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Bhogaraju Group

Structural biology of ubiquitin signalling

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

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Previous and current research

Ubiquitin is a 76-amino acid polypeptide that is one of the most abundant proteins in eukaryotic cells. It is covalently attached to other proteins in a process known as ubiquitination, which often leads to protein degradation but can also alter the function of the modified protein. Ubiquitination regulates many cellular processes, including protein turnover, DNA repair, cell division, vesicular transport, autophagy and innate immunity. The attachment of ubiquitin to proteins is carried out by a cascade of three enzymatic reactions involving E1, E2, and E3 enzymes, resulting in the attachment of the ubiquitin C-terminal carboxyl group to a lysine residue in the substrate protein via an isopeptide bond. With over 600 genes coding for E3 ubiquitin ligases in humans, these enzymes make up a significant portion of the human genome. Our group studies the structure, function, and regulation of unique, understudied ubiquitin ligase complexes of human and bacterial origin.

Exploitation of Host Ubiquitination by Legionella pneumophila: Mechanisms and Consequences

Bacteria do not have a ubiquitin system, but some pathogenic bacteria have developed virulence factors (effectors) that can manipulate the host’s ubiquitin system. During an infection, these effectors are secreted into the host cell, allowing the pathogen to evade the host’s immune system and establish a successful environment for replication in the host’s cytoplasm. Legionella bacteria are commonly found in soil and natural water sources and can cause Legionnaires’ disease, a severe form of pneumonia, if inhaled in the form of aerosols. Elderly individuals and those with compromised immune systems are at high risk of fatal complications. During an infection, Legionella secretes over 300 effectors into the host’s cytosol to control various cell-signaling events, including the ubiquitin signaling system. Our research focuses on how these Legionella effectors interact with the host’s ubiquitin system, using tools from structural biology, cell biology, and proteomics.

Our research, along with that of others, uncovered that SdeA, a toxic effector of Legionella, employs a completely different mechanism compared to the standard three enzyme cascade for attaching ubiquitin to its substrates. (Qiu et al., 2016; Bhogaraju et al., 2016). SdeA-mediated reaction results in the attachment of Arginine-42 of ubiquitin to Serines of target substrates through a phosphoribose linker. This unique process of linking ubiquitin to a substrate protein involves the formation of a phosphodiester bond instead of the typical isopeptide bond. It’s still unclear if this distinct form of ubiquitination is carried out by enzymes in other living organisms. We investigated the regulation of this unusual form of ubiquitination by SidJ, a meta-effector of Legionella. SidJ regulates the excessive toxicity caused by SdeA and its paralogues during Legionella infection in a specific and timely manner. Our research found that SidJ partners with host Calmodulin to inhibit the activity of SdeA through ATP-dependent glutamylation. The cryo-electron microscopy (Cryo-EM) structure of SidJ in complex with human Calmodulin revealed how Calmodulin activates SidJ allosterically. This structure also demonstrated that SidJ has a pseudokinase fold with two nucleotide-binding pockets (Bhogaraju et al., 2019). Recently, we used cryo-EM to determine the structure of two catalytic intermediate states of the SidJ/Calmodulin complexed with SdeA (Figure 1). This uncovered a novel mechanism of glutamylation that involves a fleeting self-modification of SidJ through Lysine AMPylation (Adams et al., 2021). We are continuing to study this unique form of ubiquitin signalling in Legionella, especially in the context of infections, through collaborations.

Investigating the Mechanisms of MAGE-RING E3 Ubiquitin Ligases in Neurodevelopment and Cancer Pathogenesis

Our research group is also exploring another aspect of ubiquitin signaling that involves a family of human proteins called MAGE (Melanoma Antigen), which is highly conserved yet largely unstudied. This family is made up of around 40 different proteins that share a common MAGE homology domain. Linked to several neurodevelopmental disorders and tumorigenesis, the MAGE proteins form complexes called MAGE-RING ligases (MRLs) with various RING ubiquitin ligases, which target specific cellular substrates for ubiquitination (Lee and Potts, 2017). The precise role of MAGE proteins in the MRL complexes needs to be better understood and it is not clear how these proteins with 40-80% sequence similarity are able to recognize a diverse range of RING proteins. The mechanism by which misregulation or mutations in a MAGE gene leads to human disease remains a mystery in most cases. Our approach to these questions involves carrying out structural and biochemical examinations on several understudied MRLs and discovering new MRLs through proteomics, which will then undergo structural and functional characterization.

Future projects and goals:

  1. Legionella has a wealth of effectors that engage with the host’s ubiquitin system, beyond the non-canonical ubiquitin system that our group has been investigating. The aim of our research is to obtain a complete picture of how the host ubiquitin system is exploited during Legionella infection and to seek out potential targets for combating Legionnaires’ disease.
  2. The aim of our research on human MRL complexes is to gain a molecular understanding of the associated human diseases, with the end goal of proposing precise therapeutic targets and validating them in model systems.
Figure 1: The structure of the SidJ (Yellow)-Calmodulin (Cyan)-SdeA (Green) complex was determined through Cryo-Electron Microscopy (Cryo-EM). The pseudokinase fold of SidJ is shown, with its C-lobe and N-lobe being labeled accordingly. The two catalytic domains of SdeA, namely the mono-ADP ribosyl transferase (mART) and the phosphodiesterase (PDE), are labeled. In a small inset, the complex of SidJ-Calmodulin-SdeA that was captured in a catalytic intermediate state is depicted.
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