Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68-and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steadystate kinetic parameters for both aminoacyl-␤-naphthylamide and unmodified dipeptide substrates over the pH range 5.0 -8.5 reveals that k cat is relatively insensitive to pH, whereas K m increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0 -5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.Human malaria is responsible for around one million deaths annually (1). Five species of the genus Plasmodium cause malaria in humans as they replicate within host erythrocytes. The cytoadherent properties of intraerythrocytic Plasmodium falciparum coupled with its ability to invade mature erythrocytes make it the most virulent of the species that infect humans. During its erythrocytic replication cycle, P. falciparum endocytoses and catabolizes over two-thirds of soluble erythrocyte proteins (2, 3), the majority of which is hemoglobin. Hemoglobin catabolism provides amino acids for protein synthesis, general metabolism, and isoleucine import (4, 5) and may also prevent premature hemolysis by reducing the colloid osmolarity of the erythrocyte (6). Blocking hemoglobin catabolism with protease inhibitors prevents parasite replication; therefore, enzymes that catalyze this process are attractive targets for the development of novel anti-malarial drugs (7).Hemoglobin is extensively catabolized by the parasite within an acidic organelle called the food vacuole or digestive vacuole. In the vacuole, numerous types of endo-and exopeptidases act in a complementary and concerted manner to catalyze the hydrolysis of the ␣-and ␤-globin chains of hemoglobin. Aspartic proteases (plasmepsin I, II, IV, and histo-aspartic protease) and cysteine endoproteases (falcipain-2, -2Ј, and -3) initiate cleavage of the globin chains and generate polypeptide fragments (8, 9). The metallopeptidase fa...

The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the food vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37°C in the pH range 5.0 -7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.Malaria remains one of the most deadly global infectious diseases with an estimated 500 million clinical cases and 2 million deaths annually (1, 2). Clinical manifestations of the disease arise as the protozoan malaria parasite replicates asexually within human erythrocytes. Five species of the genus Plasmodium infect humans. The cytoadherent properties of red blood cells infected with Plasmodium falciparum, coupled with the ability of the parasite to reach high parasitemia, make it the most virulent species. The emergence of strains of P. falciparum that are resistant to affordable anti-malarial drugs such as chloroquine has complicated efforts to manage malaria, and new drugs are urgently needed.Aminopeptidases catalyze the hydrolysis of amino acids from the amino termini of proteins and peptides. They participate in a wide range of biological processes, including peptide catabolism, protein maturation, antigen presentation on immune cells, and regulation of hormone activity. During the asexual erythrocytic replication cycle of the malaria parasite, aminopeptidases contribute to the catabolism of peptides generated by two major proteolytic pathways. One of these is initiated at the proteasome, a multifunctional protease that plays an important role in the turnover of ubiquitinated cellular proteins in the cytosol (3-5). In addition, the parasite transports host red blood cell cytosol (consisting primarily of hemoglobin) to an acidic degradat...

The M1-family aminopeptidase PfA-M1 catalyzes the last step in the catabolism of human hemoglobin to amino acids in the Plasmodium falciparum food vacuole. In this study, the structural features of the substrate that promote efficient PfA-M1-catalyzed peptide bond hydrolysis were analyzed. X-Ala and Ala-X dipeptide substrates were employed to characterize the specificities of the enzyme's S1 and S1’ subsites. Both subsites exhibited a preference for basic and hydrophobic sidechains over polar and acidic sidechains. The relative specificity of the S1 subsite was similar over the pH range 5.5 - 7.5. Substrate P1 and P1’ residues affected both Km and kcat, revealing that sidechain-subsite interactions not only drive the formation of the Michaelis complex but also influence the rates of ensuing chemical steps. Only a small fraction of the available binding energy was exploited in interactions between substrate sidechains and the S1 and S1’ subsites, which indicates a modest level of complementarity. There was no correlation between S1 and S1’ specificities and amino acid abundance in hemoglobin. Interactions between PfA-M1 and the backbone atoms of the P1’ and P2’ residues as well as the P2’ sidechain further contributed to the catalytic efficiency of substrate hydrolysis. By demonstrating the engagement of multiple, broad-specificity subsites in PfA-M1, these studies provide insight into how this enzyme is able to efficiently generate amino acids from highly sequence-diverse di- and oligopeptides in the food vacuole.

Background: M1 family aminopeptidases exhibit a diverse range of specificities. Results: Substitutions at a residue in the S1 binding pocket can induce structural changes and remodel specificity. Conclusion: Mutations in the S1 subsite contribute to the acquisition of distinct specificities. Significance: Variation of a key residue in the S1 binding pocket provides a pathway for the evolution of new specificities and functions.

For conjugated HIV-1 fusion peptide vaccine development, recombinant Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier protein to boost peptide immunogenicity. Understanding the characteristics of rTTHC is the first step prior to the peptide conjugation. A comprehensive mass spectrometry (MS) characterization was performed on E. coli expressed rTTHC during its purification process. Intact mass along with peptide mapping analysis discovered the existence of three cysteine modification forms: glutathionylation, trisulfide bond modification, and disulfide bond shuffling, in correlation to a three-peak profile during a hydrophobic interaction chromatography (HIC) purification step. Coexistence of these multiple oxidative forms indicated that the active thiols underwent redox reaction in the rTTHC material. Identity confirmation of the rTTHC carrier protein by MS analysis provided pivotal guidance to assess the purification step and helped ensure that vaccine development could proceed.