No abstract

The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum, as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5, 3.3, and 3.05 Å resolution, respectively. In the apoenzyme crystals HAP forms a tight dimer, not seen previously in any aspartic proteases. The interactions between the monomers affect the conformation of two flexible loops, the functionally important "flap" (residues 70-83) and its structural equivalent in the C-terminal domain (238-245), as well as the orientation of the helix 225-235. The flap is found in an open conformation in the apoenzyme. Unexpectedly, the active site of the apoenzyme contains a zinc ion tightly bound to His32 and Asp215 from one monomer, and to Glu278A from the other monomer, with the coordination of Zn resembling that seen in metalloproteases. The flap is closed in the structure of the pepstatin A complex, whereas it is open in the complex with KNI-10006. Although the binding mode of pepstatin A is significantly different than in other pepsin-like aspartic proteases, its location in the active site makes unlikely the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI-10006 is unusual compared to the binding of other inhibitors from the KNI series to aspartic proteases. The novel features of the HAP active site could facilitate design of specific inhibitors used in the development of antimalarial drugs.

The pathological aggregation of tau is a common feature of most of the neuronal disorders including frontotemporal dementia, Parkinson's disease, and Alzheimer's disease. The inhibition of tau aggregation is considered to be one of the important strategies for treating these neurodegenerative diseases. Curcumin, a natural polyphenolic molecule, has been reported to have neuroprotective ability. In this work, curcumin was found to bind to adult tau and fetal tau with a dissociation constant of 3.3±0.4 and 8±1 μM, respectively. Molecular docking studies indicated a putative binding site of curcumin in the microtubule-binding region of tau. Using several complementary techniques, including dynamic light scattering, thioflavin S fluorescence, 90° light scattering, electron microscopy, and atomic force microscopy, curcumin was found to inhibit the aggregation of tau. The dynamic light scattering analysis and atomic force microscopic images revealed that curcumin inhibits the oligomerization of tau. Curcumin also disintegrated preformed tau oligomers. Using Far-UV circular dichroism, curcumin was found to inhibit the β-sheets formation in tau indicating that curcumin inhibits an initial step of tau aggregation. In addition, curcumin inhibited tau fibril formation. Furthermore, the effect of curcumin on the preformed tau filaments was analyzed by atomic force microscopy, transmission electron microscopy, and 90° light scattering. Curcumin treatment disintegrated preformed tau filaments. The results indicated that curcumin inhibited the oligomerization of tau and could disaggregate tau filaments.

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...

Background: Peptide/protein hormones are stored as amyloids within endocrine secretory granules. Results: Disulfide bond cleavage enhances conformational dynamics and aggregation kinetics in somatostatin-14, resulting in amyloid fibrils with increased resistance to denaturing conditions and decreased reversibility. Conclusion: Disulfide bond could be a key modulating factor in somatostatin-14 amyloid formation associated with secretory granule biogenesis. Significance: Defective disulfide bonding might cause dysregulation of hormone storage/secretion.