Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA). Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations.

Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.

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.

M17 leucyl aminopeptidases are metal-dependent exopeptidases that rely on oligomerization to diversify their functional roles. The M17 aminopeptidases from Plasmodium falciparum (PfA-M17) and Plasmodium vivax (Pv-M17) function as catalytically active hexamers to generate free amino acids from human hemoglobin and are drug targets for the design of novel antimalarial agents. However, the molecular basis for oligomeric assembly is not fully understood. In this study, we found that the active site metal ions essential for catalytic activity have a secondary structural role mediating the formation of active hexamers. We found that PfA-M17 and Pv-M17 exist in a metal-dependent dynamic equilibrium between active hexameric species and smaller inactive species that can be controlled by manipulating the identity and concentration of metals available. Mutation of residues involved in metal ion binding impaired catalytic activity and the formation of active hexamers. Structural resolution of Pv-M17 by cryoelectron microscopy and X-ray crystallography together with solution studies revealed that PfA-M17 and Pv-M17 bind metal ions and substrates in a conserved fashion, although Pv-M17 forms the active hexamer more readily and processes substrates faster than PfA-M17. On the basis of these studies, we propose a dynamic equilibrium between monomer ↔ dimer ↔ tetramer ↔ hexamer, which becomes directional toward the large oligomeric states with the addition of metal ions. This sophisticated metal-dependent dynamic equilibrium may apply to other M17 aminopeptidases and underpin the moonlighting capabilities of this enzyme family.

M18 aspartyl aminopeptidases (DAPs) are well characterized in microbes and animals with likely functions in peptide processing and vesicle trafficking. In contrast, there is a dearth of knowledge on plant aminopeptidases with a preference for proteins and peptides with N-terminal acidic residues. During evolution of the Plantae, there was an expansion and diversification of the M18 DAPs. After divergence of the ancestral green algae from red and glaucophyte algae, a duplication yielded the DAP1 and DAP2 lineages. Subsequently DAP1 genes were lost in chlorophyte algae. A duplication of DAP2-related genes occurred early in green plant evolution. DAP2 genes were retained in land plants and picoeukaryotic algae and lost in green algae. In contrast, DAP2-like genes persisted in picoeukaryotic and green algae, while this lineage was lost in land plants. Consistent with this evolutionary path, Arabidopsis thaliana has two DAP gene lineages (AtDAP1 and AtDAP2). Similar to animal and yeast DAPs, AtDAP1 is localized to the cytosol or vacuole; while AtDAP2 harbors an N-terminal transit peptide and is chloroplast localized. His6-DAP1 and His6-DAP2 expressed in Escherichia coli were enzymatically active and dodecameric with masses exceeding 600 kDa. His6-DAP1 and His6-DAP2 preferentially hydrolyzed Asp-p-nitroanilide and Glu-p-nitroanilide. AtDAPs are highly conserved metallopeptidases activated by MnCl2 and inhibited by ZnCl2 and divalent ion chelators. The protease inhibitor PMSF inhibited and DTT stimulated both His6-DAP1 and His6-DAP2 activities suggesting a role for thiols in the AtDAP catalytic mechanism. The enzymes had distinct pH and temperature optima, as well as distinct kinetic parameters. Both enzymes had high catalytic efficiencies (kcat/Km) exceeding 1.0 x 107 M-1 sec-1. Using established molecular chaperone assays, AtDAP1 and AtDAP2 prevented thermal denaturation. AtDAP1 also prevented protein aggregation and promoted protein refolding. Collectively, these data indicate that plant DAPs have a complex evolutionary history and have evolved new biochemical features that may enable their role in vivo.

The aim of this study was to investigate if urinary glutamyl aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), Klotho and hydroxyproline can be considered as potential biomarkers of renal injury and fibrosis in an experimental model of obesity. Male Zucker lean (ZL) and obese (ZO) rats were studied from 2 to 8 months old. Kidneys from ZO rats at the end of the study (8 months old) developed mild focal and segmental glomerulosclerosis as well as moderate tubulointerstitial injury. Urinary excretion of Klotho was higher in ZO rats at 2, 5, and 8 months of study, plasma Klotho levels were reduced and protein abundance of Klotho in renal tissue was similar in ZL and ZO rats. GluAp and AlaAp urinary activities were also increased in ZO rats throughout the time-course study. ZO rats showed an augmentation of hydroxyproline content in renal tissue and a significant increase of tubulointerstitial fibrosis. Correlation studies demonstrated that GluAp, AlaAp, and Klotho are early diagnostic markers of renal lesions in Zucker obese rats. Proteinuria and hydroxyproline can be considered delayed diagnostic markers because their contribution to diagnosis starts later. Another relevant result is that GluAp, AlaAp, and Klotho are related not only with diagnosis but also with prognosis of renal lesions in Zucker obese rats. Moreover, strong predictive correlations of aminopeptidasic activities with the percentage of renal fibrosis or with renal hydroxyproline content at the end of the experiment were observed, indicating that an early increased excretion of these markers is related with a higher later extent of fibrosis in Zucker obese rats. In conclusion, GluAp, AlaAp, and Klotho are early diagnostic markers that are also related with the extent of renal fibrosis in Zucker obese rats. Therefore, they have a potential use not only in diagnosis, but also in prognosis of obesity-associated renal lesions.

Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs.

The members of the M1 aminopeptidase family share conserved domains, yet show functional divergence within the family as a whole. In order to better understand this family, this study analyzed the mammalian members in depth at exon, gene, and protein levels. The twelve human members, eleven rat members, and eleven mouse members were first analyzed in multiple alignments to visualize both reported and unreported conserved domains. Phylogenetic trees were then generated for humans, rats, mice, and all mammals to determine how closely related the homologs were and to gain insight to the divergence in the family members. This produced three groups with similarity within the family. Next, a synteny study was completed to determine the present locations of the genes and changes that had occurred. It became apparent that gene death likely resulted in the lack of one member in mouse and rat. Finally, an in-depth analysis of the exon structure revealed that nine members of the human family and eight in mouse, are highly conserved within the exon structure. Taken together, these results indicate that the M1 aminopeptidase family is a divergent family with three subgroups and that genetic evidence mirrors categorization of the family by enzymatic function.

A library of novel phosphonic acid analogues of homophenylalanine and phenylalanine, containing fluorine and bromine atoms in the phenyl ring, have been synthesized. Their inhibitory properties against two important alanine aminopeptidases, of human (hAPN, CD13) and porcine (pAPN) origin, were evaluated. Enzymatic studies and comparison with literature data indicated the higher inhibitory potential of the homophenylalanine over phenylalanine derivatives towards both enzymes. Their inhibition constants were in the submicromolar range for hAPN and the micromolar range for pAPN, with 1-amino-3-(3-fluorophenyl) propylphosphonic acid (compound 15c) being one of the best low-molecular inhibitors of both enzymes. To the best of our knowledge, P1 homophenylalanine analogues are the most active inhibitors of the APN among phosphonic and phosphinic derivatives described in the literature. Therefore, they constitute interesting building blocks for the further design of chemically more complex inhibitors. Based on molecular modeling simulations and SAR (structure-activity relationship) analysis, the optimal architecture of enzyme-inhibitor complexes for hAPN and pAPN were determined.

We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla-Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis.

Leucine aminopeptidases (LAPs) have been reported to be involved in tumor cell proliferation, invasion and angiogenesis. However, the relationship between serum leucine aminopeptidases and prognosis of hepatocellular carcinoma (HCC) patients who underwent liver transplantation (LT) was not yet reported. We aimed to evaluate the prognostic value of preoperative serum leucine aminopeptidases in these patients.

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

Designing side chain substituents complementary to enzyme binding pockets is of great importance in the construction of potent and selective phosphinic dipeptide inhibitors of metallo-aminopeptidases. Proper structure selection makes inhibitor construction more economic, as the development process typically consists of multiple iterative preparation/bioassay steps. On the basis of these principles, using noncomplex computation and modeling methodologies, we comprehensively screened 900 commercial precursors of the P1' residues of phosphinic dipeptide and dehydrodipeptide analogs to identify the most promising ligands of 52 metallo-dependent aminopeptidases with known crystal structures. The results revealed several nonproteinogenic residues with an improved energy of binding compared with the best known inhibitors. The data are discussed taking into account the selectivity and stereochemical implications of the enzymes. Using this approach, we were able to identify nontrivial structural elements substituting the recognized phosphinic peptidomimetic scaffold of metallo-aminopeptidase inhibitors.

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.

Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1 and ERAP2) are key components on the pathway that generates antigenic epitopes for presentation to cytotoxic T-lymphocytes (CTLs). Coding single nucleotide polymorphisms (SNPs) in these enzymes have been associated with pre-disposition to several major human diseases including inflammatory diseases with autoimmune etiology, viral infections, and virally induced cancer. The function of these enzymes has been demonstrated to affect CTL and natural killer cell responses toward healthy and malignant cells as well as the production of inflammatory cytokines. Recent studies have demonstrated that SNPs in ERAP1 and ERAP2 can affect their ability to generate or destroy antigenic epitopes and define the immunopeptidome. In this review, we examine the potential role of these enzymes and their polymorphic states on the generation of cytotoxic responses toward malignantly transformed cells. Given the current state-of-the-art, it is possible that polymorphic variation in these enzymes may contribute to the individual's pre-disposition to cancer through altered generation or destruction of tumor antigens that can facilitate tumor immune evasion.

Malaria remains a global health problem, and though international efforts for treatment and eradication have made some headway, the emergence of drug-resistant parasites threatens this progress. Antimalarial therapeutics acting via novel mechanisms are urgently required. Plasmodium falciparum M1 and M17 are neutral aminopeptidases which are essential for parasite growth and development. Previous work in our group has identified inhibitors capable of dual inhibition of PfA-M1 and PfA-M17, and revealed further regions within the protease S1 pockets that could be exploited in the development of ligands with improved inhibitory activity. Herein, we report the structure-based design and synthesis of novel hydroxamic acid analogues that are capable of potent inhibition of both PfA-M1 and PfA-M17. Furthermore, the developed compounds potently inhibit Pf growth in culture, including the multi-drug resistant strain Dd2. The ongoing development of dual PfA-M1/PfA-M17 inhibitors continues to be an attractive strategy for the design of novel antimalarial therapeutics.

Malaria is a parasitic disease that remains a global health burden. The ability of the parasite to rapidly develop resistance to therapeutics drives an urgent need for the delivery of new drugs. The Medicines for Malaria Venture have compounds known for their antimalarial activity, but not necessarily the molecular targets. In this study, we assess the ability of the "MMV 400" compounds to inhibit the activity of three metalloaminopeptidases from Plasmodium falciparum, PfA-M1, PfA-M17 and PfM18 AAP. We have developed a multiplex assay system to allow rapid primary screening of compounds against all three metalloaminopeptidases, followed by detailed analysis of promising compounds. Our results show that there were no PfM18AAP inhibitors, whereas two moderate inhibitors of the neutral aminopeptidases PfA-M1 and PfA-M17 were identified. Further investigation through structure-activity relationship studies and molecular docking suggest that these compounds are competitive inhibitors with novel binding mechanisms, acting through either non-classical zinc coordination or independently of zinc binding altogether. Although it is unlikely that inhibition of PfA-M1 and/or PfA-M17 is the primary mechanism responsible for the antiplasmodial activity reported for these compounds, their detailed characterization, as presented in this work, pave the way for their further optimization as a novel class of dual PfA-M1/PfA-M17 inhibitors utilising non-classical zinc binding groups.

Plasmodium falciparum alanine M1-aminopeptidase (PfA-M1) is a validated target for anti-malarial drug development. Presence of significant similarity between PfA-M1 and human M1-aminopeptidases, particularly within regions of enzyme active site leads to problem of non-specificity and off-target binding for known aminopeptidase inhibitors. Molecular docking based in silico screening approach for off-target binding has high potential but requires 3D-structure of all human M1-aminopeptidaes. Therefore, in the present study 3D structural models of seven human M1-aminopeptidases were developed. The robustness of docking parameters and quality of predicted human M1-aminopeptidases structural models was evaluated by stereochemical analysis and docking of their respective known inhibitors. The docking scores were in agreement with the inhibitory concentrations elucidated in enzyme assays of respective inhibitor enzyme combinations (r2≈0.70). Further docking analysis of fifteen potential PfA-M1 inhibitors (virtual screening identified) showed that three compounds had less docking affinity for human M1-aminopeptidases as compared to PfA-M1. These three identified potential lead compounds can be validated with enzyme assays and used as a scaffold for designing of new compounds with increased specificity towards PfA-M1.

Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial.