Why the interest in proteases?
Enzymes that operate on peptide bonds (the chemical linkages that make up proteins) have now enjoyed over two centuries of scientific attention. At first, proteases (a.k.a. peptidases) were thought to be merely non-specific and perform a "general" catabolic role, but now are accredited to also function in an exquisitely selective manner. Fundamentally, every natural protein macromolecule is subjected to the action of at least one or more peptidase during its biosynthesis, extra- or intra-cellular targeting, biological function and ultimately catabolic degradation. This ubiquitous peptidase requirement didn't go unnoticed by biopharmaceutical companies, and today proteases have been successfully targeted in various infectious organisms as well as human clinical ails. This precedent paved the way to earnestly study peptidases in parasitic systems, and is also the basis for my scientific research interests.
scientific penchant
As a parasite molecular biochemist and enzymologist, I'm fascinated by many aspects of protein catabolism in parasitic organisms. I am principally interested in parasite peptidase biochemistry, particularly in the identification, characterization and biological validation of parasite enzymes as rational targets for selective chemotherapeutic intervention. To this end, my research interests have led me to work with several parasitic protozoa and helminths (flatworms and nematodes). Currently, my principal research interests are in malaria (see, http://www.thesynapticleap.org/?q=malaria/community), Giardia, Cryptosporidium and liver flukes, the research areas are highlighted below, in three disparate but overlapping areas.
general reading
Sajid M, McKerrow JH. Cysteine Proteases of Parasitic Organisms. Molecular and Biochemical Parasitology 2002;120(1):1-21.
McKerrow JH, Caffrey C, Kelly B, Loke P and Sajid M. Proteases in Parasitic Diseases. Annual Review of Pathology; Mechanism of Disease. 2006, volume 1. 497-536
research interests
-
1. mapping and chemically targeting the peptidase active site
Peptidases are categorized in to several related groups, termed clans. Although their chemical mechanisms of catalysis may vary, they all perform a common function; that is, the all are capable of severing peptide bonds by utilsing a water molecule (hence hydrolysis) within polypeptides and/or proteins. I am keenly interested in the mechanism of substrate hydrolysis and inhibitor binding. Insights into the amino acid preferences of a given proteases can be obtained with the use of combinatorial positional scanning substrate and/or inhibitor libraries. Moreover, recent advances at UCSF in computational modeling of proteases by in silico threading on a known homologous structure, has shown great promise as a powerful approach in structure based drug development and peptidomimetic design. Indeed, where structural studies are not feasible using parasite proteases, in silico protease modeling has given us very valuable indication as to the possible substrate specificity. I also find the machinery involved in protease activation/processing from respective precursor proteins fascinating and envisage this field to yield novel avenues for the development of unique lead compounds.
further reading
Sterverding D, Caffrey C and Sajid M. Cysteine Proteinase Inhibitors as Therapy for Parasitic Diseases: Mini Reviews in Medicinal Chemistry 2006;6(9):1025-32.
Mathieu MA, Bogyo M, Caffrey CR, Choe Y, Lee J, Chapman H, Sajid M, Craik CS, McKerrow JH. Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries. Molecular Biochemical Parasitology 2002;121(1):99-105.
chemical mechanism of peptide bond hydrolysis by a cysteine protease
2. cathepsin B-like peptidase
First described in mammalian systems as lysosomal catabolic enzymes, cathepsin B-like peptidases are know to function extralysomally in a number of parasitic organisms. We have identified the most primitive cathpesin B-like enzyme from a gastrointestinal parasite, Giardia lamblia; and work is currently underway to try and understand the biological role of these enzymes in the parasite's lifecycle. The Giardia cathepsin B-like enzyme also serve as a handy tool to study the molecular, structural and functionally evolution of this class of protease.
We are also working on a number of cathepsin B-like enzymes that are found in parasitic flatworms, in particular the helminth Schistosoma mansoni, the causative agent for the disease, bilharzias. In S. mansoni we have identified and biochemically charactersied the protease termed, S. mansoni cathepsin B-like 1, or SmCB1. We believe this enzyme to be an ideal target for chemotherapy and work is ongoing to address this point.
further reading
Identification of the Major Cysteine Protease of Giardia and its Role in Encystation
DuBois KN, Abodeely M, Sakanari J, Craik CS, Lee M , McKerrow JH, Sajid M. Journal of Biological Chemistry. In press. 2008
Abdulla M-H, Lim KC, Sajid KC, McKerrow JH, Caffrey CR.
Schistosoma mansoni: novel chemotherapy using a cysteine protease inhibitor. PLoS Medicine. 2006; 2007;4(1):1-9.
Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvorak J, Hsieh I, Bahgat M, Dissous C, McKerrow JH. A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. Journal of Biological Chemistry. 2006;281(51):39316-29.
Caffrey CR, McKerrow JH, Salter JP, Sajid M. Blood 'N' Guts: an Update on Schistosome Digestive Peptidases. Trends in Parasitology 2004;20(5):241-8.
Sajid M, McKerrow JH, Hansell E, Mathieu MA, Lucas KD, Hsieh I, Greenbaum D, Bogyo M, Salter JP, Lim KC, Franklin C, Kim JH, Caffrey CR. Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase. Molecular and Biochemical Parasitology 2003;131(1):65-75.
subcellular localisations of protease activity in Giardia, using membrane permeable flourescent peptide substrates
3. Clan CD peptidases
Proteases assigned to clan CD are a collective group of enzymes that share a common ancestor, and are typified by the template protease, human caspase 3. Parasitic organisms also possess a number of clan CD enzymes and are therefore of interest to the lab. There are four clan CD proteases that are currently being studied.
I metacaspase
This enzyme represent the most recent member of the clan CD superfamily. Metacaspases are found in parasitic protozoa (Leishmania, Trypanosoma and Plasmodium) but not in humans; and as a result are attracting considerable attention from parasitologists as potential drug targets. In the malaria parasite, Plasmodium metacaspase 1 is highly regulated and is only found in the transmission stages; this may represent an essential function. There is little known about the biological function of metacaspases, however, yeast that undergo cell death (and have markers for apoptosis) activate their metacaspase, YCA1. We are interested in studying the detailed biological role of metacaspases in Plasmodium with an aim to validating metacaspases as novel targets for anti-malarial therapies.
IIasparaginyl endopeptidase
First described in leguminous plants, asparaginyl endopeptidase (AEs) have since been identified in numerous metazoa, including parasitic helminths. AEs are so named due their selective hydrolysis of substrates following asparaginyl residues. AEs have been implicated to play a number of roles including trans-processing of other proteins and proteases and therefore may represent critical targets in anthelmintic therapies. The precise biological role of AEs in S. mansoni and target validation is currently underway.
III GPI:protein transamidase
Tethering of proteins by way of a glycosylphopshatidylinositol moiety to the external surface of the plasma membrane is carried out by GPI:protein transamidase enzymes. GPI-linked proteins are particularly prevalent in parasitic organisms and are requisite to parasite survival and propagation. Validation of GPI:protein transamidase enzymes as drug targets has been hindered by the lack of availability of convenient and sensitive transamidase assay as well as the unavailability of functionally expressed enzyme; work is under way to tackle the two aforementioned hurdles (see, http://www.thesynapticleap.org/?q=node/147 )
IV. aaSeparase
Seperases are highly regulated enzymes that are vital for sister-chromatid seperation at the metaphase-anaphase transition of the cell cycle. A number of seperase ortholgues have been identified in parasitic organisms including Giardia, Leishmania, Entamoeba, Trypanasoma and Schistosoma. As a first step to target validation, we are interested in biochemically profiling the substrate preference of the parasite enzymes and comparing this to the human host orthologue.
further reading
Mottram JC, Helms MJ, Coombs GH, Sajid M. Clan CD Cysteine Peptidases of Parasitic Protozoa. Trends in Parasitology 2003;19(4):182-7.
Caffrey CR, Mathieu MA, Gaffney AM, Salter JP, Sajid M, Lucas KD, Franklin C, Bogyo M, McKerrow JH. Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris. FEBS Lett 2000;466(2-3):244-8.
collaborators
Additional links
http://www.thesynapticleap.org
http://www.thesynapticleap.org/?q=user/52