1. The use of mass spectrometry as a structural tool.
Usually thought of as an analytical technique, mass spectrometry has recently been used to obtain structural information about proteins and their interactions. We have used cross-linking reagents to trap various parts of a protein molecule in a covalent linkage. After cutting
up the protein with trypsin, one can identify which peptides are cross-linked and gain important information as to which parts of the sequence were in close proximity in the native structure. This technique has been applied to the problem of apoA-I on the surface of a discoidal HDL particle, but the applications of this technique are essentially unlimited. We are currently extending this technology to study the structure of apolipoproteins in HDL isolated from human plasma to understand their native structure and interactions. We are also studying important interactions of apoA-I with cell surface proteins such as ABCA1 and SR-BI and plasma lipid metabolizing proteins such as CETP and LCAT.
2. The structure and function of apolipoprotein A-IV and A-V.
Relatives of apoA-I, apoA-IV and apoA-V are HDL proteins that have interesting and potent functions on glucose regulation and plasma triglyceride metabolism, respectively. We have deveoloped a bacterial expression systems for these proteins and are working to undertand thier structure in detail using methods like X-ray crystallography and chemical cross-linking. The hope is that we can determine how these proteins work and then develop therapeutic ways to mimic thier function to treat disease.
3. HDL proteome and subspeciation.
HDL has over 100 distinct proteins associated with it, many involved in lipid transport, but many others with unexpected functions like protease inhibition, and complement function. These proteins seem to be distributed in distinct patterns across HDL particles with respect to size and charge. Thus, it is hard to believe that all of these particles evolved to perform a similar function. We believe that the term "HDL" refers to a broad collection of particles comprised of distint subpopulations. These subspecies have in common an abundance of phospholipid but are distinct in the complement of surface proteins they contain. Our laboratory is developing novel separation and analysis technologies to identify and characterize these HDL subspecies. Using high resolution mass spectrometry, we are characterizing the phospholipid-rich components of human plasma. The goal is to identify these particles and determine their function and how they differ between healthy and diseased individuals. We believe that certain HDL particles may be more beneficial than others when it comes to preventing metabolic disease. If we can identify them, it may be possible to design strategies to enrich these "super" particles without raising HDL cholesterol in the generic sense.
4. HDL in the immune system.
HDL contains many proteins that are intimately involved in the innate immune system. We have found that HDL can alter the lifespan of regulatory T-cells in humans and therefore may attenuate responses to foreign pathogens. Also, we are studying the role of HDL in sepsis, an uncontrolled inflammatory response to infection that causes organ failure and, all too frequently, death. We are exploring the roles of HDL subspecies in attenuating this syndrome.
Interested? Join the lab and jump on any of these or related projects!