P2 Subsite Research Articles

Carboxymethyl tethered poly(disubstituted)triazoles built on nucleoside skeletons: A unique class of ribonuclease A inhibitors designed using chemical logic

Molecular docking of N-1,4-disubstituted-1,2,3-triazole tethered carboxymethylated thymidine and uridine with ribonuclease A, indicated their possible binding with the P1, B1 and P2 subsites with varied efficiencies. This theoretical study in combination of our earlier experimental observations was used as the guiding principles for designing a range of 1,4-disubstituted 1, 2, 3- triazole tethered carboxymethylated pyrimidine nucleosides. Triazoles are biologically important molecules and at the same time easily accessible through less complicated synthetic routes as reported about two decades back in the context of “click” reactions. Regioselective propargylation of the nucleosides under controlled conditions followed by the use of CuAAC strategy afforded mono-, bis-, tris- and tetratriazolyl pyrimidine nucleosides. Although the characteristics of nucleosides were lost in these densely functionalized polyheterocycles, the catalytic efficiency of ribonuclease A was significantly reduced by these molecules which were investigated experimentally and by docking studies. Triazoles as linkers helped one or more acidic groups to reach the P1 subsite of ribonuclease A. Enzyme kinetics showed that the efficiency of inhibition reached the highest point with an optimum number of functional groups and were not linearly dependent on the number of triazole tethered carboxymethyl groups. The location of the triazole ring in the molecule affected the efficiency and nature of inhibition which were the result of the overall structure of the modified nucleosides. Thus, the tris-triazolylated thymidine derivative (T-3′, 5′, N-tris-CH2TzCH2COOH) as opposed to tetra-triazolylated uridine (U-2′, 3′, 5′, N-tetrakis-CH2TzCH2COOH) emerged as the best inhibitor with an inhibition constant value of 2.3 ± 0.05 µM.

Non-Apoptotic Caspase Activity Preferentially Targets a Novel Consensus Sequence Associated With Cytoskeletal Proteins in the Developing Auditory Brainstem.

The auditory brainstem relies on precise circuitry to facilitate sound source localization. In the chick, the development of this specialized circuitry requires non-apoptotic activity of caspase-3, for which we previously identified several hundred proteolytic substrates. Here we tested whether the sequence of the caspase cleavage site differentially encodes proteolytic preference in apoptotic and non-apoptotic contexts. We constructed a consensus sequence for caspase activity in the non-apoptotic chick auditory brainstem comprising the four residues N-terminal to the cleavage site: IX(G/R)D↓ where X represents no significant enrichment and ↓ represents the cleavage site. We identified GO terms significantly enriched among caspase substrates containing motifs found in the above consensus sequence. (G/R)D↓ was associated with the term “Structural Constituent of Cytoskeleton” (SCoC), suggesting that SCoC proteins may be specifically targeted by caspase activity during non-apoptotic developmental processes. To ascertain whether this consensus sequence was specific to the non-apoptotic auditory brainstem at embryonic day (E) 10, we used protein mass spectrometry of brainstems harvested at a time when auditory brainstem neurons undergo apoptotic cell death (E13). The apoptotic motif VD was significantly enriched among E13 cleavage sites, indicating that motif preference at the P2 subsite had shifted toward the canonical caspase consensus sequence. Additionally, Monte Carlo simulations revealed that only the GD motif was associated with SCoC substrates in the apoptotic auditory brainstem, indicating that GD encodes specificity for SCoC proteins in both non-apoptotic and apoptotic contexts, despite not being preferred in the latter. Finally, to identify candidate human non-apoptotic consensus sequences, we used Monte Carlo analyses to determine motifs and motif pairs associated with SCoC caspase substrates in the Degrabase, a database of cleavage sites in human apoptotic cell lines. We found 11 motifs significantly associated with SCoC proteolysis, including IXXD and GD. We employed a stepwise method to select motif pairs that optimized SCoC specificity for a given coverage of SCoC cleavage events, yielding 11 motif pairs likely to be preferred in SCoC-directed human non-apoptotic caspase consensus sequences. GD + IXXD was among these motif pairs, suggesting a conservation of non-apoptotic consensus sites among vertebrates.

Structure-thermodynamics-relationships of hepatitis C viral NS3 protease inhibitors

Thermodynamic parameters were determined for structurally-related inhibitors of HCV NS3 protease to assess how binding entropies and enthalpies vary with incremental changes at the P2 and P3 inhibitor subsites. Changing the heterocyclic substituent at P2 from a pyridyl to a 7-methoxy-2-phenyl-4-quinolyl group leads to a 710-fold increase in affinity. Annelating a benzene ring onto a pyridine ring leads to quinoline-derived inhibitors having higher affinities, but the individual enthalpy and entropy contributions are markedly different for each ligand pair. Introducing a phenyl group at C2 of the heterocyclic ring at P2 uniformly leads to higher affinity analogs with more favorable binding entropies, while adding a methoxy group at C7 of the quinoline ring at P2 provides derivatives with more favorable binding enthalpies. Significant enthalpy/entropy compensation is observed for structural changes made to inhibitors lacking a 2-phenyl substituent, whereas favorable changes in both binding enthalpies and entropies accompany structural modifications when a 2-phenyl group is present. Overall, binding energetics of inhibitors having a 2-phenyl-4-quinolyl group at P2 are dominated by entropic effects, whereas binding of the corresponding norphenyl analogs are primarily enthalpy driven. Notably, the reversal from an entropy driven association to an enthalpy driven one for this set of inhibitors also correlates with alternate binding modes. When the steric bulk of the side chain at P3 is increased from a hydrogen atom to a tert-butyl group, there is a 770-fold improvement in affinity. The 30-fold increase resulting from the first methyl group is solely the consequence of a more favorable change in entropy, whereas subsequent additions of methyl groups leads to modest increases in affinity that arise primarily from incremental improvements in binding enthalpies accompanied with smaller favorable entropic contributions.

Binding specificity of polypeptide substrates in NS2B/NS3pro serine protease of dengue virus type 2: A molecular dynamics Study

The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P1 and P1′ subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P5–P5′) the P1 and P2 subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of <−10kcalmol−1. Besides, the P3, P1′, P2′ and P4′ subsites showed a less contribution in binding interaction (<−2kcalmol−1). The catalytic water was detected nearby the carbonyl oxygen of the P1 reacting center of the capsid, intNS3, 2A/2B and 4B/5 peptides. These results led to the order of absolute binding free energy (ΔGbind) between these substrates and the NS2B/NS3 protease ranked as capsid>intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values.

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

3′-Oxo-, amino-, thio- and sulfone-acetic acid modified thymidines: Effect of increased acidity on ribonuclease A inhibition

A family of 3′-functionalized thymidines carrying XCH2COOH (X=O, NH, S, SO2) groups has been designed as inhibitors of RNase A. This is because it is possible to manipulate the overall acidity of this new class of nucleic ‘acids’ by changing X from oxygen to the SO2 group in the series. It is also expected that the acyclic nature of the XCH2COOH group would provide enough flexibility to the –COOH group to have maximum interactions with the catalytic subsite P1 of RNase A. As the –SO2CH2COOH substituted derivative showed better potency partially because of the increased acidity of the –COOH group, the inhibitory properties of both 5′-substituted and 3′,5′-disubstituted sulfone acetic acid modified thymidines were investigated. Two –SO2CH2COOH groups were incorporated with the expectation of targeting two phosphate binding sites simultaneously. Thus, 3′,5′-dideoxy-3′,5′-bis-S-[(carboxymethyl)sulfonyl]thymidine emerged as the best inhibitor in this series with a Ki value of 25±2μM.

Expression, extraction and activity testing of the metacaspase Scp3

Apoptosis, a fundamental characteristic of multicellular eukaryotic organisms, is a key component in maintaining organismal integrity. Proteins called metacaspases regulate apoptosis in plants, protozoans and fungi. These proteases are responsible for cleavage of other proteins during programmed cell death. Metacaspases preferentially cleave after basic residues in the P1 subsite. The substrate specificity at the P2 and P3 subsites varies for the metacaspases in plants and fungi. But, metacaspase specificity been studied in only a few plants and in two fungal organisms (S. cerevisiae and S. pombe). Our lab has been studying the five metacaspases from Schizophyllum commune, a split gill mushroom, to better understand the substrate specificity and function of these enzymes. The metacaspase Scp3 has been successfully cloned from the S. commune genome, and transformed into and expressed from Escherichia coli. Using two rounds of nickel affinity chromatography we have obtained protein that is &gt;;95% pure. Current activity assays have shown significant enzymatic activity of Scp3 with the substrate FITC‐Casein compared to a crude cell extract without Scp3. Our lab is currently working to optimize the expression of Scp3 via autoinduction in various media and at different growth temperatures. Additionally, optimization of the extraction and assay conditions for the metacaspase are being completed in conjunction with one another. Optimization of extraction will provide us with more stable and active protein that can be tested for enzymatic activity. Once optimization of all processes is completed for Scp3 the knowledge can be transferred to the other four metacaspases of S. commune.

Crystal structures of murine angiogenin‐2 and ‐3 – probing ‘structure – function’ relationships amongst angiogenin homologues

Angiogenin (Ang) is a potent inducer of neovascularization. Point mutations in human Ang have been linked to cancer progression and two neurodegenerative diseases: amyotrophic lateral sclerosis and Parkinson's disease. Intensive structural and functional analyses of Ang have been paramount in assigning functions to this novel homologue of bovine pancreatic RNase A. However, inhibitor-binding studies with crystalline Ang (for designing potential anti-cancer drugs) have been hampered as a result of the inaccessibility of the active site. Experiments with the murine homologues of Ang have not only overcome the obvious practical limitations encountered when studying the role of a human protein in healthy individuals, but also the crystal structures of murine angiogenins (mAng and mAng-4) have revealed themselves to have greater potential for the visualization of small-molecule inhibitor binding at the active site. In the present study, we report the crystal structures of two more murine Ang paralogues, mAng-2 and mAng-3, at 1.6 and 1.8 Å resolution, respectively. These constitute the first crystal structures of an Ang with a zinc ion bound at the active site and provide some insight into the possible mode of inhibition of the ribonucleolytic activity of the enzyme by these divalent cations. Both structures show that the residues forming the putative P1, B1 and B2 subsites occupy positions similar to their counterparts in human Ang and are likely to have conserved roles. However, a less obtrusive conformation of the C-terminal segment in mAng-3 and the presence of a sulfate ion in the B1 subsite of mAng-2 suggest that these proteins have the potential to be used for inhibitor-binding studies. We also discuss the biological relevance of the structural similarities and differences between the different Ang homologues.DatabaseThe atomic coordinates and structure factors for mAng-2 (3ZBV) and mAng-3 (3ZBW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/).Structured digital abstractmAng2 and mAng3 bind by x-ray crystallography (View interaction)

Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis, biochemical, and structural evaluation

Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with low μM inhibition constant (Ki) values with the ones having a triazolo linker being more potent than the ones without. The most potent of these is 1-[(β-d-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with Ki=1.6μM. The high resolution X-ray crystal structures of the RNase A in complex with three most potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B1 subsite while the triazole moiety binds at the main subsite P1, where P-O5′ bond cleavage occurs, and the ribose at the interface between subsites P1 and P0 exploiting interactions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at the inhibitory potency has been also examined and results show that any addition at this position leads to a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar RNase A—ligand complexes reveals that the triazole moiety interactions with the protein form the structural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and the ribose forms the starting point for further improvement of these inhibitors in the quest for potent ribonucleolytic inhibitors with pharmaceutical potential.

Multiple drug resistant mechanisms against darunavir, amprenavir, and nelfinavir of HIV-1 PR

Acquired immune deficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV), which is infecting more humans and is expanding faster in the world. The illness interferes with the immune system, making people with AIDS much more likely to get infections, including opportunistic infections and tumors that do not affect people with working immune systems. HIV-1 PR is one of the major targets of anti-AIDS drug discovery. It is, therefore, necessary to develop some inhibitors against HIV-1 PR. In this work, we executed molecular dynamics (MDs) simulation of HIV-1 PR with drugs darunavir (DRV), amprenavir (APV), nelfinavir (NFV), and examined the resistant mechanism of L10I, G48V, I54V, and L90M mutations of this PR, aiming at designing promising drugs. The comparative analysis suggests that the existences of dodecahydroisoquinoline ring at P1′ subsite, 4-aminophenylsulfonamide at P2′ subsite, and bis-tetrahydrofuranylurethane at P2 subsite are helpful for maintaining the high affinity of the inhibitor for the protease and exhibiting high potency against multiple drug resistance (MDR) mutant protease.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

BackgroundHemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins.ResultsAn aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'.ConclusionsBmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

On the Relevance of the Met-turn Methionine in Metzincins

The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zinc-binding site, which is structurally and spatially conserved and invariantly shows a methionine at position 3 in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating 10 mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three-dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity.

Molecular modeling studies on dengue and West Nile Virus NS2B/NS3 protease inhibitor interaction

Dengue Virus (DV) and West Nile Virus (WNV) are causes of severe public-health problems in many countries. Neither effective human vaccines nor drugs against both DV and WNV are available. NS2B (an essential cofactor) together with NS3 protease, which is a vital enzyme for the viruses in the replication cycle, is currently focused as the promising enzyme target for drug development. However, the active enzyme-inhibitor complex of DV is not yet experimentally available. Therefore, 3D structure of the DV's enzyme was built using the related WNV NS2B/NS3 structure as a template (2FP7.pdb) [1]. The DV model and 2FP7.pdb (WNV-X) were subjected to molecular dynamics (MD) simulations. MD simulations reveal the important of NS2B for interacting with both NS3 protease and P2 subsite of inhibitor. The final MD snapshot of DV model and WNV-X were employed for GRID field calculations. Results show bigger N+ and OH fields at the S1 pocket of DV model than those of WNV-X implying the importance of the electrostatic or H-bond interaction at the S1 pocket than that of WNV. On the other hand, the Dry field at the S1 pocket of WNV-X is larger than that of DV model indicating the relevance of hydrophobic interaction at this pocket for WNV. In addition, MD simulations of the NS2B/NS3 protease of WNV and DV complexed with Bz-Nle-Lys-Arg-Ala-H (most selective inhibitor, Ki = 193.00 μM for DV and IC50 = 4.6 μM for WNV) [2,3] were also carried out. Results reveal that AlaP1 of the inhibitor is not able to make favourable electrostatic interactions at the S1/S2 pockets of DV. The simulations showed also that both S1-P1 and S2-P2 interactions are important for inhibiting the DV protease whereas in case of the WNV protease the interaction at the S1-pocket is less important.

Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79's loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50'. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1' subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2' subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design.

Crystal and Molecular Structure at 0.16-nm Resolution of the Hybrid Bacillus Endo-1,3-1,4-β-D-Glucan 4-Glucanohydrolase H(A16-M)

H(A16-M) is a hybrid endo-1,3-1,4-β-D-glucan 4-glucanohydrolase from Bacillus. Its crystal structure was refined using synchrotron X-ray diffraction data up to a maximal resolution of 0.16 nm. The R value of the resulting model is 14.3% against 21032 reflections >2σ. 93% of the amino acid residues are in the most favorable regions of the Ramachandran diagram, and geometrical parameters are in accordance with other proteins solved at high resolution. As shown earlier [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287–5291], the protein folds into a compact jellyroll-type β-sheet structure. A systematic analysis of the secondary structure reveals the presence of two major antiparallel β-sheets and a three-stranded minor mixed sheet. Amino acid residues involved in catalysis and substrate binding are located inside a deep channel spanning the surface of the protein. To investigate the stereochemical cause of the observed specificity of endo-1,3-1,4-β-D-glucan 4-glucanohy-drolases towards β-1,4 glycosyl bonds adjacent to β-1,3 bonds, the high-resolution crystal structure has been used to model an enzyme-substrate complex. It is proposed that productive substrate binding to the subsites p1, p2 and p3 of H(A16-M) requires a β-1,3 linkage between glucose units bound to p1 and p2.

Properties of a non collagen-degradingBacillus subtiliskeratinase

Bacillus subtilis S14 produces a keratinase (KerS14) with non collagen-degrading activity. Indeed, this is the first keratinase described so far that does not have any detectable effect on collagen, which is a crucial property for an enzyme intended to be used in skin dehairing. Because of its importance as an industrial tanning enzyme, we report the biochemical characterization of KerS14. This protein exhibited an apparent molecular mass of 27 kDa, a pI of 6.5, and an optimum pH in the range of 8.0-9.0. The enzyme's activity was stimulated by Mn2+ (7.7-fold), Ca2+ (6.1-fold), Mg2+ (4.9-fold), and Co2+ (4.0-fold) but was inhibited by Cu2+ and Zn2+. Using p-nitroanilide and methylcoumarine derivatized peptides, we observed that KerS14 prefered Arg at subsite P1, small amino acid residues at subsite P2, and Gln or Glu at subsite P3. KerS14 presented higher keratin degradation specificity than other commercial proteases. Its high keratinolytic activity and the absence of virtually any activity against collagen remark the biotechnological potential of this enzyme to be used at larger scales in tannery dehairing processes.

Synthesis and SAR of succinamide peptidomimetic inhibitors of cathepsin S

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S ( 1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.

Biological and Structural Features of Murine Angiogenin-4, an Angiogenic Protein,

Murine angiogenin-4 (mAng-4) is a member of the pancreatic ribonuclease superfamily that is expressed in some endodermally derived organs. We now show that mAng-4 is angiogenic using a thoracic aorta assay never before applied to the angiogenins. mAng-4, human angiogenin (hAng), and murine angiogenin-1 (mAng-1) stimulate the proliferation of IGR1 melanoma cells but do not stimulate the proliferation or migration of bovine corneal endothelial cells or primary mouse embryonic fibroblasts. In addition, we report the 3-D structure of mAng-4 at 2.02-A resolution. The structure shows that the residues forming the putative B1, P1, and B2 RNA-binding subsites occupy positions similar to their hAng counterparts. The B1 subsite is obstructed by Glu115 and Ile118. The obstruction is stabilized by a novel salt bridge between the C-terminal carboxyl group and the side chain of Arg99. Through mutational studies, we identify residues critical to the angiogenic function of mAng-4. The effect of H12A and H112A mutations in the catalytic site indicates that ribonucleolytic activity is essential to angiogenesis. The consequences of a nearby E115A mutation are consistent with a significant role for Glu115 in the attenuation of enzymatic activity but also suggest that sufficient suppression of catalysis is necessary for angiogenesis. The effect of an R32A mutation in the putative nuclear localization sequence indicates that this residue is crucial for angiogenesis. In the putative cell-binding segment, the replacement of Lys59 with Asn (its counterpart at position 61 of hAng) does not abrogate enzymatic activity but abolishes angiogenic activity, the reason for which is unclear.

A comparative study of the expression of serine proteinases in quiescent seeds and in developing Canavalia ensiformis plants

An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.

Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain

Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here, the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3, and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell-permeable reagents for monitoring legumain activity in complex proteomes.