title: Exosome-related multi-pass transmembrane protein TSAP6 is a target of rhomboid protease RHBDD1-induced proteolysis.
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.
authors.affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, China.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Wan
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Shiying
authors.initials: S
authors.lastname: Miao
authors.forename: Wei
authors.initials: W
authors.lastname: Song
authors.forename: Linfang
authors.initials: L
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D055354:Exosomes / Q000378:metabolism*; D005434:Flow Cytometry; D055879:Gene Knock-In Techniques; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019169:Jurkat Cells; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases; D059748:Proteolysis; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D015513:Oncogene Proteins; D010088:Oxidoreductases; C486677:STEAP3 protein, human; C534711:RHBDD1 protein, human; D012697:Serine Endopeptidases