inhibitor of calmodulin activity). These results suggest that calmodulin is involved in the Ca²⁺-induced binding of the enzymes to muscle cytoskeleton.     

A quantitative study of the recruitment potential of all intracellular tyrosine residues on EGFR, FGFR1 and IGF1R

Receptor tyrosine kinases transmit and process extracellular cues by recruiting intracellular signaling proteins to sites of tyrosine phosphorylation. Using protein microarrays comprising virtually every human SH2 and PTB domain, we generated quantitative protein interaction maps for three well-studied receptors--EGFR, FGFR1 and IGF1R--using phosphopeptides derived from every intracellular tyrosine residue on each receptor, regardless of whether or not they are phosphorylated in vivo. We found that, in general, peptides derived from physiological sites of tyrosine phosphorylation bind to substantially more SH2 or PTB domains than do peptides derived from nonphysiological sites, supporting the idea that kinases and interaction domains co-evolve and suggesting that new sites arise predominantly through selection favoring advantageous interactions, rather than through selection disfavoring unwanted interactions. We also found substantial qualitative overlap in the recruitment profiles of these three receptors, suggesting that their different biological effects arise, at least in part, from quantitative differences in their affinities for the proteins they recruit.     

Inhibitors of catalase-amyloid interactions protect cells from beta-amyloid-induced oxidative stress and toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.     

Alpha-thrombin, epidermal growth factor, and okadaic acid activate the Na+/H+ exchanger, NHE-1, by phosphorylating a set of common sites.

The ubiquitous and amiloride-sensitive Na+/H+ exchanger (NHE-1), a plasma membrane phosphoglycoprotein that regulates intracellular pH, is rapidly activated by growth factors. We showed previously that epidermal growth factor (EGF), alpha-thrombin, or serum stimulates Na+/H+ exchange activity in growth-arrested Chinese hamster lung fibroblasts (ER22 cells) in a time-dependent manner which correlates with increased phosphorylation of NHE-1 at serine residues (Sardet, C., Counillon, L., Franchi, A., and Pouysségur, J. (1990) Science 247, 723-726). Here we show that the tumor promoter, okadaic acid, a potent in vivo inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), stimulates Na+/H+ exchange in G0-arrested ER22 cells and in exchanger-deficient fibroblasts transfected with the human NHE-1 cDNA. Okadaic acid effects are maximal at 1 microM (EC50 = 500 nM), detected in 2 min, complete within 15-20 min, and are additives when combined with EGF or alpha-thrombin. Parallel to the pHi-induced rise, okadaic acid alone or together with growth factors stimulated the phosphorylation of NHE-1. More importantly tryptic phosphopeptide maps of NHE-1, immunoprecipitated from cells treated with EGF, alpha-thrombin, or okadaic acid, show a common pattern of phosphorylation. This pattern consists of five major 32P-labeled peptides (P1-P5) present in lower amounts in resting cells. One of them, P5, barely detectable in resting cells is increased up to 15-fold in mitogen-stimulated cells. Taken together these results reinforce the notion that phosphorylation of NHE-1 controls the set point value of the exchanger and suggest that: (i) the proximate step in Na+/H+ exchange activation is mediated by as yet unidentified growth factor-activatable serine "NHE-1 kinase(s)" and (ii) this NHE-1 kinase(s), partly active in resting cells, integrate signals from receptor tyrosine kinases and G protein-coupled receptors.     

Biochemical characterization of phospholipids, sulfatide and heparin as potent stimulators for autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein in vitro

The stimulatory effects of SH (sulfatide and heparin) and two phospholipids (PI and PS) on autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein (MBP) and two synthetic MBP peptides (M86 and M156) were comparatively examined in vitro. It was found that (i) both PI and SH highly stimulated the GSK-3beta-mediated phosphorylation of MBP, but not glycogen synthase, and two MBP peptides through their direct binding to these substrates and (ii) both PI and heparin, as compared with sulfatide, highly stimulated autophosphorylation of GSK-3beta. The K(m) value of MBP for GSK-3beta was highly reduced and the V(max) value was significantly increased in the presence of these acidic modulators, which augmented further phosphorylation of MBP by the kinase. Under our experimental condition, similar stimulatory effects of PI and heparin were observed with the GSK-3beta-mediated phosphorylation of tau protein (TP) in vitro. These results presented here suggest that these two phospholipids and SH may function as effective stimulators for autophosphorylation of GSK-3beta and for the GSK-3beta-mediated high phosphorylation of SH-binding proteins, including MBP and TP, in the highly accumulated levels of these acidic and sulfated modulators in the brain.     

Signaling pathways transduced through the elastin receptor facilitate proliferation of arterial smooth muscle cells

In this report we demonstrate that soluble peptides, elastin degradation products stimulate proliferation of arterial smooth muscle cells. We show that these effects are due to generation of intracellular signals transduced through the cell surface elastin receptor, which consists of peripheral 67-kDa elastin-binding protein (EBP) (spliced variant of beta-galactosidase), immobilized to the transmembrane sialidase and the protective protein. We found that elastin receptor-transduced signaling triggers activation of G proteins, opening of l-type calcium channels, and a sequential activation of tyrosine kinases: FAK, c-Src, platelet-derived growth factor-receptor kinase and then Ras-Raf-MEK1/2-ERK1/2 phosphorylation cascade. This, in turn, causes an increase in expression of cyclins and cyclin-dependent kinases, and a consequent increase in cellular proliferation. The EBP-transduced signals also induce tyrosine kinase-dependent phosphorylation of beta-tubulin, LC3, microtubule-associated protein 1, and alpha-actin and troponin-T, which could be linked to reorganization of cytoskeleton. We have also disclosed that induction of these signals can be abolished by anti-EBP antibody or by galactosugars, which cause shedding of EBP from the cell surface. Moreover, elastin-derived peptides did not induce proliferation of EBP-deficient cells derived from patients bearing a nonsense mutation of the beta-galactosidase gene or sialidase-deficient cells from patients with congenital sialidosis.