Aberrant methylation and silencing of the SPINT2 gene in high-grade gliomas.

Hepatocyte growth factor activator inhibitor type 2 (HAI-2), encoded by the SPINT2 gene, is a membrane-anchored protein that inhibits proteases involved in the activation of hepatocyte growth factor (HGF), a ligand of MET receptor. Epigenetic silencing of the SPINT2 gene has been reported in a human glioblastoma cell line (U87) and glioblastoma-derived cancer stem cells. However, the incidence of SPINT2 methylation in tumor tissues obtained from glioma patients is unknown. In this study, we analyzed the methylation status of the SPINT2 gene of eight human glioblastoma cell lines and surgically resected glioma tissues of different grades (II, III, and IV) by bisulfite sequence analysis and methylation-specific PCR. Most glioblastoma lines (7/8) showed methylation of the SPINT2 gene with a significantly reduced level of SPINT2mRNA compared to cultured astrocytes and normal brain tissues. However, all glioblastoma lines expressed mRNA for HGF activator (HGFAC), a target protease of HAI-2/SPINT2. Forced expression of SPINT2 reduced MET phosphorylation of U87 glioblastoma cells both in vitro and in intracranial xenografts in nude mice. Methylation-specific PCR analysis of the resected glioma tissues indicated notable methylation of the SPINT2 gene in 33.3% (2/6), 71.4% (10/14), and 74.3% (26/35) of grade II, III, and IV gliomas, respectively. Analysis of RNA sequencing data in a public database indicated an increased HGFAC/SPINT2 expression ratio in high-grade compared to low-grade gliomas (P = .01). In summary, aberrant methylation of the SPINT2 gene is frequently observed in high-grade gliomas and might confer MET signaling in the glioma cells.

Role of the polycystic kidney disease domain in matriptase chaperone activity and localization of hepatocyte growth factor activator inhibitor-1.

Hepatocyte growth factor activator inhibitor-1 (HAI-1, also known as SPINT1) is an inhibitor of matriptase, a type-2 transmembrane protease widely expressed in epithelial cells. HAI-1 also functions as a chaperone to maintain the processing and localization of matriptase required for epithelial integrity. However, mechanisms underpinning the chaperone function remain to be elucidated. Here, we show that the first Kunitz domain (KD1) and the adjacent polycystic kidney disease (PKD) domain-like internal domain of HAI-1 are essential for the chaperone function. In HEK293T cells, which do not express endogenous HAI-1 or matriptase, forced matriptase overexpression was unsuccessful unless sufficient HAI-1 was co-expressed. Among mutant HAI-1 constructs, HAI-1 with inactivation mutation in KD1 (HAI-1mKD1) or HAI-1 lacking the PKD domain (HAI-1dPKD) was unable to support matriptase expression, and neither mutant formed a complex with activated matriptase. Matriptase did not localize to the cell surface when co-expressed with HAI-1dPKD. Moreover, HAI-1dPKD accumulated in the cytoplasm of HEK293T and HaCaT cells rather than localizing to the cell surface, presumably due to misfolding as judged by altered antibody recognition. On the other hand, activationlocked and activity-incompetent matriptase were stable and readily overexpressed and localized to the cell surface without HAI-1. Therefore, the observed matriptase instability was caused by its own catalytic activity in the absence of inhibitory HAI-1. The matriptase chaperone function of HAI-1 is thus mediated primarily by the inhibition of undesired intracellular matriptase activity, and the PKD domain is essential for the proper folding and trafficking of inhibitory HAI-1 and its chaperone function.

Defective Mitochondrial tRNA Taurine Modification Activates Global Proteostress and Leads to Mitochondrial Disease.

A subset of mitochondrial tRNAs (mt-tRNAs) contains taurine-derived modifications at 34U of the anticodon. Loss of taurine modification has been linked to the development of mitochondrial diseases, but the molecular mechanism is still unclear. Here, we showed that taurine modification is catalyzed by mitochondrial optimization 1 (Mto1) in mammals. Mto1 deficiency severely impaired mitochondrial translation and respiratory activity. Moreover, Mto1-deficient cells exhibited abnormal mitochondrial morphology owing to aberrant trafficking of nuclear DNA-encoded mitochondrial proteins, including Opa1. The mistargeted proteins were aggregated and misfolded in the cytoplasm, which induced cytotoxic unfolded protein response. Importantly, application of chemical chaperones successfully suppressed cytotoxicity by reducing protein misfolding and increasing functional mitochondrial proteins in Mto1-deficient cells and mice. Thus, our results demonstrate the essential role of taurine modification in mitochondrial translation and reveal an intrinsic protein homeostasis network between the mitochondria and cytosol, which has therapeutic potential for mitochondrial diseases.