Study of protease activity from Aspergillus awamori INCQS2B.361U2/1 extracellular fraction and modification of culture medium composition to isolate a novel aspartic protease.

Aspergillus awamori was cultivated in a modified Breccia medium, and the extracellular fraction was obtained, which presented 260 ± 15 µg of protein/mg and specific protease activity of 3.87 ± 0.52 mM.min-1.mg of protein-1 using Nα-p-tosyl-L-arginine methyl ester hydrochloride (L-TAME) as substrate. This fraction showed major proteins about 104 and 44 kDa and maximal protease activity at pH 5.5, 6.5, and 9.0, suggesting that A. awamori secretes acidic, neutral, and alkaline proteases with expressive thermal stability, however, aspartic protease was the most important activity. When yeast extract was supplemented to a modified Breccia medium, A. awamori protein secretion and protease activity were maximal and the affinity chromatography on pepstatin-agarose was employed to isolate the aspartic protease activity, which was called ASPA, with approximately 75 kDa. ASPA maximal activity was obtained at pH 4.5 and 6.5, and 50 °C. Pepstatin inhibited about 80% of ASPA activity, with IC50 and Ki values of 0.154 and 0.072 µM, respectively. ASPA cleaved protein and peptides substrates with the highest activity against gelatin (95 U/mg) and good peptidase activity with KM 0.0589 mM and Vmax 1.909 mM.min-1.mg protein-1, using L-TAME as substrate. A. awamori extracellular fraction is a source of proteases with important activity, and the supplementation of modified Breccia medium increased the aspartic protease production. This enzyme presented different biochemical characteristics from the previously reported A. awamori aspartic proteases. Therefore, ASPA is an excellent candidate for biotechnological application due to its important activity and thermostability.

A synergy of activity, stability, and inhibitor-interaction of HIV-1 protease mutants evolved under drug-pressure.

A clinically-relevant, drug-resistant mutant of HIV-1 protease (PR), termed Flap+(I54V) and containing L10I, G48V, I54V and V82A mutations, is known to produce significant changes in the entropy and enthalpy balance of drug-PR interactions, compared to wild-type PR. A similar mutant, Flap+(I54A) , which evolves from Flap+(I54V) and contains the single change at residue 54 relative to Flap+(I54V) , does not. Yet, how Flap+(I54A) behaves in solution is not known. To understand the molecular basis of V54A evolution, we compared nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, isothermal titration calorimetry, and enzymatic assay data from four PR proteins: PR (pWT), Flap+(I54V) , Flap+(I54A) , and Flap+(I54) , a control mutant that contains only L10I, G48V and V82A mutations. Our data consistently show that selection to the smaller side chain at residue 54, not only decreases inhibitor affinity, but also restores the catalytic activity.

Identification of a novel alkaline serine protease from gazami crab (Portunus trituberculatus) hepatopancreas and its hydrolysis of myofibrillar protein.

Serine proteases are thought to play a key role in the muscle softening of gazami crab (Portunus trituberculatus) during storage. A serine protease, Pt-sp2, was purified from the hepatopancreas of gazami crab using ammonium sulfate precipitation, anion-exchange and gel filtration chromatography, and was analyzed by mass spectrometry, transcriptome and bioinformatics. It revealed that Pt-sp2 was trypsin-like, with no 100% identical proteins in the NCBI database. The molecular weight of Pt-sp2 was approximately 37.2 kDa. Its optimum pH and temperature were 9.0 and 50 °C, respectively, using t-Butyloxy­carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide as a substrate. Pt-sp2 was activated in the presence of Ca2+. Both soybean trypsin inhibitor and Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride completely suppressed Pt-sp2 activity, while it was only partially inhibited by phenylmethylsulfonyl fluoride and EDTA. However, PMSF, Pepstatin A and cystatin inhibitor E-64 showed no inhibition on Pt-sp2 protease activity. The Km value of Pt-sp2 was 0.82 µM, and Pt-sp2 effectively hydrolyzed myofibrillar protein at 37 °C.

Pepstatin pull-down at high pH is a powerful tool for detection and analysis of napsin A.

Napsin A is an intracellular aspartic protease and biomarker of various malignancies like lung adenocarcinoma and ovarian clear cell carcinoma, but its detection is usually limited to immunohistochemical techniques gaining excellent information on its distribution but missing information about posttranslational modifications (e.g. maturation state) of the protein. We present a protocol for specific enrichment of napsin A from clinical or biological specimens, that facilitates detailed analysis of the protein. By using the exceptionally broad pH range under which napsin A binds to its inhibitor pepstatin A we achieve highly selective binding of napsin A while other aspartic proteases have negligible affinity. Using this method we demonstrate that lung napsin A in many mammals is a heterogeneous enzyme with a characteristic ladder-like appearance in SDS-PAGE that might be caused by proteolytically processed N- and/or C-termini, in contrast to the more homogeneous form found in kidneys and primary lung adenocarcinoma.

Primary structures of different isoforms of buffalo pregnancy-associated glycoproteins (BuPAGs) during early pregnancy and elucidation of the 3-dimensional structure of the most abundant isoform BuPAG 7.

Pregnancy-associated glycoproteins (PAGs) are expressed during pregnancy by the trophoectodermal cells of fetus. Presence of PAGs in dam's circulation has been widely used in pregnancy diagnosis. The present study reports the identification and characterization of different PAG isoforms in buffalo during early stages of pregnancy. The PAG mRNAs isolated from fetal cotyledons (Pregnancy stages: 45, 75 and 90 days) were successfully cloned in pJET1.2 vector and transformed in E. coli. A total of 360 random clones were sequenced and correlated with their stages of expression. A total of 12 isoforms namely, BuPAG 1, 2, 4, 6, 7, 8, 9, 13, 15, 16, 18 and one new isoform were identified. BuPAG 7 was found as the most abundant isoform in all three stages followed by BuPAG 18. Further, a large number of variants were found for most of these isoforms. Phylogenetic relationship of identified BuPAGs showed that BuPAG 2 belonged to an ancient group while other members clustered with modern group. Three-dimensional (3D) structure of BuPAG 7 was determined by homology modeling and molecular dynamic (MD) simulations which displayed a typical fold represented by other aspartic proteinase (AP) family members. Molecular docking of Pepstatin inhibitor with BuPAG 7 revealed to interact through various hydrogen bonding and hydrophobic interactions. Various amino acid substitutions were observed in peptide-binding cleft of BuPAG 7. Superimposition of BuPAG 7 with homologous structures revealed the presence of a 35-41 amino acid long insertion (alpha helix connected by two loops) near the N- terminus which seems to be a unique feature of BuPAG 7 in AP family. This is the first report on identification and sequence characterization of PAG isoforms in buffalo with unique finding that these isoforms represent many transcript variants. We also report 3D structure of the most abundant isoform BuPAG 7 for the first time.

Effects of pepsin and pepstatin on reflux tonsil hypertrophy in vitro.

There is evidence that pepsin can aggravate tonsil hypertrophy. Pepstatin is a potent inhibitor of pepsin activity and could protect patients against reflux tonsil hypertrophy by inhibiting pepsin. We examined the effects of pepstatin on the development of tonsil hypertrophy to investigate pepsin's role in the pathogenesis of tonsil lesions. We investigated whether pepstatin suppresses pepsin-mediated lymphocyte proliferation in tonsil hypertrophy. Forty-nine children with tonsil hypertrophy and twenty-two adults with tonsillitis were recruited to the study prior to surgery. Tonsil tissue from each patient was harvested and assessed for changes in the number of lymphocytes and macrophages in the presence of pepsin and pepstatin. We found that the proportions of CD4- and CD14-positive cells were significantly lower (p < 0.05), but that the proportions of CD19- and CD68-positive cells were significantly higher (p < 0.05), in children than in adults. There were significantly more CD4-positive cells after pepsin treatment, but these numbers were reduced by pepstatin. The levels of both interleukin-2 (IL-2) and interferon gamma (IFN-γ) increased significantly in response to pepsin, but were reduced when pepsin was inhibited by pepstatin. The level of IL-10 is reduced in pepsin-treated CD4 cells and the level is restored by pepstatin. IL-2 blocking reduced the increased CD4 cell number by pepsin. But, an additive or a synergic effect is not founded in combined with IL-2 blocking and pepstatin. Pepsin-positive cells did not co-localize with CD20 and CD45 cells, but they were found surrounding CD20- and CD45-positive hypertrophic tonsil cells. Pepsin-positive cells co-localized with CD68-positive cells. It is probable that pepsin from extraesophageal reflux aggravates tonsil hypertrophy and pepstatin exerts a protective effect by inhibiting pepsin activity.

Trichosporon asahii secretes a 30-kDa aspartic peptidase.

Trichosporon asahii is a fungal opportunistic pathogen that causes superficial and deep-seated infections presenting high mortality. Very little is known about the virulence attributes produced by this fungus. Herein, aspartic peptidase production was identified in Brazilian clinical isolates of T. asahii by different methodologies. Initially, T. asahii strain 250 (from skin lesion) was inoculated in both liquid and solid culture media containing bovine serum albumin (BSA) as the sole nitrogenous source. A translucent halo around the fungal colony was observed from the 5th day of culture. The cell-free culture supernatant revealed that soluble BSA was hydrolyzed along the growth, generating low molecular mass polypeptides as observed by electrophoresis. Subsequently, the secretions from four clinical strains of T. asahii were analyzed by BSA-SDS-PAGE and a single proteolytic band of 30-kDa was detected under acidic pH at 37°C. The secreted aspartic peptidase of T. asahii efficiently cleaved the cathepsin D peptide substrate, but not the substrates with specificity to HIV-1 peptidase and rennin. The capability to cleave either cathepsin D substrate in a fluorogenic assay or BSA immobilized within a gel matrix varied according to the T. asahii isolate. T. asahii extracellular peptidase activity was strongly inhibited by pepstatin A and HIV peptidase inhibitors, classifying it as an aspartic-type peptidase. Human serum albumin, mucin, non-immune immunoglobulin G and gelatin induced, in different levels, the secretion of this aspartic peptidase. With these results, T. asahii must be included in the list of many human fungal opportunistic pathogens able to secrete an aspartic-type peptidase.

Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease.

The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the 'second brain' given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrb(s-l/s-l)), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.

Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity.

UNLABELLED: Peptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a "barrier" and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target. IMPORTANCE: Pheromones are important for intraspecies communication across the tree of life. In the fungal kingdom, extracellular proteases play a key role in antagonizing pheromone signaling in multiple species. This study examines the properties and function of Candida albicans Bar1, an aspartyl protease that cleaves and thereby inactivates α pheromone. We demonstrate that Bar1 plays important roles in regulating both intra- and interspecies pheromone signaling. The fungal protease shows preferential activity on the endogenous pheromone, but, surprisingly, cleavage activity is dependent on amino acid residues distal to the scissile bond. We propose that the unusual substrate specificity of Bar1 is a direct result of coevolution with Ste2, the receptor for α pheromone, for recognition of the same peptide target. The novel specificity of Bar1 reveals the complex forces shaping the evolution of mating pathways in fungi and uncovers a protease with potentially important applications in the biotechnology industry.

Digestion of Nucleic Acids Starts in the Stomach.

The ingestion of nucleic acids (NAs) as a nutritional supplement or in genetically modified food has attracted the attention of researchers in recent years. Discussions over the fate of NAs led us to study their digestion in the stomach. Interestingly, we found that NAs are digested efficiently by human gastric juice. By performing digests with commercial, recombinant and mutant pepsin, a protein-specific enzyme, we learned that the digestion of NAs could be attributed to pepsin rather than to the acidity of the stomach. Further study showed that pepsin cleaved NAs in a moderately site-specific manner to yield 3'-phosphorylated fragments and the active site to digest NAs is probably the same as that used to digest protein. Our results rectify the misunderstandings that the digestion of NAs in the gastric tract begins in the intestine and that pepsin can only digest protein, shedding new light on NA metabolism and pepsin enzymology.

A new pepstatin-insensitive thermopsin-like protease overproduced in peptide-rich cultures of Sulfolobus solfataricus.

In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease), while the less abundant (named SsMTP-1) one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50-90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii.

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

Dynamics of thermodynamically stable, kinetically trapped, and inhibitor-bound states of pepsin.

The pepsin folding mechanism involves a prosegment (PS) domain that catalyzes folding, which is then removed, resulting in a kinetically trapped native state. Although native pepsin (Np) is kinetically stable, it is irreversibly denatured due to a large folding barrier, and in the absence of the PS it folds to a more thermodynamically stable denatured state, termed refolded pepsin (Rp). This system serves as a model to understand the nature of kinetic barriers and folding transitions between compact states. Quasielastic neutron scattering (QENS) was used to characterize and compare the flexibility of Np, as a kinetically trapped state, with that of Rp, as a thermodynamically stable fold. Additionally, the dynamics of Np were compared with those of a partially unfolded form and a thermally stabilized, inhibitor-bound form. QENS revealed length-scale-dependent differences between Np and Rp on a picosecond timescale and indicated greater flexibility in Np, leading to the conclusion that kinetic stabilization likely does not correspond to reduced internal dynamics. Furthermore, large differences were observed upon inhibition, indicating that QENS of proteins in solution may prove useful for examining the role of conformational entropy changes in ligand binding.

Structural rationale for the recognition of arginine at P3 in PEXEL motif containing proteins of Plasmodium falciparum by plasmepsin V.

The virulent form of malaria is caused by Plasmodium falciparum that infects red blood cells. In order to survive inside the host, the parasite remodels the infected erythrocytes by exporting more than 300 effector proteins outside the parasitophorous vacuole membrane into the cytosol. The main feature of all the export proteins is the presence of a pentapeptide sequence motif; RxLxE/Q/D. This sequence motif is hydrolysed between L-x and the proteins with the acetylated new N-terminus xE/Q/D are exported. The enzyme responsible for this hydrolysis is plasmepsin V which is one of the ten aspartic proteases in P. falciparum. In order to understand the structural rationale for the specificity of this protease towards cleavage of the above motif, we generated three-dimensional models of seven plasmepsins (I, V to X) for which experimental structures are not available and compared these along with the crystal structures of three P. falciparum plasmepsins (II to IV). The structure comparisons revealed the importance of Tyr13, Glu77 and Ala117 specific to plasmepsin V that facilitates the accommodation of arginine at P3 in the RxLxE/Q/D motif. Our analysis correlates the structure-function relationship of plasmepsin V.

Purification and partial characterization of ostrich skeletal muscle cathepsin D and its activity during meat maturation.

Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).

Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis.

Apoptotic defects endow tumor cells with survival advantages. Such defects allow the cellular stress response to take the path of cytoprotective autophagy, which either precedes or effectively blocks an apoptotic cascade. Inhibition of the cytoprotective autophagic response shifts the cells toward apoptosis, by interfering with an underlying molecular mechanism of cytoprotection. The current study has identified such a mechanism that is centered on the regulation of caspase-8 activity. The study took advantage of Bax(-/-) Hct116 cells that are TRAIL-resistant despite significant DISC processing of caspase-8, and of the availability of a caspase-8-specific antibody that exclusively detects the caspase-8 large subunit or its processed precursor. Utilizing these biological tools, we investigated the expression pattern and subcellular localization of active caspase-8 in TRAIL-mediated autophagy and in the autophagy-to-apoptosis shift upon autophagy inhibition. Our results suggest that the TRAIL-mediated autophagic response counter-balances the TRAIL-mediated apoptotic response by the continuous sequestration of the large caspase-8 subunit in autophagosomes and its subsequent elimination in lysosomes. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis.

Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.

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-A resolutions, respectively. In the apoenzyme crystals, HAP forms a tight dimer not seen previously in any aspartic protease. 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 (residues 238-245), as well as the orientation of 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 from that 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 with 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.

Impedance method for detecting HIV-1 protease and screening for its inhibitors using ferrocene-peptide conjugate/Au nanoparticle/single-walled carbon nanotube modified electrode.

A highly sensitive screening assay based on electrochemical impedance spectroscopy (EIS) has been developed for detecting HIV-1 protease (PR) and subsequent evaluation of its corresponding inhibitors at picomolar levels. The assay format was based on the immobilization of the thiol terminated ferrocene(Fc)-pepstatin conjugate on a single-walled carbon nanotube/gold nanoparticle (SWCNT/AuNP) modified gold electrode. The alteration of the interfacial properties of electrodes upon HIV-1 PR and Fc-pepstatin conjugate interaction was traced by EIS. On the basis of the charge transfer resistance data obtained and using a mixed kinetic and diffusion model, this procedure was capable of detecting picomolar HIV-1 PR owing to the specific binding of this enzyme to Fc modified pepstatin. A competitive inhibition assay format was then performed using four potent HIV-1 PR inhibitors. The estimated inhibition constant ( K i) attested that lopinavir/ritonavir ( K i = 20 +/- 3 pM) and saquinavir ( K i = 57 +/- 8 pM) even at 10 pM competed strongly with pepstatin for effective binding to HIV-1 PR. Indinavir ( K i = 630 +/- 22 pM) only competed well with pepstatin at a much higher concentration (1 nM). No significant inhibitory effect was observed for the fosamprenavir ( K i =11 +/- 0.5 nM) as expected from this pro-drug. Such results agreed well with the values reported in the literature. This assay format is a definite asset for the expedited development of effective HIV-1 PR inhibitors with low molecular weights.

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A.

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R(free)=21.2%) at 1.70 A resolution and 15.8% (R(free)=19.2%) at 1.85 A resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly beta-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity.

Plasmodium food vacuole plasmepsins are activated by falcipains.

Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation.