Intrinsic Disorder in Transmembrane Proteins: Roles in Signaling and Topology Prediction

Intrinsically disordered regions (IDRs) are peculiar stretches of amino acids that lack stable conformations in solution. Intrinsic Disorder containing Proteins (IDP) are defined by the presence of at least one large IDR and have been linked to multiple cellular processes including cell signaling, DNA binding and cancer. Here we used computational analyses and publicly available databases to deepen insight into the prevalence and function of IDRs specifically in transmembrane proteins, which are somewhat neglected in most studies. We found that 50% of transmembrane proteins have at least one IDR of 30 amino acids or more. Interestingly, these domains preferentially localize to the cytoplasmic side especially of multi-pass transmembrane proteins, suggesting that disorder prediction could increase the confidence of topology prediction algorithms. This was supported by the successful prediction of the topology of the uncharacterized multi-pass transmembrane protein TMEM117, as confirmed experimentally. Pathway analysis indicated that IDPs are enriched in cell projection and axons and appear to play an important role in cell adhesion, signaling and ion binding. In addition, we found that IDP are enriched in phosphorylation sites, a crucial post translational modification in signal transduction, when compared to fully ordered proteins and to be implicated in more protein-protein interaction events. Accordingly, IDPs were highly enriched in short protein binding regions called Molecular Recognition Features (MoRFs). Altogether our analyses strongly support the notion that the transmembrane IDPs act as hubs in cellular signal events.

Unraveling the Physiological Role of Capillary Morphogenesis Gene 2 in Health and Disease

Jérôme Bürgi

Capillary Morphogenesis Gene 2 (CMG2) is a 55kDa single-pass transmembrane protein best described as the main Anthrax Toxin Receptor. However, CMG2 physiological role remains elusive and understanding better its in vivo function was the main objective of my thesis. My first aim was to investigate the prevalence of intrinsically disordered domains in the human transmembrane proteome. Using prediction tools, I found that 50% of transmembrane proteins had at least one domain of minimum 30 amino-acids predicted as intrinsically disordered (IDD), these proteins being mainly involved in cell signaling and adhesion. The majority of the IDDs were localized in the cytoplasm, and the intrinsic disorder containing proteins were significantly more phosphorylated and interacted on average with more proteins than fully ordered proteins, corroborating their role in signaling. CMG2 loss-of-function mutations is the known cause of a rare inherited genetic disorder called Hyaline Fibromatosis Syndrome (HFS). HFS patients develop subcutaneous nodules, gingival hypertrophy, contracture of the large joints, and in the most severe cases diarrhea and premature death. In a second part of my thesis, I was involved in understanding the molecular defects underlying missense mutations localized in the ectodomain and a highly conserved cytoplasmic domain of CMG2. We found that CMG2 was able to bind the actin cytoskeleton through Talin and Vinculin when free of its extracellular ligand. Interestingly, the HFS mutation p.C218R, altered the cytoskeleton release and led to a CMG2 protein able to bind both the extracellular ligand and the actin cytoskeleton. Strikingly, we observed that all but one cytoplasmic missense mutation hindered actin binding, a defect potentially relevant to HFS pathogenesis. Furthermore, we showed that the Src-like kinase family are involved in the release of Talin upon ligand binding and that CMG2 binding to its extracellular ligand is critical for the correct orientation of the mitotic spindle of zebrafish epiblast cells, which allows cell division along the Animal/Vegetal axis. Through the analysis of cmg2 knock-out mice, along with the study of HFS patient samples, we aimed at better defining CMG2 function. We showed that type VI Collagen was strongly accumulating in the cmg2 KO mice uterus tissue leading to failure of parturition, an accumulation that was also observed in HFS patient nodules. In addition, we observed that CMG2 was able to interact and potentiate MT1-MMP, and that type VI Collagen is a bona fide ligand for CMG2, its binding leading to the phosphorylation of the receptor. With the scope to understand the HFS patient subcutaneous nodules pathogenesis, we investigated the gene expression profile of fibroblasts derived from non-affected and affected tissues by RNA sequencing. Fibroblasts derived from affected tissues appeared to differentially express genes involved in the TGFBeta pathway and cytoskeleton, such as alpha smooth muscle actin, a marker of myofibroblast activation. We observed the presence of myofibroblasts in the patient nodules, and showed that CMG2 was limiting their activation process, loss-of-function mutations in CMG2 leading to an exacerbated differentiation. As we observed CMG2 interaction with the TGFBeta receptor 2, we postulated that CMG2 influence the TGFBeta pathway, a process that is altered in HFS patient and potentially lead to the formation of nodules.

EPFL2015

Investigation of the neurokinin-1 receptor by fluorescence techniques

Bruno Meyer

G protein-coupled receptors (GPCR) constitute by far the largest family of transmembrane cell-surface proteins involved in signal transduction and are the most important targets for the development of novel therapeutic compounds. Many processes mediated by GPCR signal transduction are still not well understood at the molecular level and novel methods for their investigation are of general interest. The thesis presented here describes investigations on the neurokinin-1 receptor (NK1R), a member of the GPCR family, using fluorescence techniques. Fluorescence resonance energy transfer (FRET), the technique of preference in this work, is used to measure ligand-protein and protein-protein interactions in living cells. In order to reach this goal, the NK1R has been labelled with either fluorescent proteins or by using the novel post-translational ACP labelling technique. A FRET-based method for monitoring specific ligand binding to NK1R is described. In this context, a fluorescent agonist for NK1R was synthesized and characterized. The ACP labelling technique was shown to be superior for this kind of experiments compared to labelling with fluorescent proteins. The same ACP labelling approach was shown to be ideally suited to study oligomerization of membrane proteins by FRET. It was shown, that the NK1R does not form homo-dimers, as many other GPCRs do. From these experiments we furthermore conclude, that the NK1R is present in small microdomains in the membrane. Interactions between the NK1R and the different subunits of the heterotrimeric Gαqβ1γ2-complex were investigated by FRET using different instrumentation. Proximity of the NK1R and the G protein could be detected, but no change in FRET was observed after activation of the receptor. Total internal reflection fluorescence (TIRF), was used to develop an assay for studying ligand binding in vitro. Here, the NK1R was immobilized directly on a surface using a cell membrane preparation. This method was shown to be highly sensitive.

EPFL2005

Unraveling the Physiological Role of Capillary Morphogenesis Gene 2 in Health and Disease

Jérôme Bürgi

EPFL2015

Investigation of the neurokinin-1 receptor by fluorescence techniques

Bruno Meyer

EPFL2005