The Role And Mechanism Of Lysosomal Protein Transmembrane 5 (LAPTM5) In Lipid Metabolism

Background Non-alcoholic steatohepatitis has received great attention due to its extremely high incidence.As a leading cause culpable for the progression of cirrhosis and hepatocellular carcinoma,NASH is estimated to affect approximately hundreds of millions of the world adult population according to recent reports.Unfortunately,effective therapeutic measures to protect against the development and progression of NASH remain limited,and so far no FDA approved pharmacological therapy has been available.Lysosomal Protein Transmembrane family is reported to regulate cellular homeostasis and metabolism,and LAPTM5 was screened associated with the NASH progression through extensive bioinformatical analysis.However,the role and underlying regulatory mechanism of LAPTM5 in NASH progression are unclear and require further research.ObjectiveTo explore the biological function of LAPTM5 in the progression of NASH,and investigate the molecular mechanism of LAPTM5 in regulating NASH,so as to provide a new theoretical basis and research target for the treatment of NASH in clinical practice.Methods1.Establishing the liver sample bank of NASH and detecting the expression changes of LAPTM5 in liver tissues.Collecting the liver samples from patients with NASH and non-NASH.At the same time,to consturct the mouse NASH models and collect the liver samples.Then detecting the protein and m RNA expression changes of LAPTM5.2.Combining in vitro cell experiments and in vivo experiments in mice to investigate the biological function of LAPTM5 in NASH.Constructing liver-specific Laptm5 gene knockout and overexpression mice.Isolating and culturing the primary hepatocytes of mice in vitro under the stimulation of PAOA.And the mouse NASH models were established by high-fat high-cholesterol diet.Lipid deposition and inflammatory responses in cells and liver are then measured.Finally,RNA-seq sequencing was performed on cells and livers,and gene expression and pathway enrichment were analyzed to comprehensively evaluate the biological function of LAPTM5 in NASH.3.Screening the molecular pathway and downstream target of LAPTM5 in regulating NASH progression.The RNA-seq results of primary hepatocytes and mouse liver tissues were combined to screen for the significantly altered signaling pathway after Laptm5-KO.Combined with mass spectrometry and molecular biological experiments to screen the downstream target of LAPTM5 in regulating NASH,and verifying the expression changes of relevant signaling pathway.Results1.The hepatic LAPTM5 protein levels were found to be significantly downregulated under the stimulation of lipid,and the protein levels of LAPTM5 were negatively correlated with the NAS score.2.After the knockout of Laptm5,the lipid deposition,inflammatory response in hepatocytes and interstitial fibrosis were significanty aggravated.However,these symptoms were significanty ameliorated after the overexpression of Laptm5.Which means LAPTM5 plays a protective role in NASH.3.Laptm5 knockout most significantly altered the MAPK signaling pathway.And mass spectrometry experiments showed that CDC42 was a downstream regulatory molecule of LAPTM5.4.CDC42 mediated the effect of LAPTM5 on lipid deposition and inflammation in hepatocytes.And LAPTM5 suppressed activation of MAPK signaling pathway by promoting the lysosomal degradation of CDC42.5.Adenovirus-mediated hepatic Laptm5 over-expression was an efficacious treatment for NASHConclusionsLAPTM5 plays an important protective effect in NASH progression.Hepatocyte-specific depletion of Laptm5 exacerbated hepatic steatosis,inflammation and fibrosis in mouse NASH models.In contrast,Laptm5 overexpression in hepatocytes exerted diametrically opposite effects.Mechanistically,LAPTM5 interacted with CDC42 and promoted its degradation through a lysosome-dependent manner,thus inhibiting activation of the mitogen-activated protein kinase signaling pathway.Finally,adenovirus-mediated hepatic Laptm5 overexpression ameliorated aforementioned symptoms in NASH models.In this study,we proved that LAPTM5 was an efficacious treatment for NASH and could potentially serve as a biological marker indicative of NASH progression.