| During the past few years, complete genomes of multiple organisms have been sequenced. This avalanche of genetic information is a starting point for the more global task of defining function for all proteins within an organism. Although global in scope, genomics and proteomics techniques provide only indirect information about protein function. In fact, in many cases enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility. We have engineered chemical probes that can be used to broadly track activity of the Papain family of cysteine proteases.; A series of functional proteomics tools and methods was developed to identify and analyze the Papain family (CA/CB Clan) of enzymes within any complex cellular “proteome”. These tools can be used to determine the relative activity levels of active proteases and rapidly map the dynamics of activity changes throughout a normal or pathological biological process. Moreover, these tools also allow for the screening of small molecule inhibitor libraries for any Papain family target using either pure enzyme or even cellular lysate. A general method for rapid colorimetric analysis of large data sets, generated from library screening of multiple targets in crude cell extracts, was developed. This approach allows rapid comparison of inhibitors as well as targets based on similarities in structure-function relationships.; These methods were used to study protease function in several biological systems. Using the activity-based cysteine protease affinity probes, a calcium-dependent cysteine protease, Lp82, was found to be the key protease in cataract generation using a mouse model of cataractogenesis. This protease was found to be hyperactivated without any concomitant increase in protein level, validating the use of activity-based techniques for studying enzyme function. Furthermore, evidence is presented that Falcipain 1, a cysteine protease in P. falciparum , is involved in the invasion pathway of red blood cells. Falcipain 1 function was determined by thorough understanding of protease activity and localization during the life cycle of the parasite coupled with phenotypic analysis of protease inhibition using specific Falcipain 1 specific inhibitors. |
