Putative Protein Interactome of the Rhomboid Protease RHBDL4

Abstract

The physiological functions of the rhomboid-related protein 4 (RHBDL4) are emerging, but their molecular details remain unclear. Because increased expression of RHBDL4 has been clinically linked to poorer outcomes in cancer patients, this association urgently demands a better understanding of RHBDL4. To elucidate the molecular interactions and pathways that RHBDL4 may be involved in, we conducted proximity-dependent biotin identification (BioID) assays. Our analyses corroborated several of the expected protein interactors such as the transitional endoplasmic reticulum (ER) ATPase VCP/p97 (TERA), but they also described novel putative interactors including IRS4, PGAM5, and GORS2. Using proximity-ligation assays, we validated VCP/ p97, COPB, and VRK2 as proteins that are in proximity to RHBDL4. Overall, our results support the emerging functions of RHBDL4 in ER quality control and also point toward putative RHBDL4 functions in protein membrane insertion and membrane organization and trafficking.

Eta-secretase-like processing of the amyloid precursor protein (APP) by RHBDL4

“…Likewise, RHBDL4 was suggested to organize GPCR-mediated trafficking events like for the signaling molecule TGFα (Wunderle et al , 2016). Using BioID proximity assays, it was indeed shown that RHBDL4 interacts with dozens of other proteins likely through interactions not related to proteolysis further supporting non-catalytic functions of RHBDL4 (Hsiao et al , 2023; Ikeda & Freeman, 2019). Remarkably, we found an increased catalytic activity of RHBDL4 at low cellular cholesterol levels (Paschkowsky et al , 2018), while β- and γ-secretases show an increased activity at high cellular cholesterol levels (Holmes et al , 2012; Kalvodova et al , 2005; Marquer et al , 2011; Osenkowski et al , 2008).…”

“…A remarkable feature of APP is its processing by a myriad of proteases, besides α-, βand γ-secretases, multiple others such as MT5-MMP, RHBDL4, HtrA2, meprin-β, and possibly MT3-MMP. However, it remains elusive which pathways are active in which cell types at what stage and how they may be regulated (Ahmad et al, 2006;Bien et al, 2012;Huttunen et al, 2007;Park et al, 2006;Zhang et al, 2015). The relevance of Aη peptides in Alzheimer's disease is also unclear.…”

Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer’s disease mouse model

“…On the one hand, RHDBL4 could directly bind to ubiquitinated β-catenin via its UIM and target it for proteasomal degradation. As RHBDL4 is in part located in the peri-nuclear areas of the ER (Lastun et al, 2022) and our BioD data show that RHBDL4 interacts with nuclear pore complex components like nucleoporin NDC1 and integral proteins of the inner nuclear membrane such as MAN1 (Hsiao et al, 2023), it is possible that RHBDL4 encounters β-catenin while it translocates to the nucleus. Our BioID data also revealed α-catenin as a putative interactor of RHBDL4 (Hsiao et al, 2023).…”

“…As RHBDL4 is in part located in the peri-nuclear areas of the ER (Lastun et al, 2022) and our BioD data show that RHBDL4 interacts with nuclear pore complex components like nucleoporin NDC1 and integral proteins of the inner nuclear membrane such as MAN1 (Hsiao et al, 2023), it is possible that RHBDL4 encounters β-catenin while it translocates to the nucleus. Our BioID data also revealed α-catenin as a putative interactor of RHBDL4 (Hsiao et al, 2023). Interestingly, α-catenin is a known negative regulator of the Wnt/β-catenin signalling pathway via direct cytosolic sequestering of β-catenin or interaction with adenomatous polyposis coli (APC), a member of the destruction complex responsible for β-catenin degradation.…”

“…More recently, it was shown that RHBDL4 also targets aggregated proteins that do not have a transmembrane domain for degradation through the proteasome via interactions with partners, Erlin1/2, that bring aggregated targets directly to RHBDL4 (Bock et al, 2022). The diversity of RHBDL4’s substrates and interactors suggests it plays a major role in maintaining ER homeostasis (Hsiao et al, 2023; Lastun et al, 2022; Tang et al, 2022). In the context of AD, the UPR is upregulated due to ER stress induced by oxidative stress, metabolic restrictions, and misfolded protein accumulation (Ajoolabady et al, 2022; Hoozemans et al, 2012).…”