Val Bond Research Articles

Native Chemical Ligation at Valine

Peptide ligation at hydrophobic sites is possible with a ligation–desulfurization strategy in which penicillamine serves as a precursor of valine. The β,β-dimethylcysteine peptides reacted surprisingly fast in native chemical ligation reactions. Even the sterically crowded and unpolar Leu–Val bond can be formed in high yield.

Selective Modulation of Matrix Metalloproteinase 9 (MMP-9) Functions via Exosite Inhibition

Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.

Triple-helical transition state analogues: a new class of selective matrix metalloproteinase inhibitors.

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P1-P1' subsites of the triple-helical peptide. Inhibition studies revealed Ki values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).

ATP signaling site in the ARM domain of atrial natriuretic factor receptor guanylate cyclase

Atrial natriuretic factor (ANF) receptor guanylate cyclase (ANF-RGC) is a single transmembrane spanning modular protein. It binds ANF to its extracellular module and activates its intracellular catalytic module located at its carboxyl end. This results in the accelerated production of cyclic GMP, which acts as a critical second messenger in decreasing blood pressure. Two mechanistic models have been proposed for the ANF signaling of ANF-RGC. One is ATP-dependent and the other ATP-independent. In the former, ATP works through the ARM (ATP-regulated transduction module) of ANF-RGC. This model has recently been challenged [Antos et al. (2005) J Biol Chem 280:26928-26932] in support of the ATP-independent model. The present in-depth study analyzes the major principles of this challenge and concludes that the challenge lacks merit. The study then moves on to dissect the ATP mechanism of ANF signaling of ANF-RGC. It shows that the ATP photoaffinity probe, [gamma(32)P]-8-azido-ATP, reacts with Cys(634) residue in the ATP-binding pocket of ARM, and also signals the ANF-dependent activation of ANF-RGC. The target site of the 8-azido (nitrene) group is between the Cys(634) and Val(635) bond of the ATP-binding pocket. Thus, the study experimentally validates the ARM model-predicted role of Val(635) in the folding pattern of the ATP-binding pocket. And, it also identifies another residue Cys(634) that along with eight already identified residues is a part of the fold around the adenine ring of the ATP pocket. This information establishes the direct role of ATP in ANF signal transduction model of ANF-RGC, and provides a significant advancement on the mechanism by which the ATP-dependent transduction model operates.

Unusual Proteolytic Activation of Pro-hepatocyte Growth Factor by Plasma Kallikrein and Coagulation Factor XIa

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an alpha-chain containing four Kringle domains (K1-K4) and a serine protease domain-like beta-chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg(494)-Val(495) bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC(50)) of (125)I-labeled pro-HGF during a 4-h period were 10 and 17 nm. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg(494)-Val(495) and the novel site Arg(424)-His(425) located in the K4 domain of the alpha-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg(494) --> Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg(494)-Val(495) site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved alpha-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/c-Met pathway, such as tissue repair and angiogenesis.

Engineered Recombinant Enteropeptidase Catalytic Subunit: Effect of N-Terminal Modification

Enteropeptidase (enterokinase) is a serine protease highly specific for recognition and cleavage of the target sequence of Asp–Asp–Asp–Asp–Lys (D4K). The three-dimensional structure of the enteropeptidase shows that the N-terminal amino acid is buried inside the protein providing molecular interactions necessary to maintain the conformation of the active site. To determine the influence of the N-terminal amino acid of enteropeptidase light chain (EKL) on the enzymatic activity, we constructed various mutants including 17 different single amino acid substitutions and three different extensions at the N-terminal end. The mutants of recombinant enteropeptidase (rEKL) were expressed in Saccharomyces cerevisiae and secreted into culture medium. Among 20 different mutants tested, the only mutant with the Ile → Val substitution exhibited significant activity. The kinetic properties of the mutant protein were very similar to those of the wild-type rEKL. Based on the three-dimensional structure where the N-terminal Ile is oriented into hydrophobic pocket, the results suggest that Val could substitute Ile without affecting the active conformation of the enzyme. The results also explain why all trypsin-like serine proteases carry either Ile or Val at the N-termini and none other amino acid residues are found. Moreover, this finding provides a mental framework for expressing the N-terminally engineered enteropeptidase in Escherichia coli, utilizing the known property of the methionine aminopeptidase that exhibits poor activity toward the N-terminal Met–Ile bond, but offers efficient cleavage of the Met–Val bond.

Human Cationic Trypsinogen: Arg117 IS THE REACTIVE SITE OF AN INHIBITORY SURFACE LOOP THAT CONTROLS SPONTANEOUS ZYMOGEN ACTIVATION

Mutation of Arg(117), an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg(117) was investigated in wild-type cationic Tg and in the activation-resistant Lys(15) --> Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg(117) can be examined selectively. We found that trypsinolytic cleavage of the Arg(117)-Val(118) bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca(2+), at pH 8.0, the hydrolysis equilibrium (K(hyd) = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca(2+) reduced the rate of cleavage at Arg(117) at least 20-fold, and shifted K(hyd) to 0.7. These observations indicate that the Arg(117)-Val(118) bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated K(i) of 80 microm, while no inhibition was observed with K15Q-Tg carrying the Arg(117) --> His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg(117). Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg(117) results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg(117)-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg(117) --> His mutation.

Macromolecular Ensembles of External and Internal Fibrinolysis: Ways to Enhance the Thrombolytic Effect (A Review)

Cardiovascular disorders are among the most widespread pathologies in both industrial and developing countries [1, 2]. An important role in the chemotherapy of cardiovascular disorders belongs to thrombolytic drugs, the administration of which significantly decreases the lethal outcome in patients suffering from myocardial infarction [3, 4]. Effective preparations for thrombolytic therapy are the physiological plasminogen activators representing serine proteases of the tissue (tissue plasminogen activator, t-PA) or urokinase (urokinase, UK; prourokinase, proUK) types and bacterial proteins (streptokinase, Strk; staphylokinase, Stph) acquiring biocatalytic properties upon interaction with plasminogen (Pmg) or plasmin (Pm). These drugs, usually referred to simply as plasminogen activators (PAs), catalyze the hydrolysis of Pmg at the Arg – Val bond with the formation of active Pm. Action of the latter agent leads to degradation of the insoluble fibrin clot which is the reinforcing (framework) basis of thrombi. Characterized by a short lifetime (halflife in the blood stream, 0.1 sec), Pm is effectively inhibited by 2-antiplasmin ( 2-AP) and (to a lesser extent) by 2-macroglobulin ( 2-MG), while the tissue and urokinase PAs in the plasma are inhibited by a plasminogen activator inhibitor of the first type (PAI-1). Fibrin (Fb) serves not only as a substrate for Pm but as a surface for the specific adsorption of Pmg and t-PA as well. Under the action of pulsating pressure in the blood stream, Fb can be saturated with PAs that even do not possess significant affinity with respect to Fb. Fibrinolytic transformations on Fb protect components of the fibrinolytic system from the action of plasma inhibitors and enhance the Pmg activation. This is a general (Fig. 1) scheme of the possible interactions of PAs involved in exogenous thrombolysis. DEVELOPMENT OF NEW EFFECTIVE ACTIVATORS

Conversion of Glu-Plasminogen to Lys-Plasminogen Is Necessary for Optimal Stimulation of Plasminogen Activation on the Endothelial Cell Surface

When Glu-plasminogen is bound to cells, plasmin (Pm) formation by plasminogen (Pg) activators is markedly enhanced compared with the reaction in solution. It is not known whether the direct activation of Glu-Pg by Pg activators is promoted on the cell surface or whether plasminolytic conversion of Glu-Pg to the more readily activated Lys-Pg is necessary for enhanced Pm formation on the cell surface. To distinguish between these potential mechanisms, we tested whether Pm formation on the cell surface could be stimulated in the absence of conversion of Glu-Pg to Lys-Pg. Rates of activation of Glu-Pg, Lys-Pg, and a mutant Glu-Pg, [D646E]Glu-Pg, by either tissue Pg activator (t-PA) or urokinase (u-PA) were compared when these Pg forms were either bound to human umbilical vein endothelial cells (HUVEC) or in solution. ([D646E]Glu-Pg can be cleaved at the Arg(561)-Val(562) bond by Pg activators but does not possess Pm activity subsequent to this cleavage because of the mutation of Asp(646) of the serine protease catalytic triad.) Glu-Pg activation by t-PA was enhanced on HUVEC compared with the solution phase by 13-fold. In contrast, much less enhancement of Pg activation was observed with [D646E]Glu-Pg ( approximately 2-fold). Although the extent of activation of Lys-Pg on cells was similar to that of Glu-Pg, the cells afforded minimal enhancement of Lys-Pg activation compared with the solution phase (1.3-fold). Similar results were obtained when u-PA was used as activator. When Glu-Pg was bound to the cell in the presence of either t-PA or u-PA, conversion to Lys-Pg was observed, but conversion of ([D646E]Glu-Pg to ([D646E]Lys-Pg was not detected, consistent with the conversion of Glu-Pg to Lys-Pg being necessary for optimal enhancement of Pg activation on cell surfaces. Furthermore, we found that conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was markedly enhanced ( approximately 20-fold) on the HUVEC surface, suggesting that the stimulation of the conversion of Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.

A new family of serine protease inhibitors (Bombyx family) as established from the unique topological relation between the positions of disulphide bridges and reactive site.

The positions of the reactive site and the disulfide bridges in fungal protease inhibitor F (FPI-F) from silkworm (Bombyx mori), which has a unique amino acid sequence and inhibitory specificity, were investigated. At pH 3.0, subtilisin BPN', which is one of target proteases of this inhibitor, specifically cleaved the peptide bond of the inhibitor at Thr(29)-Val(30). The cleaved bond was regenerated by subtilisin BPN' at pH 8.0. These results indicate that the Thr(29)-Val(30) bond of the inhibitor is the reactive site. The locations of disulfide bridges were determined to be Cys(3)-Cys(35), Cys(14)-Cys(27), Cys(18)-Cys(55), and Cys(37)-Cys(49). Based on the positions of the reactive site and the disulfide bridges, FPI-F is considered to be a member of a new family of serine protease inhibitors. We propose the designation Bombyx family for the new inhibitor family of which FPI-F is a member.

Proteolytic Processing of Farnesylated Peptides: Assay and Partial Purification from Pig Brain Membranes of an Endopeptidase Which Has the Characteristics of EC 3.4.24.15

The proteolytic processing of farnesylated peptides was investigated with the tritiated C-terminal heptapeptide of mouse N-Ras protein, Propionyl-Gly-Ser-Pro-(farnesyl-Cys)-[3H]Val-Leu-Met, as substrate. Using two rapid and sensitive methods, the peptidase which cleaves the (farnesyl-Cys)-[3H]Val bond was purified more than 100 times from a microsomal fraction of pig brain. The determination of its molecular mass (about 70 kDa) and its pH range for optimum activity (neutral pH) as well as the results obtained with various inhibitors indicate that this [3H]Val-Leu-Met releasing enzyme resembles endopeptidase 24.15 : a thiol-dependent zinc metallopeptidase. Parallel and comparative inhibition assays on partially purified enzyme and pure "24.15" confirm this similarity.

Kinetics of tryptic hydrolysis of the arginine-valine bond in folded and unfolded ribonuclease T1.

We have used ribonuclease T1 and its chemically modified derivatives as substrates, and trypsin as proteinase, to investigate the kinetics of proteolysis of a specific peptide bond in the folded and unfolded conformations of a protein. Steady-state kinetic studies showed that Km = 0.27 mM and Kcat. = 2.45 s-1 for the tryptic hydrolysis of the Arg(77)-Val(78) peptide bond in unfolded ribonuclease T1. This Km is somewhat lower than, and the kcat. value similar to, values found for the tryptic hydrolysis of comparable bonds in small peptides. Our data for the initial velocity of hydrolysis of the Arg(77)-Val(78) bond in a solution of the folded protein indicate that the bond is at least 1700 times less rapidly hydrolysed in the folded than in the unfolded conformation of ribonuclease T1, and do not exclude the possibility that the bond is completely resistant to hydrolysis in the folded protein.

Potent new inhibitors of human renin.

The renal acid protease renin selectively cleaves its plasma substrate angiotensinogen to release the decapeptide angiotensin I, which in turn is cleaved by a carboxydipeptidase converting enzyme to yield the pressor octapeptide angiotensin II1. It is generally accepted that the renin–angiotensin system has a physiological role in blood pressure and electrolyte homeostasis2, and that abnormalities of the system contribute to certain forms of hypertension3. We previously showed that reduction of the Leu10–Leu11 scissile peptide bond in the (6–13) octapeptide sequence 2 of equine angiotensinogen produced potent and selective inhibitors of canine plasma renin5. We have now used the same approach to modify the recently elucidated N-terminal sequence6 of human angiotensinogen and report here the production of highly active and species-specific in vitro inhibitors of endogenous human renin cleaving renin substrate in human plasma. Whereas the (6–13) octapeptide H-112 of human angiotensinogen is only a weak inhibitor of human plasma renin (IC50 = 313 µM), the analogue H-113, in which the scissile Leu–Val bond has been reduced, has IC50 = 0.19 µM. Extension of H-113 with Pro at the N-terminus and with Lys at the C-terminus7, giving the decapeptide derivative H-142, increases inhibitory potency further (IC50 = 10 nM). We believe that the high in vitro inhibitory potencies of H-113 and H-142 are due to the ability of the reduced moiety —CH2—NH— to act as a non-hydrolysable analogue of the tetrahedral transition state formed during hydrolysis of the (10–11) peptide bond. H-113 and H-142 are strongly species specific, and are highly specific for renin among acid proteases: H-142 has no inhibitory effect in vitro on human cathepsin D or renal acid protease at a concentration of 712 µM.

Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.