Organisms have sophisticated subcellular compartments containing enzymes that function in tandem. These confined compartments ensure effective chemical transformation and transport of molecules, and the elimination of toxic metabolic wastes1,2. Creating functional enzyme complexes that are confined in a similar way remains challenging. Here we show that two or more enzymes with complementary functions can be assembled and encapsulated within a thin polymer shell to form enzyme nanocomplexes. These nanocomplexes exhibit improved catalytic efficiency and enhanced stability when compared with free enzymes. Furthermore, the co-localized enzymes display complementary functions, whereby toxic intermediates generated by one enzyme can be promptly eliminated by another enzyme. We show that nanocomplexes containing alcohol oxidase and catalase could reduce blood alcohol levels in intoxicated mice, offering an alternative antidote and prophylactic for alcohol intoxication.

The endoplasmic reticulum (ER) chaperone protein glucose-regulated protein 78 (GRP78/BiP) is a master regulator of ER homeostasis and stress response which are implicated in pathogenesis of metabolic disorders. By applying the LoxP-Cre strategy, we generated mice with liver specific GRP78 loss. Our studies using this novel mouse model revealed that liver GRP78 was required for neonatal survival and more than 50% loss of GRP78 in the adult liver caused ER stress response and dilation of ER compartment, accompanied by onset of apoptosis, suggesting critical involvement of GRP78 in maintaining hepatocyte ER homeostasis and viability. Further, these mice exhibited elevation of serum alanine aminotransferase and fat accumulation in the liver, and were sensitized to a variety of acute and chronic hepatic disorders by alcohol, high fat diet, drugs and toxins, which were alleviated by simultaneous administration of the molecular chaperone, 4-phenylbutyrate. Analysis by microarray followed by 2D protein profile revealed major perturbation of unfolded protein response (UPR) targets and common enzymes/factors in lipogenesis as well as new factors including liver major urinary proteins, fatty acid binding proteins, adipose differentiation-related protein, cysteine-rich with EGF-like domains 2 (Creld2), nuclear protein 1, and growth differentiation factor 15 (gdf15) possibly contributing to liver steatosis or fibrosis under ER stress. Our findings underscore the importance of GRP78 in managing the physiological client protein load and suppressing apoptosis in hepatocytes, supporting the pathologic role of ER stress in the evolution of fatty liver and disease under adverse conditions of drugs, diet, toxins and alcohol.

A portion of HIV-infected patients under therapy with protease inhibitors (HIV PIs) concomitantly consume or abuse alcohol leading to hepatic injury. The underling mechanisms are not known. We hypothesize that HIV PI aggravates alcohol-induced liver injury through an endoplasmic reticulum (ER) stress mechanism. To address this, we treated mice and primary mouse and human hepatocytes (PMH and PHH respectively) with alcohol and the two HIV PIs: ritonavir and lopinavir. In mice, ritonavir and lopinavir (15 mg/kg body weight each) induced mild ER stress and inhibition of Sarco/ER Calcium-ATPase (SERCA) without significant increase in serum ALT levels. However, a single dose of alcohol by gavage (5g/kg body weight) plus the two HIV PIs caused a greater than 5-fold increase in serum ALT, a synergistic increase in alcohol-induced liver lipid accumulation and ER stress response, and a decrease of SERCA. Mice treated with chronic HIV PIs and alcohol developed moderate liver fibrosis. In PMH, the HIV drugs plus alcohol also inhibited SERCA expression and increased expression of GRP78, CHOP, SREBP1c and phosphorylated JNK2, which were accompanied by a synergistic increase in cell death compared to alcohol or the HIV drugs alone. In PHH, ritonavir and lopinavir or alcohol alone treatment increased mRNA of spliced Xbp1 and decreased SERCA, which were accompanied by reduced levels of intracellular calcium. Alcohol combined with the HIV drugs significantly reduced intracellular calcium levels and potentiated cell death, which was comparable to the cell death caused by the SERCA inhibitor-thapsigargin. Our findings suggest the possibility that HIV PIs potentiate alcohol-induced ER stress and injury through modulation of SERCA and maintaining calcium homeostasis should be a therapeutic aim for a better care of HIV patients.

Mortality from liver cancer in humans is increasingly attributable to heavy or long-term alcohol consumption. The mechanisms by which alcohol exerts its carcinogenic effect are not well understood. In this study, the role of alcohol-induced endoplasmic reticulum (ER) stress response in liver cancer development was investigated using an animal model with a liver knockout (KO) of the chaperone BiP and under constitutive hepatic ER stress. Long-term alcohol and high fat diet feeding resulted in higher levels of serum alanine aminotransferase, impaired ER stress response, and higher incidence of liver tumor in older (aged 16 months) KO females than in either middle-aged (6 months) KOs or older (aged 16 months) wild type females. In the older KO females, stronger effects of the alcohol on methylation of CpG islands at promoter regions of genes involved in the ER-associated degradation (ERAD) were also detected. Altered expression of ERAD factors including derlin 3, Creld2 (cysteine-rich with epidermal growth factor-like domains 2), Herpud1 (homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member), Wfs1 (Wolfram syndrome gene), and Yod1 (deubiquitinating enzyme 1) was co-present with decreased proteasome activities, increased estrogen receptor α variant (ERα36), and enhanced phosphorylations of ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) and STAT3 (the signal transducers and activators of transcription) in the older KO female fed alcohol. Our results suggest that long-term alcohol consumption and aging may promote liver tumorigenesis in females through interfering with DNA methylation and expression of genes involved in the ERAD.

Background Certain anti-HIV drugs alone or in combination are often associated with liver damages, which are frequently worsened by alcohol consumption. We previously found an endoplasmic reticulum (ER) stress mechanism for the drug- and alcohol-induced hepatic injuries in animal models and in vitro hepatocytes. However, it is unknown whether anti-HIV drugs and alcohol induce similar cellular stress responses and injuries in liver nonparenchymal cells. Methods Primary mouse hepatocytes (PMH), kupffer cells (KC), and hepatocellular stellate cells (HSC) were freshly isolated from mouse liver and treated with DMSO, stress-inducing pharmaceutical agents, alcohol alone, or in combination with antiviral ritonavir (RIT), lopinavir (LOP), or efavirenz (EFV). Expression of cellular stress markers, protein colocalization, and cell death were analyzed with immunoblotting, immunocytochemistry, and positive double staining with Sytox green and Hoechst blue, respectively. Results Expression of the ER stress markers of BiP, CHOP, and SERCA and the autophagy marker LC3 was significantly changed in PMH in response to combined alcohol, RIT, and LOP, which was companied by increased cell death compared with control. In contrast, although pharmaceutical agents induced ER stress and cell death, no significant ER stress or cell death was found in KC treated with alcohol, RIT, LOP, and EFV singly or in combination. In HSC, alcohol, RIT, LOP, or EFV induced BiP, but not CHOP, SERCA, or cell death compared with vehicle control. Further in PMH, RIT and LOP or in combination with alcohol-induced dose-dependent inhibition of β-actin. Inhibition of β-actin by RIT and LOP was companied with an inhibited nuclear expression of the antioxidant response regulator Nrf2 and reduced GST downstream of Nrf2. Ascorbic acid treatment reduced the alcohol-, RIT-, and LOP-induced cell death. Conclusions The data suggest for the first time that sensitivities of hepatocytes and nonparenchymal cells to alcohol and anti-HIV drugs in vitro are different in terms of cellular stress response and cell death injury. Oxidative stress mediated by Nrf2 contributes to the alcohol- and drug-induced toxicity in the hepatocytes.