The Salk Institute
10010 Torrey Pines Road
, CA 92037
2008 Searle Scholar
An important aspect of chemical biology is the elucidation of the molecular mechanisms that underlie important biological and pathological processes. Global metabolite profiling promises accelerate our understanding of these mechanisms by providing access to a portion of biomolecular space currently inaccessible by other methods. Our lab is focused on the development and application of liquid chromatography-mass spectrometry (LC-MS) based global metabolite profiling methods through research at the interface of chemistry and biology.
Endogenous biochemical characterization of enzymes
Enzymes play important roles in the regulation (production and degradation) of biologically active metabolites in vivo. Understanding the biochemical role of an enzyme in vivo requires a detailed understanding of the metabolic substrates of that enzyme. Traditionally, the substrate scope of an enzyme is defined through in vitro assays. However, these assays fail to account for important aspects of in vivo biology, such as post-translational modifications, competing metabolic pathways, and the existence of uncharacterized metabolites. This difficulty can be overcome through the use of a comparative metabolite profiling strategy to identify the endogenous substrates of an enzyme by quantitatively measuring changes in metabolite levels that result from the inhibition (i.e. chemical inhibition or genetic knockout) of the enzyme in vivo). Application of this approach to the study of biomedically important family enzymes will uncover the relevant endogenous substrates of these enzymes and will lay the foundation for future experiments designed to understand the impact of these enzymatic nodes in cellular and physiological processes.
Protein-Metabolite Interactions (PMIs)
The assignment of enzyme-substrate interactions in vivo underscores the capacity of global metabolite profiling to establish important connections between the proteome and metabolome in complex biological settings. Of course, biology provides us with a multitude of other important protein-metabolite interactions (PMIs), such as the binding of metabolites to receptors and transport proteins. Thus, extending our metabolomics platform to include these other important, yet hard to identify, PMIs will be of tremendous value. However, unlike the enzymatic conversion of a substrate to a product the binding of a ligand to a receptor lacks a detectable metabolic signature. As a result, binding information between ligands and receptors is overlooked by our current metabolite profiling protocols and the challenge remains to refine our methodology to identify these important interactions. The key to identifying endogenous PMIs is the isolation of endogenous protein metabolite complexes from cells and tissues. The lab will approach this problem using two different and complementary strategies for the identification of endogenous protein metabolite interactions.
Integrated molecular profiling
A complete molecular understanding of human physiology and pathology requires new technologies that enable the large-scale analysis of genes, proteins, and metabolites in complex biological systems. The application of integrated genomic, proteomic, and metabolomic methods will facilitate the coordinated profiling and functional characterization of biomolecules in complex biological systems (Figure 2). Such an integrated molecular profiling platform would retain all the advantages of more classical genomics/proteomics approaches, while at the same time gaining the capacity to elucidate endogenous biochemical activities for genes/proteins through the inclusion of metabolite profiling. To test this method we plan to study the process of adipocyte differentiation, which is of interest due to the health problems associated with obesity, such as coronary heart disease and diabetes. A complete inventory of the biomolecules that show altered expression and activity during adipogenesis would provide key insights into the metabolic and signaling pathways that support this cellular process. We anticipate that this study will yield an unprecedented boon of molecular information that, when viewed as a whole, will illuminate new biochemical networks that support the preadipocyte to adipocyte transition.
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