An Oxygen-resolved Proteomic Approach to Elucidate the Influence of Post-translational Modifications on Thermogenesis in Brown Adipocytes
Abstract
The aim of this proposal is to acquire a system for the evaluation of the effect of various oxygen tensions on the human brown adipocyte proteome as it relates to non-shivering thermogenesis. The proposed system is composed of both a dedicated tissue culture facility capable of tissue culture from anoxia to hyperoxia, mitochondrial proteome sample preparation under a controlled atmosphere, and a liquid chromatography-mass spectrometry system for high resolution bottom-up proteomic analysis.-Body heat maintenance during extended cold exposure is critical to the safety and success of deep-water diving operations, and a diver#s intrinsic thermogenesis plays a role in combating heat loss. Recently, functional brown adipose tissue, capable of non-shivering thermogenesis by regulated mitochondrial membrane uncoupling by uncoupling protein 1 (UCP1), has been identified in adults, an organ previously unknown to be maintained into adulthood. Functional brown adipose tissue represents a potentially untapped intrinsic thermal regulation mechanism, yet little is known about the biochemistry of human brown adipose tissue.-Regulation of mitochondrial uncoupling via UCP1 has been proposed to involve reactive oxygen species in mice, which target UCP1 and post-translationally modify a matrix exposed cysteine conserved among mammals. This observation suggests that if similar post-translational modifications occur in human mitochondria, the oxygen concentration and pressure could have significant effects on thermogenesis, parameters that can vary dramatically across diving depths.-Two major questions remain unresolved: are cysteine post-translational modifications of UCP1 linked to functional outcomes that dictate thermogenic regulation, and how might these responses be distinct in the human and mouse UCP1? The proposed system, in concert with ongoing functional assay development work that motivates this proposal, will allow for a detailed analysis of post-translational modifications, both in vivo and in vitro, with sufficient resolution to identify and quantitate structural effects on functional outcomes of modified UCP1. This structure-function relationship will shed light on the regulatory function of reactive oxygen species in thermogenesis and provide a context from which to interpret thermogenesis under they hyperbaric conditions associated with deep-water diving.-The proposed system will be initially focused on identifying and rationalizing posttranslational modifications in brown adipose tissue, but the potential applications of the equipment are much broader, and will strengthen the research within the Department of Defense, the Office of Naval Research, and the University of California, Santa Barbara. By comparative analysis, we can examine proteomic changes during browning of white adipose tissue, the proteomic response to long-term cold/norepinephrine exposure, and the subsequent recovery. Beyond adipose tissue, the proposed equipment is suitable for alternative tissue culture studies where hypoxia or hyperoxia are of interest. Given this generality, we will develop an educational hands-on module for proteomic mass spectrometry through an upper-division undergraduate biochemistry lab course, to train the next generation of bioenergeticists in the most cutting-edge tools of the trade.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Feb 06, 2023
- Source ID
- N000142312197
Entities
People
- Brandon Greene
Organizations
- Office of Naval Research
- United States Navy
- University of California, Santa Barbara