SMOCs: supramolecular organizing centres that control innate immunity

This Opinion article proposes that higher-order protein complexes — referred to as supramolecular organizing centres (SMOCs) — form on specific organelles by nucleated polymerization downstream of innate immune receptors to amplify the signal and reach a response threshold.

Abstract

The diverse receptor families of the innate immune system activate signal transduction pathways that are important for host defence, but common themes to explain the operation of these pathways remain undefined. In this Opinion article, we propose — on the basis of recent structural and cell biological studies — the concept of supramolecular organizing centres (SMOCs) as location-specific higher-order signalling complexes in which increased local concentrations of signalling components promote the intrinsically weak allosteric interactions that are required for enzyme activation. We suggest that SMOCs are assembled on various membrane-bound organelles or other intracellular sites, which may assist signal amplification to reach a response threshold and potentially define the specificity of cellular responses that are induced in response to infectious and non-infectious insults.

Introduction

Perhaps no area of immunology has benefitted more from the sequencing of the human (and mouse) genome than that of innate immunity. Modern studies of innate immunity received widespread attention with the discovery in the late 1990s that Toll-like receptors (TLRs) link microbial detection with the induction of adaptive immunity¹. Because TLRs and their associated families of signalling proteins have sequence homology, surveying the human and mouse genomes for uncharacterized orthologous proteins became a common approach to study these biological processes. Thus, within a few years of the discovery of cell-surface and endosomal TLRs^{1, 2, 3}, more than 100 genes had been identified that regulate the signalling pathways induced by these receptors^{4, 5, 6, 7, 8}, as well as the functionally related, cytosolic NOD-like receptors (NLRs), RIG-I-like receptors (RLRs) and others⁹ (Fig. 1; Table 1). Individual members of these pattern recognition receptor (PRR) families detect conserved pathogen-associated molecular patterns (PAMPs) that are present on bacteria, viruses and fungi, or recognize intrinsic damage-associated molecular patterns (DAMPs) that are elicited by cellular injury. Upon ligand binding, these receptors activate numerous cellular responses to fight infection and restore homeostasis.

Figure 1: SMOC formation for TLRs, RLRs and NLRs.

Depicted are the best-studied supramolecular organizing centres (SMOCs), including the ligands and regulatory proteins that promote their assembly, and the downstream biological activities induced by these protein complexes. The figure does not show the exact stoichiometry of the protein components in each signalling complex. Binding of lipopolysaccharide (not shown) activates Toll-like receptor 4 (TLR4), leading to assembly of a Myddosome on the plasma membrane. By contrast, unmethylated CpG-containing DNA oligonucleotides (not shown) promote TLR9 to assemble an endosomal Myddosome. The TLR-specific sorting adaptor Toll/IL-1R domain-containing adaptor protein (TIRAP) facilitates Myddosome assembly. TIRAP has an amino-terminal lipid-binding domain that interacts promiscuously with acidic phosphoinositides and phosphatidylserine. For example, TIRAP is depicted as binding phosphatidylinositol-3-phosphate (PtdIns3P) and phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂) on the endosomal membrane and the plasma membrane, respectively. Binding of cardiolipin, which translocates to the outer mitochondrial membrane upon mitochondrial dysfunction, relieves the autoinhibited state of NOD-, LRR- and pyrin domain-containing 3 (NLRP3). This in turn may promote NLRP3 inflammasome assembly through downstream pyrin domain (PYD)–PYD and caspase activation and recruitment domain (CARD)–CARD interactions. Activation of RIG-I-like receptors — retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5) — leads to the formation of higher-order oligomers of mitochondrial antiviral signalling protein (MAVS). IFN, interferon; IκBα, NF-κB inhibitor-α; IL, interleukin; IRAK, IL-1 receptor-associated kinase; IRF, IFN-regulatory factor; LRR, leucine-rich repeat; MYD88, myeloid differentiation primary response protein 88; NOD, nucleotide-binding oligomerization domain; NF-κB, nuclear factor-κB; TAB1, TAK1-binding protein 1; TAK1, TGFβ-associated kinase 1; TRAF, tumour necrosis factor receptor-associated factor.

The success of using bioinformatics and reverse genetics to study innate immune signalling pathways came at the expense of alternative strategies to address these areas. As such, studies of the biochemistry, cell biology and dynamics of these signalling pathways have been much less common. In fact, most early studies of TLRs and their associated signalling proteins did not include any analysis of the subcellular localization of the newly identified protein(s). Thus, although we know the identity of many genes that are involved in innate immunity, the functional mechanisms of the proteins encoded by these genes, and how they interact in space and time, are poorly understood.

 

The lack of knowledge on the specific activities of the proteins that control innate immunity has given rise to biological models that do not address many aspects of the signalling process, such as the subcellular site where a given signalling event occurs, or the dynamics of putative protein–protein interactions. Current models of TLR, NLR or RLR signalling rather depict a series of arrows connecting receptors with downstream signalling proteins, yet we have little understanding of what these arrows actually represent. Do they represent direct protein–protein interactions? If so, are these interactions constitutive or are they induced upon microbial encounter? How are these interactions regulated and where in the cell do they occur? As described below, recent biochemical and cell biological studies have provided important insight into these questions. These new studies indicate that numerous protein regulators of innate immunity are organized into higher-order signalling complexes that define the subcellular sites and specificity of innate immune signal transduction.

 

In this Opinion article, we propose that these higher-order signalling complexes function as 'supramolecular organizing centres' (SMOCs) that control cellular responses induced by specific families of upstream receptors. We discuss how SMOCs can operate from various locations within the cell, and describe how they consist of proteins that either sense the activation of upstream receptors or elicit specific downstream effector responses. We further propose that these complexes include context-dependent components, which may be cell type-specific or organelle-specific regulators, such that a given SMOC can elicit diverse cellular responses depending on the stimulus. A benefit of coordinating innate immune signalling pathways around a set of organizing centres may be the modularity of the system, whereby numerous upstream stimuli can be directed into a common downstream module. Indeed, this is the case when considering the operation of other non-membranous organizing centres in mammalian cells, such as the microtubule organizing centre (MTOC) and the proteasome. In these examples, a large protein complex coordinates an entire biological process that may be needed to address diverse cellular needs.

 

Figure 2: Structures of SMOCs that are formed by the mechanism of nucleated polymerization.

a | A ribbon diagram of the electron cryomicroscopic structure of polymerized ASC pyrin domain (ASC^PYD) filaments in inflammasomes (shown in red, purple and orange for each of the three helical strands), in complex with polymerized absent in melanoma 2 PYD (AIM2^PYD) filaments (shown in blue) formed upon double-stranded DNA (dsDNA) stimulation. b | A ribbon diagram of the electron cryomicroscopic structure of polymerized mitochondrial antiviral signalling protein caspase activation and recruitment domain (MAVS^CARD) in the RIG-I-like receptor (RLR) pathway (shown in purple), in complex with a retinoic acid-inducible gene I (RIG-I) double CARD (RIG-I^2CARD) tetramer (shown in blue, cyan, green and yellow) upon viral RNA stimulation. c | Proposed mechanism of nucleated polymerization for the formation of ASC^PYD and MAVS^CARD SMOCs. Receptors (for example, AIM2 and RIG-I) are shown in red wedges for monomers and in red disks for oligomerized forms. Adaptors (for example, ASC and MAVS) are shown in blue wedges for monomers and in blue cylinders for filaments.

Conclusions and future perspectives

Examples now exist for the three major families of PRRs — TLRs, RLRs and NLRs — that SMOC assembly occurs on membranes, even when the upstream receptors are cytosolic proteins. In each documented example, a membrane protein seeds the formation of a higher-order signalling complex that activates specific innate immune responses. A biophysical explanation for the apparent membrane localization of SMOCs may be the marked energetic enhancement of protein–protein interactions on a two-dimensional membrane surface compared with those in a three-dimensional cellular milieu⁵⁷. For this reason, we propose that future studies of the biochemical mechanisms of SMOC assembly and function would benefit from a greater consideration of the subcellular sites where this assembly occurs. This analysis may also help to address the question of why SMOCs have evolved to operate from specific organelles. Additional studies of the relationship between organelle function and SMOC assembly should address the possibility that organelles are not solely needed as a scaffold for SMOC assembly, but rather that a metabolic or biochemical activity of the organelle may contribute as well. This cell biological analysis may reveal important means of controlling and manipulating SMOC assembly and subsequent inflammatory responses.

 

Nature Reviews Immunology | Perspectives | Opinion

Jonathan C. Kagan^1, Venkat Giri Magupalli^1, Hao Wu^1,

Published online 31 October 2014      

Corrected online 03 November 2014