Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis

Johnson, Nicholas; Březinová, Jana; Stephens, Elaine; Burbridge, Emma; Freeman, Matthew; Adrain, Colin; Střı́šovský, Kvido

doi:10.1038/s41598-017-07556-3

Cited by 47 publications

(38 citation statements)

References 39 publications

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“…A number of native and model rhomboid substrates are single‐pass membrane proteins (Urban et al , ; Strisovsky et al , ; Riestra et al , ; Johnson et al , ; Saita et al , ). The cleavage of the transmembrane core of MgtE by YqgP described here adds to growing evidence that rhomboid proteases do engage in cleaving polytopic membrane proteins under some conditions (Erez & Bibi, ), although the features of transmembrane domains that they recognise still need to be properly structurally understood.…”

Section: Discussionmentioning

confidence: 99%

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

et al. 2020

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Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major highaffinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn 2+ /Zn 2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn 2+ and Zn 2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of "ERAD" in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins.

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Section: Discussionmentioning

confidence: 99%

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

et al. 2020

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“…RHBDL2 also cleaves EGF, but likely only to modulate its signaling, a process that may nevertheless be deregulated in certain cancer cells (Adrain et al , ). More recently, substrate proteomics identified several additional proteins to be cleaved by RHBDL2 (Table ) (Johnson et al , ). RHBDL4 has been linked to the ER‐associated degradation (ERAD) pathway (Fleig et al , ) and has been suggested to act as a non‐canonical sheddase of APP (Paschkowsky et al , ).…”

Section: Hardware: Non‐canonical Sheddasesmentioning

confidence: 99%

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments

2018

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Proteolytic removal of membrane protein ectodomains (ectodomain shedding) is a post-translational modification that controls levels and function of hundreds of membrane proteins. The contributing proteases, referred to as sheddases, act as important molecular switches in processes ranging from signaling to cell adhesion. When deregulated, ectodomain shedding is linked to pathologies such as inflammation and Alzheimer's disease. While proteases of the "a disintegrin and metalloprotease" (ADAM) and "beta-site APP cleaving enzyme" (BACE) families are widely considered as sheddases, in recent years a much broader range of proteases, including intramembrane and soluble proteases, were shown to catalyze similar cleavage reactions. This review demonstrates that shedding is a fundamental process in cell biology and discusses the current understanding of sheddases and their substrates, molecular mechanisms and cellular localizations, as well as physiological functions of protein ectodomain shedding. Moreover, we provide an operational definition of shedding and highlight recent conceptual advances in the field. While new developments in proteomics facilitate substrate discovery, we expect that shedding is not a rare exception, but rather the rule for many membrane proteins, and that many more interesting shedding functions await discovery.

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“…300493, Cell Lines Service, Germany) were cultured in DMEM medium (Invitrogen). To deplete endogenous RHBDL2, HaCaT cells were transduced with a recombinant lentivirus expressing the shRNA #01 targeting RHBDL2 (16) as described (15) and selected for puromycin resistance.…”

Section: Cell Culturementioning

confidence: 99%

“…All superfamily members share the basic transmembrane fold, and it is thus important to establish whether regulatory dimerization is a common property of the superfamily or whether it is peculiar only to some subgroups. Here we zoom in on the well-characterized human rhomboid protease RHBDL2 (15)(16)(17)(18)(19)(20), which is localized to the plasma membrane and consists of seven transmembrane helices, and analyze its oligomeric status in living cells.…”

Section: Introductionmentioning

confidence: 99%

Dimerization of rhomboid protease RHBDL2 in lipid membranes addressed by FRET with MC simulations

Škerle

Humpolíčková

Rampírová

et al. 2018

Preprint

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Many membrane proteins are thought to function as oligomers, but measuring membrane protein dimerization in native lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are non-invasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. The effects inherent to such two-dimensional systems, such as excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and FCS we introduce methods to measure surface protein density and to estimate kappa squared, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined 2D environments. We then use FRET and FCS to analyze the dimerization of human rhomboid protease RHBDL2 in its native lipid membranes.While previous reports have proposed that rhomboid proteases dimerize and this allosterically activates them, we find no evidence for stable oligomers of RHBDL2 in lipid membranes of human cells. This indicates that the rhomboid transmembrane core may be intrinsically monomeric.Finally, our findings will find use in the application of FRET and FCS for the analysis of oligomerization of transmembrane proteins in lipid membranes.

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Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis

Cited by 47 publications

References 39 publications

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments

Dimerization of rhomboid protease RHBDL2 in lipid membranes addressed by FRET with MC simulations

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