Abstract
We have previously reported that rhomboid domain containing 1 (RHBDD1), a mammalian rhomboid protease highly expressed in the testis, can cleave the Bcl-2 protein Bik. In this study, we identified a multi-pass transmembrane protein, tumor suppressor activated pathway-6 (TSAP6) as a potential substrate of RHBDD1. RHBDD1 was found to induce the proteolysis of TSAP6 in a dose- and activity-dependent manner. The cleavage of TSAP6 was not restricted to its glycosylated form and occurred in three different regions. In addition, mass spectrometry and mutagenesis analyses both indicated that the major cleavage site laid in the C-terminal of the third transmembrane domain of TSAP6. A somatic cell knock-in approach was used to genetically inactivate the endogenous RHBDD1 in HCT116 and RKO colon cancer cells. Exosome secretion was significantly elevated when RHBDD1 was inactivated in the two cells lines. The increased exosome secretion was verfied through the detection of certain exosomal components, including Tsg101, Tf-R, FasL and Trail. In addition, the elevation of exosome secretion by RHBDD1 inactivation was reduced when TSAP6 was knocked down, indicating that the role of RHBDD1 in regulating exosomal trafficking is very likely to be TSAP6-dependent. We found that the increase in FasL and Trail increased exosome-induced apoptosis in Jurkat cells. Taken together, our findings suggest that RHBDD1 is involved in the regulation of a nonclassical exosomal secretion pathway through the restriction of TSAP6.
Highlights
Over the past few decades, studies have found that regulated intramembrane proteolysis (RIP) plays an important role in various kinds of cellular processes, including cell signaling, gene transcription and apoptosis [1,2]
The N-terminal region (1–214 aa, mainly composed of the 59–214 aa rhomboid domain) of rhomboid domain containing 1 (RHBDD1) was sufficient for the proteolysis of tumor suppressor activated pathway-6 (TSAP6), but its activity was much weaker than full-length RHBDD1 (Fig. 1D)
In order to determine whether elevated exosome secretion caused by RHBDD1 inactivation was reproducible in other types of cells, we introduced the same mutations into endogenous RHBDD1 in RKO colon cancer cells
Summary
Over the past few decades, studies have found that regulated intramembrane proteolysis (RIP) plays an important role in various kinds of cellular processes, including cell signaling, gene transcription and apoptosis [1,2]. Regulated intramembrane proteolysis is a process through which proteases cleave substrates within their transmembrane regions. The mitochondrial rhomboid protease PARL has been found to regulate mitochondrial membrane remodeling and apoptosis [6,7]. Few substrates have been identified for the rhomboid proteases, and the identification of these substrates will play a key role in understanding the function of this intriguing family of intramembrane proteases. Only single transmembrane proteins have been identified as substrates of rhomboid proteases [9,10,11]. Because so many transmembrane proteins are multi-pass membrane proteins, there is a question regarding whether any multi-pass transmembrane proteins are substrates for rhomboid proteases
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