The role of Pex16 and TMEM135 in peroxisome abundance

Abstract

Peroxisomes are essential in cellular lipid metabolism and detoxification, and the mechanisms of regulating their abundance and function have attracted increasing interest. Pex16 is a well-established factor involved in peroxisome biogenesis, while TMEM135 is a peroxisomal membrane protein with emerging regulatory functions. In this study, I investigated the molecular mechanisms underline both peroxisome biogenesis and degradation. In the first part, depletion of PEX16 in human retinal pigment epithelial-1 cells led to a decrease of peroxisome abundance and function. Evidence for activated pexophagy upon PEX16 knockdown was supported by two key observations (i) prevention of peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cells, and (ii) increased autophagy flux and co-localization of p62 with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. And second part, I investigated how TMEM135 regulates peroxisome abundance through depleting and overexpressing TMEM135 expression level. In TMEM135 knockout mice, peroxisome number and function were increased, accompanied by the upregulated peroxisome biogenesis genes. In contrast, overexpression of TMEM135 in AML12 cells caused a decrease of peroxisome number through the enhanced pexophagy mediated by p62 and NBR1. Interestingly, peroxisome loss results from torin1, a known pexophagy inducer, was attenuated in TMEM135 depleted cells while TMEM135 depletion itself did not affect pexophagy. Taken together, these data complement the roles of PEX16 and TMEM135 in maintaining peroxisome integrity. PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes. TMEM135 functions as a key regulator in balancing peroxisome biogenesis and degradation depending on its expression level, which may serve as a potential target for therapeutic intervention in peroxisome-associated disorders. ABBRIVIATIONS AAA complex – AAA-ATPase complex ABCD3 – ATP Binding Cassette Subfamily D Member 3 ATG5 – Autophagy-deficient autophagy-related gene 5 DAPI – 4,6-diamidino-2-phenylindole DCA – Dicarboxylic acids DHA – Docosahexaenoic acid DMEM – Dulbecco’s modified Eagle medium DPA – Docosapentaenoic acid EHHADH – Peroxisomal L-bifunctional enzyme EPA – Eicosapentaenoic acid ER – Endoplasmic reticulum FBS – Fetal Bovine Serum GC-MS – Gas Chromatography Mass Spectrometry GNPAT – Glyceronephosphate O-acyltransferase KO – Knockout MARCH5 – Membrane-associated ring-CH-type finger 5 MEF – Mouse embryonic fibroblasts NBR1 – Neighbor of BRCA1 gene 1 PEX – Peroxin PMPs – Peroxisomal membrane proteins PUFA – Polyunsaturated fatty acid ROS – Reactive oxygen species RPE – Retinal pigment epithelial siRNA – small interfering RNA TMEM135 – Transmembrane protein 135 USP30 – Ubiquitin specific peptidase 30 VLCFAs – Very-long-chain fatty acids WT – Wild-type