RESUMEN
Niemann-Pick type C disease (NP-C) is a progressive lysosomal lipid storage disease caused by mutations in the NPC1 and NPC2 genes. NPC1 is essential for transporting cholesterol and other lipids out of lysosomes, but little is known about the mechanisms that control its cellular abundance and localization. Here we show that a reduction of TMEM97, a cholesterol-responsive NPC1-binding protein, increases NPC1 levels in cells through a post-transcriptional mechanism. Reducing TMEM97 through RNA-interference reduces lysosomal lipid storage and restores cholesterol trafficking to the endoplasmic reticulum in cell models of NP-C. In TMEM97 knockdown cells, NPC1 levels can be reinstated with wild type TMEM97, but not TMEM97 missing an ER-retention signal suggesting that TMEM97 contributes to controlling the availability of NPC1 to the cell. Importantly, knockdown of TMEM97 also increases levels of residual NPC1 in NPC1-mutant patient fibroblasts and reduces cholesterol storage in an NPC1-dependent manner. Our findings propose TMEM97 inhibition as a novel strategy to increase residual NPC1 levels in cells and a potential therapeutic target for NP-C.
Asunto(s)
Proteínas Portadoras/genética , Colesterol/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Enfermedad de Niemann-Pick Tipo C/genética , Animales , Células CHO , Proteínas Portadoras/biosíntesis , Colesterol/metabolismo , Cricetulus , Retículo Endoplásmico/genética , Fibroblastos/metabolismo , Fibroblastos/fisiología , Técnicas de Silenciamiento del Gen , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/metabolismo , Lisosomas/patología , Glicoproteínas de Membrana/biosíntesis , Mutación , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Proteínas de Transporte VesicularRESUMEN
Elevated plasma cholesterol levels are considered responsible for excess cardiovascular morbidity and mortality. Cholesterol in plasma is tightly controlled by cholesterol within cells. Here, we developed and applied an integrative functional genomics strategy that allows systematic identification of regulators of cellular cholesterol levels. Candidate genes were identified by genome-wide gene-expression profiling of sterol-depleted cells and systematic literature queries. The role of these genes in cholesterol regulation was then tested by targeted siRNA knockdown experiments quantifying cellular cholesterol levels and the efficiency of low-density lipoprotein (LDL) uptake. With this strategy, 20 genes were identified as functional regulators of cellular cholesterol homeostasis. Of these, we describe TMEM97 as SREBP target gene that under sterol-depleted conditions localizes to endo-/lysosomal compartments and binds to LDL cholesterol transport-regulating protein Niemann-Pick C1 (NPC1). Taken together, TMEM97 and other factors described here are promising to yield further insights into how cells control cholesterol levels.