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Matthew Bogyo

Publications and source records attributed to Matthew Bogyo.

46 records · Page 3Linked to original sources
PubMed2003

Sequential autolytic processing activates the zymogen of Arg-gingipain.

Most proteases are synthesized as inactive precursors to protect the synthetic machinery of the cell and allow timing of activation. The mechanisms used to render latency are varied but tend to be conserved within protease families. Proteases belonging to the caspase family have a unique mechanism mediated by transitions of two surface loops, and on the basis of conservation of mechanism one would expect this to be preserved by caspase relatives. We have been able to express the full-length precursor of the Arg-specific caspase relative from the bacterium Porphyromonas gingivalis, Arg-gingipain-B, and we show that it contains N- and C-terminal extensions that render a low amount of latency, meaning that the zymogen is substantially active. Three sequential autolytic processing steps at the N and C terminus are required for full activity, and the N-propeptide may serve as an intramolecular chaperone rather than an inhibitory peptide. Each step in activation requires the previous step, and an affinity probe reveals that incremental activity enhancements are achieved in a stepwise manner.

Adhesins, Bacterial↗
PubMed2003

Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase.

Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.

Animals↗
PubMed2003

Chemical proteomics and its application to drug discovery.

The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better ways to treat human disease. The multidisciplinary science of chemical proteomics can be used to distill this flood of new information. This approach makes use of synthetic small molecules that can be used to covalently modify a set of related enzymes and subsequently allow their purification and/or identification as valid drug targets. Furthermore, such methods enable rapid biochemical analysis and small-molecule screening of targets thereby accelerating the often difficult process of target validation and drug discovery.

Chemistry, Pharmaceutical↗
PubMed2002

Pathways accessory to proteasomal proteolysis are less efficient in major histocompatibility complex class I antigen production.

Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal beta-subunits (beta(1), beta(2), and beta(5)) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2D(b) molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways.

Animals↗
PubMed2002

A role for the protease falcipain 1 in host cell invasion by the human malaria parasite.

Cysteine proteases of Plasmodium falciparum are required for survival of the malaria parasite, yet their specific cellular functions remain unclear. We used a chemical proteomic screen with a small-molecule probe to characterize the predominant cysteine proteases throughout the parasite life cycle. Only one protease, falcipain 1, was active during the invasive merozoite stage. Falcipain 1-specific inhibitors, identified by screening of chemical libraries, blocked parasite invasion of host erythrocytes, yet had no effect on normal parasite processes such as hemoglobin degradation. These results demonstrate a specific role for falcipain 1 in host cell invasion and establish a potential new target for antimalarial therapeutics.

Animals↗
PubMed2002

Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries.

Asparaginyl endopeptidases, or 'legumains' have been identified and characterized in plants, the blood fluke parasite Schistosoma, and mammals. The legumains are a novel family of cysteine proteases and display restricted specificity for peptide hydrolysis on the carboxyl side of asparagine residues. Two forms of recombinant asparaginyl endopeptidase from Schistosoma mansoni (C197 Sm32 and N197C Sm32), expressed in Pichia pastoris, have been analyzed for substrate specificity using a positional-scanning synthetic combinatorial library (PS-SCL). We first screened Sm32 using a P1-diverse library. This library demonstrated the absolute specificity of Sm32 for asparagine at P1. To determine the P2-P3 preferences of Sm32, we constructed a library with asparagine fixed at P1, and the P2-P3 positions randomized. The library was screened using the two forms of Sm32, human asparaginyl endopeptidase, and to confirm its diversity, cruzain from Trypanosoma cruzi. The schistosome legumain showed a preference for P3: Thr>Ala>Val>Ile, and P2: Ala>Thr>Val>Asn, with an overall broader specificity at P3 than at P2. Both human and schistosome legumain can accommodate Thr and Ala at P2 and P3. However, optimal substrate sequences differ, with Sm32 preferring Thr-Ala-Asn, and human legumain preferring Pro-Thr-Asn. Predictions of substrate specificity from the library screen were confirmed using single peptide substrates for kinetic assays.

Amino Acid Sequence↗
PubMed2002

Probing structural determinants distal to the site of hydrolysis that control substrate specificity of the 20S proteasome.

The 20S proteasome is a large multicomponent protease complex. Relatively little is known about the mechanisms that control substrate specificity of its multiple active sites. We present here the crystal structure at 2.95 A resolution of a beta2-selective inhibitor (MB1) bound to the yeast 20S proteasome core particle (CP). This structure is compared to the structure of the CP bound to a general inhibitor (MB2) that covalently modified all three (beta1, beta2, beta5) catalytic subunits. These two inhibitors differ only in their P3 and P4 residues, thereby highlighting binding interactions distal to the active site threonine that control absolute substrate specificity of the complex. Comparisons of the CP-bound structures of MB1, MB2, and the natural products epoxomycin and TMC-95A also provide information regarding general binding modes for several classes of proteasome inhibitors.

Binding Sites↗
PubMed2002

Small molecule affinity fingerprinting. A tool for enzyme family subclassification, target identification, and inhibitor design.

Classifying proteins into functionally distinct families based only on primary sequence information remains a difficult task. We describe here a method to generate a large data set of small molecule affinity fingerprints for a group of closely related enzymes, the papain family of cysteine proteases. Binding data was generated for a library of inhibitors based on the ability of each compound to block active-site labeling of the target proteases by a covalent activity based probe (ABP). Clustering algorithms were used to automatically classify a reference group of proteases into subfamilies based on their small molecule affinity fingerprints. This approach was also used to identify cysteine protease targets modified by the ABP in complex proteomes by direct comparison of target affinity fingerprints with those of the reference library of proteases. Finally, experimental data were used to guide the development of a computational method that predicts small molecule inhibitors based on reported crystal structures. This method could ultimately be used with large enzyme families to aid in the design of selective inhibitors of targets based on limited structural/function information.

Algorithms↗
PubMed2002

Chemical approaches for functionally probing the proteome.

With the availability of complete genome sequences, emphasis has shifted toward the understanding of protein function. We have developed a functional proteomic methodology that makes use of chemically reactive fluorescent probes to profile and identify enzymes in complex mixtures by virtue of their catalytic activity. This methodology allows a comparison of changes in activity of multiple enzymes under a variety of conditions using a single two-dimensional separation. The probes can also be used to localize active enzymes in intact cells using fluorescence microscopy. Furthermore, the probes enable screens for selective small molecule inhibitors of each enzyme family member within crude lysates or intact cells. Ultimately, this technology allows the rapid identification of potential drug targets and small molecule lead compounds targeted to them.

Affinity Labels↗