Figure 1: Mechanism of E1, E2-independent ubiquitination by SdeA class of enzymes.

Figure 1: Mechanism of E1, E2-independent ubiquitination by SdeA class of enzymes.

The Bhogaraju group uses structural and cell biology-based approaches to study ubiquitination pathways in normal physiology and disease.

Previous and current research

Ubiquitination of proteins is involved in virtually every cellular process including protein turnover, DNA-repair, cell-cycle, vesicular transport, autophagy and innate immunity. Ubiquitination proceeds through a universally conserved process involving 3 enzyme families (E1, E2 and E3) and ATP resulting in the attachment of the C-terminal carboxyl group of ubiquitin to a lysine residue in substrate protein via an isopeptide bond. Recently, a string of exciting studies have shown that the SidE family of toxic proteins (SdeA, SdeB, SdeC & SidE) from pathogenic Legionella pneumophila can attach ubiquitin to substrates independent of E1, E2 enzymes (Qiu et al., 2016; Bhogaraju et al., 2016). My postdoctoral work has elucidated the comprehensive mechanism of SdeA-mediated ubiquitination wherein the mono-ADPribosyl transfer (mART) domain in SdeA ADPribosylates ubiquitin on Arg42. This is followed by phosphodiesterase (PDE) domain-driven cleavage of the pyrophosphate bond between α and β phosphates of ADP-ribose, linked to ubiquitin. The PDE reaction is accompanied by the attachment of ubiquitin to serines of target substrates by a phosphoribosyl linker on Arg42 (Bhogaraju et al., 2016) (Figure 1). This unique bridging of ubiquitin to substrate protein involves a phosphodiester bond instead of the canonical isopeptide bond. These studies have opened an exciting new facet of ubiquitin biology that largely remains to be explored.

Future projects and goals

  • Our lab will focus on exploring the functional and mechanistic aspects of this unique phosphoribosyl-dependent ubiquitination further. We are especially interested in several poorly studied proteins that are involved in regulating this post-translational modification.
  • One of the focuses of the lab will be to study a highly conserved but largely unexplored family of human proteins called MAGE (Melanoma Antigen). This family consists of around 60 different proteins that share a conserved MAGE homology domain. Mutations or deregulation of MAGE family of proteins are the cause of several neurodevelopmental disorders. Erroneous expression of MAGEs also actively drive tumorigenesis underlining the potent oncogenic properties of these genes (Lee and Potts, 2017). MAGE proteins are proposed to interact with various RING ubiquitin ligases to form complexes called MAGE-RING ligases (MRLs) that target specific cellular substrates for ubiquitination. Most of the cognate RING partners for MAGE proteins are unknown and importantly it is also not known how these proteins with sequence similarity of 40-80% are able to recognize a diverse set of RING proteins.  Our aim is to answer these questions by structural and biochemical studies of various MRLs. We will also focus on identifying new MRLs through mass spectrometry followed by their functional and structural characterization.