How do immune regulatory enzymes recognise substrates inside the lipid bilayer?

Project type

Honours and/or PhD

Supervisor(s)	Division	Email
Dr Matthew Call (Primary)	Structural Biology	.(JavaScript must be enabled to view this email address)
Dr Melissa Call (Co-supervisor)	Structural Biology	.(JavaScript must be enabled to view this email address)

Details of project

Lymphocyte cell-surface molecules involved in adhesion and intercellular communication are regulated at many different levels. We most often think about regulation at the levels of transcription and translation, but membrane proteins are also heavily regulated through endocytosis and intracellular trafficking pathways. A recently discovered family of enzymes known as the membrane-associated RING-CH (MARCH) proteins regulates immune functions by controlling the endocytosis of receptors and adhesion molecules. They are expressed in a wide variety of immune cells, and their regulatory power is so potent that several viruses have evolved to express homologous proteins that actively impair immune responses to infection (see project references 1-3).

MARCH proteins are E3 ubiquitin ligases, enzymes that mark targets for regulation by attaching the small protein ubiquitin to exposed sequences. Of more than 300 E3 ubiquitin ligases in the human genome, the MARCHs appear to be the only ones that are attached to the membrane through a-helical transmembrane (TM) domains. While the ubiquitin conjugation reaction occurs between cytosolic domains, there is strong evidence that substrate recognition occurs as a consequence of interactions between the TM domains of enzyme and substrate (see figure below; reviewed in project reference 4). This type of intra-membrane substrate recognition is almost completely unstudied, and we are applying several different techniques to determine the structural basis of MARCH-substrate complex formation.

A student taking on this project will be involved in the design and production of protein complexes representing the interacting TM fragments, the collection and analysis of solution NMR data for structure determination, and functional analysis in cellular assays. The results will provide important new insights into mechanisms of normal immune regulation, viral immune evasion and the pathways through which membrane proteins are controlled in general.

<p>Schematic illustration of MARCH-substrate interactions in the plasma membrane. Different MARCH family members have different substrate preferences, and not all membrane proteins are subject to regulation by MARCHs. While there is no known “substrate-recognition motif” for these enzymes, a non-substrate protein can be converted to a substrate protein simply by replacing its TM domain with that of a known substrate. We are working to understand how this mode of intra-membrane substrate recognition works and what features of the enzyme and substrate TM domains are responsible.
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Project references

Ishido S, Choi JK, Lee BS, Wang C, DeMaria M, Johnson RP, Cohen GB, Jung JU. Inhibition of natural killer cell-mediated cytotoxicity by Kaposi’s Sarcoma-associated herpesvirus K5 protein. Immunity. 2000 Sept;13:365-374. PMID:11021534.
Bartee E, Mansouri M, Hovey Nerenberg BT, Gouveia K, Früh K. Downregulation of major histocompatibility complex class I by human ubiquitin ligases related to viral immune evasion proteins. J Virol. 2004 Feb;78(3):1109-20. PMID:14722266.
Young LJ, Wilson NS, Schnorrer P, Proietto A, ten Broeke T, Matsuki Y, Mount AM, Belz GT, O'Keeffe M, Ohmura-Hoshino M, Ishido S, Stoorvogel W, Heath WR, Shortman K, Villadangos JA. Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells. Nat Immunol. 2008 Nov; 9(11):1244-52. PMID:18849989.
Ohmura-Hoshino M, Goto E, Matsuki Y, Aoki M, Mito M, Uematsu M, Hotta H, Ishido S. A novel family of membrane-bound E3 ubuquitin ligases. J. Biochem. 2006 Aug;140(2):147-54. PMID: 16954532

Research interests

Research in the Call laboratory is broadly focussed on understanding how cellular signalling and molecular recognition events take place in the context of the plasma membrane. Cells of the immune system must exchange a vast amount of information with each other and with their surroundings in order to direct appropriate responses to potential pathogens, cancerous cells and injured tissues. This information exchange is achieved primarily through the actions of a broad array of cell-surface receptors expressed on lymphocytes and other immune cells that integrate many different types of signals to regulate immune functions.

Decades of research into immune signalling pathways have produced a large base of knowledge about the molecules involved, but in most cases we still do not understand the mechanics of information transmission across the lipid bilayer. Our laboratory has taken a particular interest in how the structure and dynamics of the membrane-embedded portions of receptors and their associated proteins contribute to immune regulation. These transmembrane domains turn out to be much more than mere anchors to the lipid bilayer – they organise complex membrane protein interactions and participate in dynamic regulatory processes. They also represent the only direct physical link between ligand-binding and signalling domains across the cell membrane, and we therefore have a keen interest in obtaining the detailed structural information required to accurately describe their roles in signalling.

Our lab combines expertise in protein engineering, biophysical methods (primarily solution NMR) and molecular immunology to unravel the mechanics of transmembrane signalling in the immune system. Our aim is to understand both how ligand binding to a receptor is physically communicated through the lipid bilayer and how the unique characteristics of the membrane environment contribute to and regulate this communication.

Selected publications

Call ME, Chou JJ. A view into the blind spot: solution NMR provides new insights into signal transduction across the lipid bilayer. Structure. 2010 Dec 8;18(12):1559-1569. PMID: 21134635
Wucherpfennig KW, Gagnon E, Call MJ, Huseby ES, Call ME. Structural biology of the T-cell receptor: insights into receptor assembly, ligand recognition, and initiation of signaling. Cold Spring Harb Perspect Biol. 2010 Apr 1;2(4):a005140 PMID: 20452950
Call ME, Wucherpfennig KW. The T cell receptor: critical role of the membrane environment in receptor assembly and function. Ann Rev Immunol. 2005; 23:101-25. PMID: 15771567

Research theme

Infectious diseases

Scientific discipline

Biochemistry
Immunology
Systems Biology

Keywords

ubiquitin ligase, transmembrane domains, immune regulation, molecular recognition

The Walter and Eliza Hall Institute of Medical Research