Mapping the substrate specificity of the Plasmodium M1 and M17 aminopeptidases.

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. Overall, the P. vivax and P. berghei enzymes had a faster substrate turnover rate than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

Aminopeptidases in Cardiovascular and Renal Function. Role as Predictive Renal Injury Biomarkers.

Aminopeptidases (APs) are metalloenzymes that hydrolyze peptides and polypeptides by scission of the N-terminus amino acid and that also participate in the intracellular final digestion of proteins. APs play an important role in protein maturation, signal transduction, and cell-cycle control, among other processes. These enzymes are especially relevant in the control of cardiovascular and renal functions. APs participate in the regulation of the systemic and local renin-angiotensin system and also modulate the activity of neuropeptides, kinins, immunomodulatory peptides, and cytokines, even contributing to cholesterol uptake and angiogenesis. This review focuses on the role of four key APs, aspartyl-, alanyl-, glutamyl-, and leucyl-cystinyl-aminopeptidases, in the control of blood pressure (BP) and renal function and on their association with different cardiovascular and renal diseases. In this context, the effects of AP inhibitors are analyzed as therapeutic tools for BP control and renal diseases. Their role as urinary biomarkers of renal injury is also explored. The enzymatic activities of urinary APs, which act as hydrolyzing peptides on the luminal surface of the renal tubule, have emerged as early predictive renal injury biomarkers in both acute and chronic renal nephropathies, including those induced by nephrotoxic agents, obesity, hypertension, or diabetes. Hence, the analysis of urinary AP appears to be a promising diagnostic and prognostic approach to renal disease in both research and clinical settings.

Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases.

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.

Redox regulation of methionine aminopeptidase 2 activity.

Protein translation is initiated with methionine in eukaryotes, and the majority of proteins have their N-terminal methionine removed by methionine aminopeptidases (MetAP1 and MetAP2) prior to action. Methionine removal can be important for protein function, localization, or stability. No mechanism of regulation of MetAP activity has been identified. MetAP2, but not MetAP1, contains a single Cys(228)-Cys(448) disulfide bond that has an -RHStaple configuration and links two ß-loop structures, which are hallmarks of allosteric disulfide bonds. From analysis of crystal structures and using mass spectrometry and activity assays, we found that the disulfide bond exists in oxidized and reduced states in the recombinant enzyme. The disulfide has a standard redox potential of -261 mV and is efficiently reduced by the protein reductant, thioredoxin, with a rate constant of 16,180 m(-1) s(-1). The MetAP2 disulfide bond also exists in oxidized and reduced states in glioblastoma tumor cells, and stressing the cells by oxygen or glucose deprivation results in more oxidized enzyme. The Cys(228)-Cys(448) disulfide is at the rim of the active site and is only three residues distant from the catalytic His(231), which suggested that cleavage of the bond would influence substrate hydrolysis. Indeed, oxidized and reduced isoforms have different catalytic efficiencies for hydrolysis of MetAP2 peptide substrates. These findings indicate that MetAP2 is post-translationally regulated by an allosteric disulfide bond, which controls substrate specificity and catalytic efficiency.

TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions.

A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses (∼35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

Census of cytosolic aminopeptidase activity reveals two novel cytosolic aminopeptidases.

Activation of CD8(+) cytotoxic T cells is crucial for the adaptive immune response against viral infections and the control of malignant transformed cells. Together with activation of costimulatory molecules like CD3 and CD28, CD8(+) T cells need activation of their unique T cell receptor via recognition of foreign peptide epitopes in combination with major histocompatibility complexes class I on the cell surface of professional antigen-presenting cells. Presentation of pathogen-associated proteins is the result of a complex proteolytic process. It starts with the breakdown of proteins by a cytosolic endopeptidase, the proteasome, and is continued by subsequent N-terminal trimming events in the cytosol and/or the endoplasmic reticulum. Analysis of the proteolytic aminopeptidase activity in the former cellular compartment showed that the cytosol harbors a multitude of aminopeptidases that have singular specificities, but on the other hand also show redundancy in the trimming of N-terminal residues. The observed pattern of the overall trimming in the cytosol is reflected by the activity of the four identified aminopeptidases, and the administration of protease inhibitors made it possible to assign specificity of cleaving of proteinogenic amino acids to one or more identified aminopeptidase. The only exception was the cleavage of aspartic acid, which is performed by one yet unidentified enzyme.

Structural basis for the unusual specificity of Escherichia coli aminopeptidase N.

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.

Structure analysis of peptide deformylases from Streptococcus pneumoniae, Staphylococcus aureus, Thermotoga maritima and Pseudomonas aeruginosa: snapshots of the oxygen sensitivity of peptide deformylase.

Peptide deformylase (PDF) has received considerable attention during the last few years as a potential target for a new type of antibiotics. It is an essential enzyme in eubacteria for the removal of the formyl group from the N terminus of the nascent polypeptide chain. We have solved the X-ray structures of four members of this enzyme family, two from the Gram-positive pathogens Streptococcus pneumoniae and Staphylococcus aureus, and two from the Gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa. Combined with the known structures from the Escherichia coli enzyme and the recently solved structure of the eukaryotic deformylase from Plasmodium falciparum, a complete picture of the peptide deformylase structure and function relationship is emerging. This understanding could help guide a more rational design of inhibitors. A structure-based comparison between PDFs reveals some conserved differences between type I and type II enzymes. Moreover, our structures provide insights into the known instability of PDF caused by oxidation of the metal-ligating cysteine residue.

Mapping of the active site zinc ligands of peptide deformylase.

A set of 50 site-directed mutants of the Escherichia coli fms gene was constructed to delineate the residues of the active site of peptide deformylase, including the ligands of the zinc ion. In particular, because zinc is usually coordinate by Asp, Cys, Glu or His residues, all the corresponding codons were individually changed. The functional consequence of the substitutions was assessed by complementation of a fms-null strain with the help of vectors expressing the mutate genes. In addition to the mutations of the Cys90 codon, only those of the three conserved residues of the 132HEXXH136 motif of peptide deformylase prevented the indicator strain growing. Most enzyme variants were purified to homogeneity in a second step. Their characterization in vitro showed that the defects in complementation as observed in vivo corresponded to huge decreases of deformylation efficiency. The change of Glu88 also led to a significant decrease in catalytic rate. Unexpectedly, upon substitutions of Glu79 or of Glu83, the enzymes exhibited a strongly increased catalytic efficiency. The measurement of the content of zinc in each purified variant indicated that Cys90, His132 and His136 bound the metal ion. Zinc-free variants mutated at these positions were obtained and shown to display an increased sensitivity to proteolytic attack. Altogether, the data showed that both the presence of zinc and the conserved residues of the HEXXH motif were crucial for the activity of deformylase. This behaviour identified the enzyme as a member of the zinc metalloproteases superfamily. However, the unexpected participation in the binding of the zinc atom of Cys90, upstream from the HEXXH motif, suggested that peptide deformylase could be representative of a new sub-family, distinct from those of thermolysin and astacin.

Distinctive features of the two classes of eukaryotic peptide deformylases.

Peptide deformylases (PDFs) are essential enzymes of the N-terminal protein processing pathway of eubacteria. The recent discovery of two types of PDFs in higher plants, PDF1A and PDF1B, and the detection of PDF1A in humans, have raised questions concerning the importance of deformylation in eukaryotes. Here, we have characterized fully in vitro and compared the properties of the two classes of eukaryotic PDFs, PDF1A and PDF1B, using the PDFs from Arabidopsis thaliana and Lycopersicon esculentum. We have shown that the PDFs of a given class (1A or 1B) all display similar features, independently of their origin. We also observed similar specificity of all plant PDFs for natural substrate peptides, but identified a number of biochemical differences between the two classes (1A or 1B). The main difference lies at the level of the bound cofactor, iron for PDF1B-like bacterial PDFs, and zinc for PDF1A. The nature of the metal cation has important consequences concerning the relative sensitivity to oxygen of the two plant PDFs. Investigation of the specificity of these enzymes with unusual substrates revealed additional differences between the two types of PDFs, enabling us to identify specific inhibitors with a lower affinity against PDF1As. However, the two plant PDFs were inhibited equally strongly in vitro by actinonin, an antibiotic that specifically acts on bacterial PDFs. Uptake of actinonin by A. thaliana seedlings was used to investigate the function of PDFs in the plant. Because it induces an albino phenotype, we conclude that deformylation is likely to play an essential role in the chloroplast.

Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain.

We evaluated the subcellular distribution of four membrane-bound aminopeptidases in the human and rat brain cortex. The particulate enzymes under study--puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), pyroglutamyl-peptidase I (PG I) and aspartyl-aminopeptidase (Asp-AP)--were fluorometrically measured using beta-naphthylamide derivatives. Membrane-bound aminopeptidase activity was found in all the studied subcellular fractions (myelinic, synaptosomal, mitochondrial, microsomal and nuclear fractions), although not homogenously. Human PSA showed highest activity in the microsomal fraction. APN was significantly higher in the nuclear fraction of both species, while PG I showed highest activity in the synaptosomal and myelinic fractions of the human and rat brain. The present results suggest that in addition to inactivating neuropeptides at the synaptic cleft, these enzymes may participate in other physiological processes. Moreover, these peptidases may play specific roles depending on their activity levels at the different subcellular structures where they are localized.

Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin.

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.

Aminopeptidases and angiogenesis.

A number of proteases, including matrix metalloproteinases and plasminogen activators, have been shown to be involved in angiogenesis. In addition, recent reports suggest that aminopeptidases also play roles in angiogenesis. These peptidases regulate the N-terminal modification of proteins and peptides required in processes such as maturation, activation, or degradation, and thereby they are related to a variety of physiological and pathological processes. At least three aminopeptidases are reported to be involved in angiogenesis, namely, type 2 methionine aminopeptidase, aminopeptidase N, and adipocyte-derived leucine aminopeptidase/puromycin-insensitive leucyl-specific aminopeptidase. This review will focus on the possible role of these aminopeptidases in angiogenesis.

Membrane bound members of the M1 family: more than aminopeptidases.

In mammals the M1 aminopeptidase family consists of nine different proteins, five of which are integral membrane proteins. The aminopeptidases are defined by two motifs in the catalytic domain; a zinc binding motif HEXXH-(X18)-E and an exopeptidase motif GXMEN. Aminopeptidases of this family are able to cleave a broad range of peptides down to only to a single peptide. This ability to either generate or degrade active peptide hormones is the focus of this review. In addition to their capacity to degrade a range of peptides a number of these aminopeptidases have novel functions that impact on cell signalling and will be discussed.

Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

BACKGROUND: The N-terminal protein processing mechanism (NPM) including N-terminal Met excision (NME) and N-terminal acetylation (N(α)-acetylation) represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α)-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α)-acetylated) proteins. Most proteins (47, >81%) are subjected to N(α)-acetylation following the N-terminal removal of Met, indicating that N(α)-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α)-acetylation (NPM) to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs) and N-terminal acetyltransferase (Nat) enzymes in poplar. The N(α)-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α)-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α)-acetylation of proteins in poplar.

Characterization of the biochemical properties of two methionine aminopeptidases of Cryptosporidium parvum.

We identified two methionine aminopeptidases of Cryptosporidium parvum (CpMetAP1 and CpMetAP2) and characterized the biochemical properties of the recombinant enzymes. CpMetAP1 and CpMetAP2 belong to the type I and type II MetAP subfamilies, respectively. Both CpMetAPs have typical amino acid residues essential for metal binding and substrate binding sites, which are conserved in the MetAP family. Bacterially expressed recombinant CpMetAP1 and CpMetAP2 showed similar biochemical properties including a broad optimal pH range (pH 7.5-8.5) with maximum activity at pH 8.0. The two enzymes were stable under neutral and alkaline pHs but were relatively unstable under acidic conditions. The activities of CpMetAP1 and CpMetAP2 increased highly in the presence of Mn(2+) and Co(2+). CpMetAP1 and CpMetAP2 were effectively inhibited by the metal chelators, EDTA and 1,10-phenanthroline, and were partially inhibited by the aminopeptidase inhibitors, amastatin and bestatin. Fumagillin also showed an inhibitory effect on both CpMetAPs.

Metallo-aminopeptidase inhibitors.

Aminopeptidases are enzymes that selectively hydrolyze an amino acid residue from the N-terminus of proteins and peptides. They are important for the proper functioning of prokaryotic and eukaryotic cells, but very often are central players in the devastating human diseases like cancer, malaria and diabetes. The largest aminopeptidase group include enzymes containing metal ion(s) in their active centers, which often determines the type of inhibitors that are the most suitable for them. Effective ligands mostly bind in a non-covalent mode by forming complexes with the metal ion(s). Here, we present several approaches for the design of inhibitors for metallo-aminopeptidases. The optimized structures should be considered as potential leads in the drug discovery process against endogenous and infectious diseases.