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Lysine acetylation

Lysine acetylation and methylation were among the first epigenetic modifications reported on histones. Using advanced proteomic technologies, we showed that lysine acetylation targets thousands of histone and non-histone proteins (Choudhary et al., Science, 2009). By combining genetic and proteomic approaches, we showed that acetylation in bacteria occurred through a novel mechanism involving the metabolite acetyl-phosphate (AcP) that can acetylate proteins non-enzymatically (Weinert et al., Mol Cell, 2013). We further showed that acetylation in eukaryotic cells is controlled distinctly in a sub-cellular compartment-dependent manner; and that acetylation of most mitochondrial proteins likely occurs through a non-enzymatic mechanism involving exposure to high concentration of acetyl-CoA in this compartment (Weinert et al., Mol Syst Biol, 2014). We measured the stoichiometry of acetylation using a novel method and showed that most acetylation occurs at a low level, mitochondrial acetylation correlates with the fluctuations in acetyl-CoA levels, and that SIRT3 suppress non-enzymatic acetylation in mitochondria (Weinert et al., EMBO J, 2015). We also investigated the substrates of lysine deacetylases using chemical and genetic approaches (Scholz et al, Nat Biotech, 2015). This work revealed unexpected specificities of several widely used KDAC inhibitors and a possible mechanism by which recently revoked drug bufexamac caused pro-inflammatory adverse effects in patients. Very recently, we combined genetic knockout cells and CBP/p300-specific inhibitors to reveal the landscape of CBP/p300-regulated acetylome (Weinert and Narita et al., Cell, 2018). Our time-resolved acetylome analysis showed that CBP/p300-catalzyed rapid acetylation kinetics is a driver of target gene transcription. Current research in the laboratory is focused on investigating the dynamics and regulatory mechanisms of acetylation.


DNA damage signaling

DNA is the blueprint of life and thus repair of damaged DNA is essential for organismal health and for protection from devastating disease, such as cancer. DNA damage repair (DDR) signaling involves an elaborate network of protein-protein interactions and a variety of PTMs, such as phosphorylation, acetylation, and ubiquitylation. Our group used quantitative proteomics to identify the global changes in phosphorylation, acetylation, and protein abundance in response to DDR signaling (Beli et al., Mol Cell, 2012). We further investigated the dynamics of ubiquitylation in response to UV damage and identified UV-induced ubiquitylation of several proteins, identifying a role of PAF15 ubiquitylation DNA damage bypass (Povlsen et al., Nat Cell Biol, 2012). Recently, the Group developed a promiximity-based network mapping (PROX-NET) approach to map the networks of endogenous DNA damage repair networks (Gupta et al., Cell, 2018). Importantly, this work identified the novel protein complex shieldin that play key roles in regulating NHEJ, antibody class switching, and sensitivity to PARP inhibitors. Additionally, the Group has contributed in implicating several novel proteins and PTMs in DNA damage signaling. We continue to focus our efforts in this area for identifying novel regulators in genome maintenance, as well as identifying novel regulatory PTMs on proteins with known function in DNA damage signaling.


Ubiquitin signaling

The ubiquitin proteasome system is a major regulator of protein homeostasis in eukaryotic cells. In recent years ubiquitylation has emerged as a key regulatory PTM in cell signaling. When ubiquitylated proteins are proteolysed with trypsin, the two C-terminal glycine (di-Gly) residues of substrate-proximal ubiquitin remain attached to the target lysine. Our group developed a streamlined di-Gly profiling approach and provided the first detailed, quantitative map of ubiquitylation sites in mammalian cells and tissues (Wagner et al., Mol Cell Proteomics, 2011 & 2012). These studies painted a broad picture of the ubiquitylation landscape and revealed extensive overlap between ubiquitylation and acetylation sites. We further applied this method to investigate the dynamics of ubiquitylation in response to UV irradiation and identified many UV-regulated ubiquitylation sites (Povlsen and Beli et al., 2012, Nat Cell Biol). We also investigated the functions of anaphase promoting complex (APC/C)-associated E2 ubiquitin enzymes UBE2C and UBE2S, which generate K11 ubiquitin linkages in mitosis. Our research that APC/C function is supported by overlapping functions of three E2 enzymes- UBE2C, UBE2S, and UBE2D, and identified the molecular logic for the essentiality of the spindle assembly checkpoint for viability of human cells. Our current research in this area is focused on investigating ubiquitylation dynamics in response to different cellular perturbations.


Immune receptor signaling

Cells express diverse receptors to respond external and internal cues. Activation of receptor signaling involves the assembly of signaling complexes and modification of proteins with distinct PTMs. Our group uses quantitative proteomics to simultaneously study the dynamics of receptor signaling complexes and downstream PTMs. Using multilayered proteomic approaches we have investigated signaling downstream of B cell receptor (BCR) and tumor necrosis factor alpha (TNF-a) receptor. This work provided a global view of ligand-induced signaling complexes, as well as downstream phosphorylation and ubiquitylation. We showed that BCR signaling induces phosphorylation of RAB7, which impairs its interaction with the effector proteins and results in the dissociation of RAB7 from the endosomes. We further showed that BCR signaling involves TRAF6 and LUBAC-dependent linear ubiquitylation of BCL10, and this is required for full activation of NF-kappaB signaling. Our research identified SPATA2 as novel adaptor that links deubiquitylase CYLD to the TNF receptor signalosome and play an important role in TNF-a-induced necropotosis. The current research in the laboratory is focused on further developing the technologies for integrated signaling analyses and applying them to diverse signaling systems, in particular to immune receptor signaling.


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