Preliminary Study On The Subcellular Distribution Of PNPLA7 Protein

Nonalcoholic Fatty Liver Disease(NAFLD)refers to excess fat accumulation in the livers of patients with no history of alcohol abuse or other causes of secondary hepatic steatosis.NAFLD represents a complex spectrum of hepatic damage ranging from simple steatosis and Nonalcoholic Steatohepatitis(NASH),progressing to fibrosis and ultimately cirrhosis.NAFLD is the most common cause of chronic liver dysfunction and is one of the most common public health problems worldwide.Recent epidemiology studies suggest that NAFLD is present in 12-38%of the general population and NASH affects 3-15%.In China,NAFLD is becoming a greater health concern,with increasing rates of metabolic disturbances,such as obesity,type 2 diabetes mellitus,and dyslipidemia.The prevalence of NAFLD in patients with type 2 diabetes is 28-55%and in those with hyperlipidemia is 27%-92%.NAFLD is regarded as a hepatic manifestation of metabolic syndrome and is strongly associated with obesity and insulin resistance.The PNPLA family protein contains a "patatin-like phospholipase domain"consisting of nine members.Patatin-like phospholipid domain-containing protein 3(PNPLA3)is an ER-and LD-associated protein.It is one of the few examples of a protein that has been validated in several populations to be conclusively shown to be associated with NAFLD,particularly the I148M(rs738409 C/G)variant.As a member of the PNPLA family,PNPLA3 is most closely related to PNPLA2(adipose triglyceride lipase,ATGL),the major cellular TAG lipase.PNPLA7 is one of the member of PNPLA family protein has been shown that has lysophosphatase activity and can hydrolyzes LPA(lysobisphosphatidic acides,LPA)and LPC(lysophosphatidylcholine,LPC)in vitro,however the function of PNPLA7 is still unknown in vivo.The sub-cellular localization of protein is closely associated with the function of protein.The aim of this study is to elucidate the subcellular localization of PNPLA7 by using biochemical fractionaltion and immunofluoresence approchs.According to the result of bioinfromatics analsysis which predicted that PNPLA7 is an ER membrane protein.Based on the prediction,we generated different trunctated constructs of PNPLA7 to explore its subcellular localization.After detailed immunofluorescence analysis,we found that PNPLA7 protein is located on the ER·The aa35-60 residues of PNPLA7 are critical transmembrane region to determin the subcellular localization of PNPLA7 on ER.Meanwhile,we also found that PNPLA7 has the characteristics of encapsulated mitochondria.It is consistant with the result of biochemical fractionation analysis which showed that PNPAL7 was also distributed on the mitochondrial-associated membrane and the ER.Overall,PNPLA7 is a single transmembrane protein on the ER.Furthermore,PNPLA7 is also distributed on mitochondrial-associated membranes and encapsulated mitochondria.The aa35-60 residues ofPNPLA7 not only affect the localization of PNPLA7 on the ER but also influence encapsulated mitochondria characteristics of PNPLA7.ER and mitochondria are important intracellular organelles of energy metabolism.The subcellualr localization of PNPLA7 protein on the ER and mitochondira associated membrane indicated that the PNPLA7 protein may play a role in energy metabolism and correlated with organelle communication between ER and mitochondria.