Characterising the rhomboid-like protein TMEM115

Thesis

The rhomboid-like superfamily of proteins comprises transmembrane proteins with an ancient evolutionary origin. They include both intramembrane serine proteases and inactive pseudoproteases, and they have diverse cellular and pathophysiological functions. These include regulation of growth factor signalling, protein quality control, trafficking, and mitochondrial dynamics. TMEM115, a recently recognised enzymatically inactive member of the rhomboid-like superfamily (confirmed in this thesis), is conserved from yeast to human and is ubiquitously expressed in all tissues. The absence of TMEM115 in both Drosophila and mice causes severe phenotypes. These compelling preliminary data indicate that TMEM115 has important cellular functions. To capitalise on these preliminary observations, the overall aim of my PhD project was to characterise the mammalian TMEM115 both structurally and functionally.

Structurally, I performed a topology study for TMEM115, and with a combination of bioinformatic and experimental analysis, proved that TMEM115 indeed has a six TMD structure. Using the HHpred and Phyre algorithms, which identify structural similarity among proteins, a high degree of homology was identified between the TMD regions of TMEM115 and other rhomboid-like proteins. The above analysis together with the topology of TMEM115 definitively positions TMEM115 in the rhomboid-like superfamily.

To elucidate the biological role of TMEM115, I started with a proteomic approach, a BioID proximity screen, to identify novel interactors for TMEM115 under different physiologically relevant conditions. A number of binding partners were identified in the BioID screen and validated with co-immunoprecipitation. These indicate intriguing possible functions of TMEM115, including regulating lipid biology, protein trafficking, protein degradation and ion channels. Given that several candidates are involved in protein trafficking, I used a secretome profile analysis method, the SPECS, for identifying TMEM115 dependent secreted proteins.

A role of TMEM115 in regulating the fundamental de novo lipogenesis pathway, the SCAP-SREBP pathway (Freeman lab unpublished data), had been identified during the course of my project. Pursuing this, I investigated the possible relationship between TMEM115, SCAP and the novel interactor, p62; the results suggest that TMEM115 may be involved in regulating lipid homeostasis by modulating SCAP levels through the proteasomal degradation machinery.

Authors: Shen, X

• DOI: 10.5287/ora-n145yojzq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford