Proteomics approaches for the study of adipose tissue biology: TRIB3 and beyond

Abstract

Adipose tissue (AT) regulates whole-body metabolism and energy homeostasis through the production and secretion of adipose-specific factors, the so-called: adipokines. Dysregulation of adipose tissue due to excessive and prolonged exposure to an over-caloric diet is a hallmark in the development of obesity and obesity-associated diseases like Type II Diabetes and cancer. Since the global obesity epidemic has not shown signs of declining, understanding the factors and signals that regulate adipose tissue function remains of critical importance for the scientific community. The three main players of this thesis are introduced in Chapter 1. Adipose tissue, pseudoenzymes and their role in biology, and finally, mass spectrometry-based proteomics which is the connecting thread throughout the rest of the thesis. In Chapter 2, the role of peroxisome proliferator-activated receptor gamma (PPARg), a member of the nuclear receptor superfamily, is summarized. With special emphasis on the different molecular mechanisms behind its function in different cellular contexts, such as adipocytes and immune cells. In addition, the role PPARg in cancer is explained in detail. Chapter 3 is focused on Tribbles and their interactome. Tribbles are highly conserved pseudokinases that play a role in many aspects of biology, from metabolism to immune cell differentiation. We hypothesized that the different set of proteins that interacts with specific Tribbles in a given biological context is what determines their role in that cellular context. We used a mass-spectrometry-based approach to study the interactome of these proteins and we discover and validate that TRIB3 functions as a transcriptional repressor. In Chapter 4 we use a multidisciplinary approach to study the role of TRIB3 in AT. We characterized the phenotype of the Trib3 knock-out mice, and we used a combination of -omics technologies (transcriptomics, proteomics, and lipidomics) to look at the molecular mechanism. Similarly, in Chapter 5, we combined in-vivo with in-vitro techniques to understand the role of FHL2 in adipocytes. We found that FHL2 interacts with NFAT5 in early adipogenesis and that this signaling complex represses the adipogenic transcriptional program. Next, in Chapter 6 we validated the interaction of TRIB3 with the WRAD complex. The WRAD complex is the catalytic subunit of the histone writer SET1/MLL complex. This complex is responsible for histone H3 lysine-4 three-methylation (H3K4me3), a histone mark found in active promoters and link of transcriptional activation. We found that TRIB3 can regulate PPARg expression through the interactions with the WRAD complex and this signaling pathway might open the door to future therapeutic interventions in breast cancer patients. Finally, in Chapter 7 we studied the long-known interaction between TRIB3 and AKT. We used a combination of in-silico computational modeling and an in-vitro approach to map this interaction. We found a motif in the C-Terminal tail of TRIB3 that is pivotal for the interaction with AKT and might explain some of the controversies described during previous years. Lastly, some of the conclusions and future directions of the work presented in this thesis are described in Chapter 8.