Page 19 - N. Metabolism and metabolic diseases

USP15 promotes non-alcoholic fatty liver disease by regulation of lipid droplet-associated proteins

Jung-Hwan Baek , Myung-Sup Kim 1, 2 and Kyung-Hee Chun 1, 2, *
1, 2
1 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, Republic of Korea
2 Brain Korea 21 PLUS Project for Medical Science, Yonsei University
BACKGROUND AIM

As an important metabolic organ in the human, the live plays a major role in the regulation of lipid metabolism and The ubiquitin specific peptidases (USPs) consists of 54 members (USP1-USP54) that can fuction as
glucose metabolism such as gluconeogenesis. Hepatic steatosis is an early pathological step of the liver diseases deubiquitination of each target proteins and promotes their stabilities. Among USP members, USP15 plays
and can cause steatohepatitis, cirrhosis, hepatocellular carcinoma, and serious cardiovascular diseases. Some role as a major component of the transforming growth factor-β (TGF-β) signaling pathway. It binds to R-
evidence indicates that hepatic steatosis occurs in individuals with obesity and insulin resistance. . Recently, TRAF3, SMADs and promotes TGF-beta cell signal. . In the recent years, USP10 interacts with AMPK, which is
which has E3 ligase function11 and is involved in ubiquitination, induces hepaticsteatosis and insulin resistance in associated with insulin resistance, and regulates glucose metabolism. Addtionally, USP7 interacts with
hepatocyte by interacitng with TAK1. Although extensive research about NAFLD has been conducted, the specific histone acetyltransferase TIP60 and regulates early adipogenesis. However, correlation between USP15 and
and complex mechansim of causing and progressing NAFLD are not fully understood. metabolic disease is poorly understood.
In this study, we demonstrated that USP15 interacts Perilipin then increased protein stability of them by
deubiquitination. Therefore, we proposed that ablation of USP15 expression can ameliorate lipid
accumulation in liver.
METHODS
USP15 is a hydrolase that removes conjugated ubiquitin from target proteins.There have been a number of USP15 studies as to cancer and cancer immunology. However, correlation between USP15 and metabolic diseases is poorly
understood.
In this study, we demonstrate that considerably high expression level of USP15 in liver of steatosis patients. Knock down of USP15 expression retarded lipid accumulation in hepatocyte. In contrast, overexpression of USP15 increased
lipid accumulation in hepatocyte. We also confirmed that liver specific USP15 KO mice have less hepatic lipid accumulation than that of WT. Moreover, we determined that USP15 interacted with lipid droplet-associated proteins and
then up-regulated their protein expressions. Additionally, we identified that up-regulated protein stability of lipid droplet-associated proteins was due to retardation of ubiquitination by USP15. Taken together, USP15 induces
deubiquitination of lipid droplet-associated proteins and protein stability of them is upregulated. We proposed that USP15 has positive role of lipid accumulation in hepatocyte and ablation of USP15 expression prohibits NAFLD.

RESULTS

Figure 1. USP15 is upregulated in fatty liver. (A, B) USP15 mRNA and protein expression in liver of Figure 2. USP15 interact with perilipin and FABP4. (A) Interaction between endogenouse USP15 and
NFD fed mice and HFD fed mice. mRNA level was detected by qRT-PCR. Protein level was detected by
western blotting. β-actin was used as a loading control. (C) Expression of USP15 in patients with perilipin. AML12 lysates were immunoprecipitated using USP15 antibody. (B) Interaction of USP15 and Figure 3. USP15 increases protein stability of perilipin and FABP4. (A) Upregulation of perilipin protein
nonalcoholic fatty liver disease. mRNA level was detected by qRT-PCR. β-actin was used as a loading perilipin. HEK293 was transfected with Flag-USP15 and HA-perilipin plasmid. Cell lysates were by USP15 overexpression. HEK293 was transfected with HA-perilipin and Flag-USP15. (B) Effect on
control (D) Representative immunohistochemical staining for USP15 in NFD fed (n=4) and HFD fed immunoprecipitated using Flag antibody (C, D) Expression of perilipin in patients with nonalcoholic fatty protein level of perilipin by USP15 depletion. USP15 silenced AML12 was treated 20uM MG132 for 8h.
liver disease. Representative images of human nonalcoholic fatty liver disease with low (0), intermediate
mice (n=4) liver tissues. Scale bar shown is 100 µm. (E) Correlation between USP15 level and (1+), and strong (2+). (E) Correlation between USP15 and perilipin in nonalcoholic fatty liver specimens (C) Half-life of perilipin protein by USP15 depletion. AML12 was transfected with USP15 siRNA followed
progression of NAFLD Representative images of human nonalcoholic fatty liver disease with low (0), by treatment 100uM CHX for up to 18h.
intermediate (1+), and strong (2+). (Pearson’s x 2 test = 7.483, p = 0.006).

Figure 4. USP15 regulates protein stability of perilipin and FABP4 through deubiquitination (A, B) (D) Deubiquitination of
perilipin and FABP4 by USP15.
Figure 5. Hepatocyte-specific USP15 knockout improves HFD-induced liver steatosis (A,B) (C,D) Representative pictures for liver of WT and USP15 LKO mice
after feeding with HFD for 12 weeks.(E) Body weight, liver weight and liver to body weight ratio of WT (n=9) and USP15 LKO (n=7) mice fed HFD for 12 weeks.
(F) qPCR analysis of expression of genes involved in fatty acid metabolism in liver of WT and USP15 LKO mice after feeding with HFD for 12 weeks (G)
Representative immunohistochemical staining for F4/80 in liver sections and qPCR analysis of expression of genes involved pro-inflammatory cytokines and
macrophage marker in liver WT and USP15 LKO mice after feeding with HFD for 12 weeks.
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