Abstract
With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
Serine hydrolases play important roles in numerous developmental and tissue-specific events in vivo, including blood coagulation, inflammation, angiogenesis, neural plasticity, peptide hormone processing, and T-lymphocyte-mediated cytotoxicity. Additionally, several human diseases are associated with dysfunctions in serine proteases and/or their endogenous inhibitory proteins, including hemorrhagic disorders, emphysema, and cancer. The large number of mammalian serine hydrolases identified to date is both impressive and perplexing, with the endogenous functions of many members of this enzyme family remaining unknown. As ORFs encoding putative serine hydrolases continue to accumulate in public databases , the need for alternative experimental methods to study these enzymes is evident. One attractive approach for the analysis of serine hydrolase function would be to characterize these enzymes collectively, rather than individually. In particular, the majority of serine hydrolases are potently and irreversibly inhibited by fluorophosphonate/fluorophosphate (FP) derivatives like diisopropyl fluorophosphate , whereas cysteine, aspartyl, and metallohydrolases are for the most part inert to such agents. Moreover, the reactivity of FPs with serine hydrolases requires that the enzymes be in a catalytically active state. Accordingly, we hypothesized that an FP linked to a small molecule reporter group might serve as a potent and selective probe for monitoring simultaneously the activities of multiple serine hydrolases. In this manner, serine hydrolases could be visualized on a systems level of analysis, greatly accelerating the assignment of potential functions and malfunctions to members of this enzyme family. Here, we report the synthesis and characterization of a biotinylated long-chain fluorophosphonate, referred to as FP-biotin and highlight its utility as an agent for profiling dynamics in serine hydrolase expression and function.
Resource: http://www.ncbi.nlm.nih.gov
Resource: http://www.nutritionforest.com/
With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
Serine hydrolases play important roles in numerous developmental and tissue-specific events in vivo, including blood coagulation, inflammation, angiogenesis, neural plasticity, peptide hormone processing, and T-lymphocyte-mediated cytotoxicity. Additionally, several human diseases are associated with dysfunctions in serine proteases and/or their endogenous inhibitory proteins, including hemorrhagic disorders, emphysema, and cancer. The large number of mammalian serine hydrolases identified to date is both impressive and perplexing, with the endogenous functions of many members of this enzyme family remaining unknown. As ORFs encoding putative serine hydrolases continue to accumulate in public databases , the need for alternative experimental methods to study these enzymes is evident. One attractive approach for the analysis of serine hydrolase function would be to characterize these enzymes collectively, rather than individually. In particular, the majority of serine hydrolases are potently and irreversibly inhibited by fluorophosphonate/fluorophosphate (FP) derivatives like diisopropyl fluorophosphate , whereas cysteine, aspartyl, and metallohydrolases are for the most part inert to such agents. Moreover, the reactivity of FPs with serine hydrolases requires that the enzymes be in a catalytically active state. Accordingly, we hypothesized that an FP linked to a small molecule reporter group might serve as a potent and selective probe for monitoring simultaneously the activities of multiple serine hydrolases. In this manner, serine hydrolases could be visualized on a systems level of analysis, greatly accelerating the assignment of potential functions and malfunctions to members of this enzyme family. Here, we report the synthesis and characterization of a biotinylated long-chain fluorophosphonate, referred to as FP-biotin and highlight its utility as an agent for profiling dynamics in serine hydrolase expression and function.
Resource: http://www.ncbi.nlm.nih.gov
Resource: http://www.nutritionforest.com/
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