The Processing of Human Rhomboid Intramembrane Serine Protease RHBDL2 Is Required for Its Proteolytic Activity

Cornell University, Итак, New York, United States
Journal of Molecular Biology (Impact Factor: 4.33). 10/2009; 394(5):815-25. DOI: 10.1016/j.jmb.2009.10.025
Source: PubMed


RHBDL2, a human homolog of the rhomboids, belongs to a unique class of serine intramembrane proteases; little is known about its function and regulation. Here, we show that RHBDL2 is produced as a proenzyme and that the processing of RHBDL2 is required for its cellular protease activity. The processing of RHBDL2 was shown by both Western blot and immunofluorescence analysis. We have demonstrated that a highly conserved Arg residue on loop 1 of RHBDL2 plays a critical role in the activation of the proenzyme. The activation of RHBDL2 is catalyzed by a protease that is sensitive to a class of sulfonamide compounds. Furthermore, endogenous RHBDL2 exists as the processed form and treatment of cells with a sulfonamide inhibitor led to an accumulation of the full length of RHBDL2. Therefore, this study has demonstrated that RHBDL2 activity is regulated by proenzyme activation, revealed a role for the conserved WR residues in loop 1 in RHBDL2 activity, and provided critical insights into the regulation and function of this human rhomboid protease.

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    • "To investigate whether homeless-induced RHBDL2 upregulation is critical in anoikis resistance, a serine protease inhibitor for RHBDL2 (DCI) [19] and specific shRNA for RHBDL2 were used. For HeLa S3 cells cultured in the suspension condition and treated with various doses of DCI for 48 hours, the protein levels of cleaved caspase 3 were upregulated in a dose-dependent pattern (Figure 3(a)). "
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    ABSTRACT: Anoikis resistance allows metastatic tumor cells to survive in a homeless environment. Activation of epithelial growth factor receptor (EGFR) signaling is one of the key mechanisms for metastatic tumor cells to resist anoikis, yet the regulation mechanisms of homeless-triggered EGFR activation in metastatic tumor cells remain unclear. Rhomboid-like-2 (RHBDL2), an evolutionally conserved intramembrane serine protease, can cleave the EGF ligand and thus trigger EGFR activation. Herein, we demonstrated that RHBDL2 overexpression in human epithelial cells resulted in promotion of cell proliferation, reduction of cell adhesion, and suppression of anoikis. During long-term suspension cultures, increased RHBDL2 was only detected in aggressive tumor cell lines. Treatment with the rhomboid protease inhibitor or RHBDL2 shRNA increased cleaved caspase 3, a marker of apoptosis. Finally, inhibition of EGFR activation increased the cleaved caspase 3 and attenuated the detachment-induced focal adhesion kinase phosphorylation. Taken together, these findings provide evidence for the first time that RHBDL2 is a critical molecule in anoikis resistance of malignant epithelial cells, possibly through the EGFR-mediated signaling. Our study demonstrates RHBDL2 as a new therapeutic target for cancer metastasis.
    Full-text · Article · May 2014
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    • "Bacterial rhomboids are known to be active in their full-length form [20À23] and do not require any proteolytic activation. Although there is some evidence that mammalian rhomboids might undergo proteolytic processing [42], it is not yet clear how significant that observation is for catalysis. An important and established factor governing Rhomboid-1 mediated proteolysis in vivo is regulated trafficking of the substrate [15] [16], as introduced in section Reaction and specificity. "

    Full-text · Article · Dec 2013
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    • "Since these experiments were performed in vitro, it remains to be seen if this phenomenon has any role in a physiological context. The human rhomboid RHBDL2 is expressed as an inactive proenzyme and its activity is regulated by proteolysis [24], but it is unclear if other members of the family are regulated in this way. The cytosolic regions of the proteases may also play a role in regulating rhomboid activity. "
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    ABSTRACT: The rhomboid-like proteins constitute a large family of intramembrane serine proteases that are present in all branches of life. First studied in Drosophila, these enzymes catalyse the release of the active forms of proteins from the membrane and hence trigger signalling events. In protozoan parasites, a limited number of rhomboid-like proteases have been investigated and some of them are associated to pathogenesis. In Apicomplexans, rhomboid-like protease activity is involved in shedding adhesins from the surface of the zoites during motility and host cell entry. Recently, a Toxoplasma gondii rhomboid was also implicated in an intracellular signalling mechanism leading to parasite proliferation. In Entamoeba histolytica, the capacity to adhere to host cells and to phagocytose cells is potentiated by a rhomboid-like protease. Survey of a small number of protozoan parasite genomes has uncovered species-specific rhomboid-like protease genes, many of which are predicted to encode inactive enzymes. Functional investigation of the rhomboid-like proteases in other protozoan parasites will likely uncover novel and unexpected implications for this family of proteases.
    Full-text · Article · Dec 2011 · Molecular and Biochemical Parasitology
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