Many plant aspartic proteases contain an additional sequence of ϳ100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plantspecific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å , revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus. Aspartic proteases (APs)2 are characterized by a common bilobal tertiary structure containing two catalytic aspartic acid residues (Asp 32 and Asp 215 in pepsin) within an active site cleft(1, 2). They are found in all higher organisms, and their respective roles are well established, although structural and functional characteristics of APs in plants are least understood. Of practical interest among plant APs are their roles in plant pathogen resistance (3) as well as in senescence and postharvest physiology (4, 5). Plant APs share the common AP bilobal structure; however, some contain an additional sequence of ϳ100 residues inserted within the C-terminal primary structure. These additional amino acids unique to plant APs (6 -8) create an extra domain protruding from the canonical AP molecule (9 -11). This structural oddity among APs is called the plantspecific insert (PSI), also known as the plant-specific sequence, which belongs to the saposin-like protein (SAPLIP) family (12, 13). Plant APs are found in either monomeric or heterodimeric forms (9, 14); the latter result from post-translational proteolysis, which includes the removal of part or all of the PSI, whereas the PSI is retained in monomeric plant APs (6,8).In general, members of the SAPLIP family have various physiological functions, all of which entail membrane interaction (14 -16) manifested in three principal ways: membrane binding, membrane perturbation without permeabilization, and membrane permeabilization (15). Examples of SAPLIP functions include roles in exohydrolase degradation of sphingolipids in the...

Saccharomyces cerevisiae proteinase A (saccharopepsin; EC 3.4.23.25) is a member of the aspartic proteinase superfamily (InterPro IPR001969), which are proteolytic enzymes distributed among a variety of organisms. Targeted to the vacuole as a zymogen, its activation at acidic pH can occur by two different pathways, a one-step process to release mature proteinase A, involving the intervention of proteinase B, or a step-wise pathway via the autoactivation product known as pseudo-proteinase A. Once active, S. cerevisiae proteinase A is essential to the activities of other yeast vacuolar hydrolases, including proteinase B and carboxypeptidase Y. The mature enzyme is bilobal, with each lobe providing one of the two catalytically essential aspartic acid residues in the active site. The crystal structure of free proteinase A reveals that the flap loop assumes an atypical position, pointing directly into the S 1 pocket of the enzyme. With regard to hydrolysis, proteinase A has a preference for hydrophobic residues with Phe, Leu or Glu at the P1 position and Phe, Ile, Leu or Ala at P1 , and is inhibited by IA 3 , a natural and highly specific inhibitor produced by S. cerevisiae. This review is the first comprehensive review of S. cerevisiae PrA.

By mid-century, the world population will surpass 9 billion people, meaning higher demand for available food, water, arable land and environmental impacts. Food safety issues, nutrition deficiencies, postharvest losses, regulation inconsistencies and consumer attitudes are all striking challenges which must be met in maintaining food security and sustainability. Possible solutions include advancements in food processing technologies, nanotechnology, innovative food formulations and the use of genomic approaches manifested in examples such as alternative protein sources, insect flour, nutrigenomics, 3D food printing, biomimicry, food engineering and merging technology. International organizations like the International Union of Food Science and Technology also play important roles in securing the world's food supplies by providing expertise through their respective country memberships. The present review addresses the food science and technology roles in meeting current challenges and investigates possible solutions to feed the world in the near future.

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Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies. Refolding conditions were optimized and zymogen was processed into a mature form via cleavage at the Asn80-Ala81 peptide bond. Mature plasmepsin V exhibited a pH optimum of 5.5-7.0 with Km and kcat of 4.6 μM and 0.24s(-1), respectively, at pH 6.0 using the substrate DABCYL-LNKRLLHETQ-E(EDANS). Furthermore, the prosegment of proplasmepsin V was shown to be nonessential for refolding and inhibition. Unexpectedly, unprocessed proplasmepsin V was enzymatically active with slightly reduced substrate affinity (∼ 2-fold), and similar pH optimum as well as turnover compared to the mature form. Both zymogenic and mature plasmepsin V were partially inhibited by pepstatin A as well as several KNI aspartic protease inhibitors while certain metals strongly inhibited activity. Overall, the present study provides the first report on the nonessentiality of the prosegment for plasmepsin V folding and activity, and therefore, subsequent characterization of its structure-function relationships of both zymogen and mature forms in the development of novel inhibitors with potential antimalarial activities is warranted.