Signal-CF: a subsite-coupled and window-fusing approach for predicting signal peptides

We have developed an automated method for predicting signal peptide sequences and their cleavage sites in eukaryotic and bacterial protein sequences. It is a 2-layer predictor: the 1st-layer prediction engine is to identify a query protein as secretory or non-secretory; if it is secretory, the process will be automatically continued with the 2nd-layer prediction engine to further identify the cleavage site of its signal peptide. The new predictor is called Signal-CF, where C stands for "coupling" and F for "fusion", meaning that Signal-CF is formed by incorporating the subsite coupling effects along a protein sequence and by fusing the results derived from many width-different scaled windows through a voting system. Signal-CF is featured by high success prediction rates with short computational time, and hence is particularly useful for the analysis of large-scale datasets. Signal-CF is freely available as a web-server at http://chou.med.harvard.edu/bioinf/Signal-CF/ or http://202.120.37.186/bioinf/Signal-CF/.     

Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion

Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen''s long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1′ recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.     

Enkephalin-containing polypeptides are potent inhibitors of enkephalin degradation

Enkephalin-containing polypeptides derived from pro-enkephalin A, pro-enkephalin B, or pro-opiomelanocortin were inhibitors of enkephalin degradation by aminoenkephalinases purified from cytosol or membranes. Of the peptides, Arg°-Met-enkephalin was the most potent inhibitor for the aminoenkephalinases, with an IC₅₀ of about 0.6 μM, it was more effective than bestatin (IC₅₀=0.8–1.0 μM). This inhibition was partly due to substrate competition. Arg°-Met-enkephalin was hydrolyzed by aminoenkephalinases to form Arg, Tyr, and Gly-Gly-Phe-Met in a substrate-inhibited manner. The hexapeptide also inhibited the breakdown of Arg- and Tyr-β-naphthylamide by the membrane aminoenkephalinase. Since Arg°-Met-enkephalin did not inhibit leucine aminopeptidase, it was a more selective inhibitor than bestatin of Met-enkephalin breakdown by aminopeptidases. Arg°-Met-enkephalin inhibited enkephalin breakdown by synaptosomal plasma membranes but not by brain slices. Our data suggest that in addition to their possible role as opioids, the enkephalin-containing polypeptides may be regulators of enkephalin levels.     

Substrate specificity and inhibition of human kallikrein-related peptidase 3 (KLK3 or PSA) activated with sodium citrate and glycosaminoglycans

We report the enzymatic properties and substrate specificity of human recombinant KLK3 in the presence of glycosaminoglycans (GAGs) and sodium citrate. This salt is highly concentrated in prostate and in its presence KLK3 had a similar hydrolytic efficiency as chymotrypsin. In contrast to the latter peptidase, KLK3 activated by sodium citrate efficiently hydrolyzed substrates containing R, H and P at the P1 position. Activated KLK3 also cleaved peptides derived from the bradykinin domain of human kininogen at the same sites as human kallikrein KLK1, but presented low kininogenase activity. Angiotensin I has several sites for hydrolysis by KLK3; however, it was cleaved only at the Y-I bond (DRVY↓IHPFHL). Sodium citrate modulated KLK3 conformation as observed by alterations to the intrinsic fluorescence of phenylalanines and tryptophans. Activated KLK3 was reversibly inhibited by Z-Pro-Prolinal and competitively inhibited by ortho-phenantroline. Together, these are noteworthy observations for the future design of specific non-peptide inhibitors of KLK3 and to find natural substrates.     

Rapid Evolution of the Trophoblast Kunitz Domain Proteins (TKDPs)—A Multigene Family in Ruminant Ungulates

The trophoblast Kunitz domain proteins (TKDPs) are products of the outer cells (trophoblasts) of the placenta of cattle, sheep, and related species. Most are expressed abundantly for only a few days during the time at which the ruminant conceptus is first establishing intimate contacts with the uterine lining. The TKDPs are secretory proteins that possess a carboxyl-terminal peptidase inhibitory domain related to the Kunitz family of serine peptidase inhibitors. On the amino-terminal end are one or more highly unusual regions that are unique to the TKDP genes and have no apparent similarity to any other known sequences. The TKDPs are a rather divergent family that exhibits a good deal of variation among the members. To better understand the reason for such variation, the rates of synonymous (dS) and nonsynonymous (dN), as well as radical (p _NR ) and conservative (p _NC ), substitutions were assessed. Phylogenetic trees revealed that the Kunitz domains represented three related groups, whereas the amino-terminal domains formed four groupings. Pairwise comparisons between Kunitz and amino-terminal domain groups demonstrated that dN was consistently greater than dS. In addition, nonsynonymous substitutions in the Kunitz domains tended to be radical (changing charge or polarity), while those in the amino-terminal domains exhibited neither a preponderance of conservative nor radical substitution rates. In summary, the rapid evolution of the TKDPs, coupled with their restricted temporal expression during development, likely reflects the establishment of protein-protein interactions that have evolved to serve the unusual synepitheliochorial placenta of ruminant ungulates. [Reviewing Editor: Dr. Martin Kreitman]     

Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme

Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.     

Decay of proteinase and peptidase activities of human and rabbit erythrocytes during cellular aging

Variations in activity of the membrane-bound and cytosolic proteinases and peptidases were analyzed in human and rabbit erythrocytes at various stages of their life-span. The patterns observed with human erythrocytes were the following. (a) The acidic endopeptidase activity associated with the membranes undergoes a substantial decline during cellular aging, with an estimated half-life of 65 days. Concomitantly it appears to become progressively more latent. (b) All cytosolic proteinase and peptidase activities described previously (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Benatti, U., Morelli, A. and De Flora, A. (1980) Eur. J. Biochem. 110, 421–430) decline exponentially throughout the erythrocyte life-span, with the exception of dipeptidyl aminopeptidase III. The calculated half-lives were: 60 days for the neutral endopeptidase; 87 days for the total acidic endopeptidase activity which is accounted for by three distinct enzymes; 49 days for aminopeptidase B and 133 days for a second aminopeptidase with broad substrate specificity; 84 days for dipeptidyl aminopeptidase II. The results obtained with the rabbit erythrocytes were: (a) no significant decline of leucine aminopeptidase, dipeptidyl aminopeptidase II and III activities in the transition from reticulocytes to mature erythrocytes; (b) very limited decline of aminopeptidase B activity; (c) a pronounced age-dependent decay, in increasing order, of neutral endopeptidase, aminopeptidase A, carboxypeptidase and acidic endopeptidase activities.     

Chondroitin Sulfate Promotes Activation of Cathepsin K

Cathepsin K (CatK), a major lysosomal collagenase produced by osteoclasts, plays an important role in bone resorption. Evidence exists that the collagenase activity of CatK is promoted by chondroitin sulfate (CS), a sulfated glycosaminoglycan. This study examines the role of CS in facilitating CatK activation. We have demonstrated that chondroitin 4-sulfate (C4-S) promotes autoprocessing of the pro-domain of CatK at pH ≤ 5, leading to a fully matured enzyme with collagenase and peptidase activities. We present evidence to demonstrate this autoactivation process is a trans-activation event that is efficiently inhibited by both the covalent cysteine protease inhibitor E-64 and the reversible selective CatK inhibitor L-006,235. During bone resorption, CatK and C4-S are co-localized at the ruffled border between osteoclast bone interface, supporting the proposal that CatK activation is accomplished through the combined action of the acidic environment together with the presence of a high concentration of C4-S. Formation of a multimeric complex between C4-S and pro-CatK has been speculated to accelerate CatK autoactivation and promote efficient collagen degradation. Together, these results demonstrate that CS plays an important role in contributing to the enhanced efficiency of CatK collagenase activity in vivo.     

Allosteric Inhibition of the Neuropeptidase Neurolysin

Neuropeptidases specialize in the hydrolysis of the small bioactive peptides that play a variety of signaling roles in the nervous and endocrine systems. One neuropeptidase, neurolysin, helps control the levels of the dopaminergic circuit modulator neurotensin and is a member of a fold group that includes the antihypertensive target angiotensin converting enzyme. We report the discovery of a potent inhibitor that, unexpectedly, binds away from the enzyme catalytic site. The location of the bound inhibitor suggests it disrupts activity by preventing a hinge-like motion associated with substrate binding and catalysis. In support of this model, the inhibition kinetics are mixed, with both noncompetitive and competitive components, and fluorescence polarization shows directly that the inhibitor reverses a substrate-associated conformational change. This new type of inhibition may have widespread utility in targeting neuropeptidases.