Does prenylation predict progression in NAFLD?

Non-alcoholic fatty liver disease (NAFLD) often develops in concert with related metabolic diseases, such as obesity, dyslipidemia and insulin resistance. Prolonged lipid accumulation and inflammation can progress to non-alcoholic steatohepatitis (NASH). Although factors associated with the development of NAFLD are known, triggers for the progression of NAFLD to NASH are poorly understood. Recent findings published in The Journal of Pathology reveal the possible regulation of NASH progression by metabolites of the mevalonate pathway. Mevalonate can be converted into the isoprenoids farnesyldiphosphate (FPP) and geranylgeranyl diphosphate (GGPP). GGPP synthase (GGPPS), the enzyme that converts FPP to GGPP, is dysregulated in humans and mice during NASH. Both FPP and GGPP can be conjugated to proteins through prenylation, modifying protein function and localization. Deletion or knockdown of GGPPS favors FPP prenylation (farnesylation) and augments the function of liver kinase B1, an upstream kinase of AMP-activated protein kinase (AMPK). Despite increased AMPK activation, livers in Ggpps-deficient mice on a high-fat diet poorly oxidize lipids due to mitochondrial dysfunction. Although work from Liu et al provides evidence as to the potential importance of the prenylation portion of the mevalonate pathway during NAFLD, future studies are necessary to fully grasp any therapeutic or diagnostic potential. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Doxorubicin‐induced normal breast epithelial cellular aging and its related breast cancer growth through mitochondrial autophagy and oxidative stress mitigated by ginsenoside Rh2

Clinical dose of doxorubicin (100 nM) induced cellular senescence and various secretory phenotypes in breast cancer and normal epithelial cells. Herein, we reported the detailed mechanism underlying ginsenoside Rh2‐mediated NF‐κB inhibition, and mitophagy promotion were evaluated by antibody array assay, western blotting analysis, and immunocytostaining. Ginsenoside Rh2 suppressed the protein levels of TRAF6, p62, phosphorylated IKK, and IκB, which consequently inactivated NF‐κB activity. Rh2‐mediated secretory phenotype was delineated by the suppressed IL‐8 secretion. Senescent epithelial cells showed increased level of reactive oxygen species (ROS), which was significantly abrogated by Rh2, with upregulation on SIRT 3 and SIRT 5 and subsequent increase in SOD1 and SOD2. Rh2 remarkably favored mitophagy by the increased expressions of PINK1 and Parkin and decreased level of PGC‐1α. A decreased secretion of IL‐8 challenged by mitophagy inhibitor Mdivi‐1 with an NF‐κB luciferase system was confirmed. Importantly, secretory senescent epithelial cells promoted the breast cancer (MCF‐7) proliferation while decreased the survival of normal epithelial cells demonstrated by co‐culture system, which was remarkably alleviated by ginsenoside Rh2 treatment. These data included ginsenoside Rh2 regulated ROS and mitochondrial autophagy, which were in large part attributed to secretory phenotype of senescent breast epithelial cells induced by doxorubicin. These findings also suggested that ginsenoside Rh2 is a potential treatment candidate for the attenuation of aging related disease.     

The three ‘P’s of mitophagy: PARKIN,PINK1, and post-translational modifications

Two Parkinson''s disease (PD)-associated proteins, the mitochondrial kinase PINK1 and the E3-ubiquitin (Ub) ligase PARKIN, are central to mitochondrial quality control. In this pathway, PINK1 accumulates on defective mitochondria, eliciting the translocation of PARKIN from the cytosol to mediate the clearance of damaged mitochondria via autophagy (mitophagy). Throughout the different stages of mitophagy, post-translational modifications (PTMs) are critical for the regulation of PINK1 and PARKIN activity and function. Indeed, activation and recruitment of PARKIN onto damaged mitochondria involves PINK1-mediated phosphorylation of both PARKIN and Ub. Through a stepwise cascade, PARKIN is converted from an autoinhibited enzyme into an active phospho-Ub-dependent E3 ligase. Upon activation, PARKIN ubiquitinates itself in concert with many different mitochondrial substrates. The Ub conjugates attached to these substrates can in turn be phosphorylated by PINK1, which triggers further cycles of PARKIN recruitment and activation. This feed-forward amplification loop regulates both PARKIN activity and mitophagy. However, the precise steps and sequence of PTMs in this cascade are only now being uncovered. For instance, the Ub conjugates assembled by PARKIN consist predominantly of noncanonical K6-linked Ub chains. Moreover, these modifications are reversible and can be disassembled by deubiquitinating enzymes (DUBs), including Ub-specific protease 8 (USP8), USP15, and USP30. However, PINK1-mediated phosphorylation of Ub can impede the activity of these DUBs, adding a new layer of complexity to the regulation of PARKIN-mediated mitophagy by PTMs. It is therefore evident that further insight into how PTMs regulate the PINK1–PARKIN pathway will be critical for our understanding of mitochondrial quality control.     

Structural and Functional Impact of Parkinson Disease‐Associated Mutations in the E3 Ubiquitin Ligase Parkin

Mutations in the PARKIN/PARK2 gene that result in loss‐of‐function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early‐onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure–function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.     

MicroRNA-33/33* inhibit the activation of MAVS through AMPK in antiviral innate immunity

Innate immunity plays a prominent role in the host defense against pathogens and must be precisely regulated. As vital orchestrators in cholesterol homeostasis, microRNA-33/33* have been widely investigated in cellular metabolism. However, their role in antiviral innate immunity is largely unknown. Here, we report that VSV stimulation decreased the expression of miR-33/33* through an IFNAR-dependent manner in macrophages. Overexpression of miR-33/33* resulted in impaired RIG-I signaling, enhancing viral load and lethality whereas attenuating type I interferon production both in vitro and in vivo. In addition, miR-33/33* specifically prevented the mitochondrial adaptor mitochondrial antiviral-signaling protein (MAVS) from forming activated aggregates by targeting adenosine monophosphate activated protein kinase (AMPK), subsequently impeding the mitophagy-mediated elimination of damaged mitochondria and disturbing mitochondrial homeostasis which is indispensable for efficient MAVS activation. Our findings establish miR-33/33* as negative modulators of the RNA virus-triggered innate immune response and identify a previously unknown regulatory mechanism linking mitochondrial homeostasis with antiviral signaling pathways.     

耐力训练对帕金森模型小鼠中脑线粒体自噬相关基因表达的影响

目的:探讨耐力训练对帕金森病(PD)模型小鼠中脑线粒体自噬的影响,揭示运动预防帕金森病的分子生物学机制。方法:10周龄雄性C57BL/6小鼠32只,随机分为安静组(C),运动组(E),帕金森模型安静组(P)及帕金森模型运动组(PE),每组8只。E组及PE组小鼠进行为期8周跑台耐力训练,跑台速度15m/min,坡度为0%,每天运动持续50min,每周6次,周日休息。训练结束后,P组和PE组小鼠腹腔注射MPTP(30mg/kg),C组和E组小鼠注射等量生理盐水,持续3d。注射结束1周后,处死各组小鼠,取双侧中脑及纹状体,采用实时PCR及Western blot技术检测线粒体自噬相关基因及TH蛋白表达,HPLC技术检测中脑及纹状体组织内DA含量。结果:与C组比,E组小鼠中脑TH蛋白上调(P0.05),中脑及纹状体DA含量均增加(P0.05),P组和PE组小鼠TH蛋白及DA含量均显著下降(P0.01),而P组较PE组相应指标下降更明显(P0.05)。与C组比,E组小鼠中脑PINK、PARK2 m RNA及PINK1蛋白表达上调(P0.05),LC3Ⅱ/Ⅰ比值增加,P组小鼠PINK1m RNA表达下调(P0.05),PE组小鼠PINK1 m RNA、Parkin蛋白含量及LC3Ⅱ/Ⅰ比值较P组增加。结论:为期3d的MPTP处理可导致小鼠中脑及纹状体TH及DA含量下降,诱导帕金森病发生,耐力训练可缓解这一现象;MPTP诱导的帕金森小鼠模型中脑线粒体自噬水平下降,而耐力训练可作用于线粒体自噬相关基因的表达,抑制MPTP的神经毒性作用。     

Pharmacological inhibition of TLR4/NF‐κB with TLR4‐IN‐C34 attenuated microcystin‐leucine arginine toxicity in bovine Sertoli cells

This study investigated the pharmacological inhibition of the toll‐like receptor 4 (TLR4) genes as a measure to attenuate microcystin‐LR (MC‐LR) reproductive toxicity. Bovine Sertoli cells were pretreated with TLR4‐IN‐C34 (C34) for 1 hour. Thereafter the pretreated and non‐pretreated Sertoli cells were cultured in medium containing 10% heat‐activated fetal bovine serum + 80 μg/L MC‐LR for 24 hours to assess the ability of TLR4‐IN‐C34 to attenuate the toxic effects of MC‐LR. The results showed that TLR4‐IN‐C34 inhibited MC‐LR‐induced mitochondria membrane damage, mitophagy and downregulation of blood‐testis barrier constituent proteins via TLR4/nuclear factor‐kappaB and mitochondria‐mediated apoptosis signaling pathway blockage. The downregulation of the mitochondria electron transport chain, energy production and DNA replication related genes (mt‐ND2, COX‐1, COX‐2, ACAT, mtTFA) and upregulation of inflammatory cytokines (interleukin [IL]‐6, tumor necrosis factor‐α, IL‐1β, interferon‐γ, IL‐4, IL‐10, IL‐13 and transforming growth factor β1) were modulated by TLR4‐IN‐C34. Taken together, we conclude that TLR4‐IN‐C34 inhibits MC‐LR‐related disruption of mitochondria membrane, mitophagy and downregulation of blood‐testis barrier proteins of the bovine Sertoli cell via cytochrome c release and TLR4 signaling blockage.     

保肾通络方含药血清调节线粒体自噬对高糖诱导下足细胞氧化损伤的影响

目的 观察保肾通络方含药血清对高糖培养下足细胞氧化损伤的影响,并对保肾通络方的作用机制进行探讨。方法 以体外培养的肾小球足细胞为研究对象,给予高浓度葡萄糖刺激,采用CCK-8法分析足细胞活力以确定最佳模型及给药浓度,分为正常对照组、高糖组、保肾通络方组,采用鬼笔环肽进行细胞骨架染色,Western blotting法检测足细胞裂孔膜蛋白nephrin,自噬相关蛋白5(autophagy-related protein 5,ATG5)、ATG7、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)的表达,流式细胞术检测足细胞活性氧(reactive oxygen species,ROS)及线粒体膜电位,免疫荧光共标LC3B及线粒体,观察各组足细胞内线粒体自噬泡的生成情况。结果 30 mmol/L高糖干预48 h可显著降低足细胞活力(P＜0.05),保肾通络方含药血清可使高糖诱导的足细胞活力明显提升(P＜0.01);与正常对照组相比,高糖组足细胞骨架褶皱呈多边形,应力纤维束被破坏,ROS生成显著增多(P＜0.01),足细胞nephrin、ATG5、ATG7表达显著降低,LC3Ⅱ/LC3Ⅰ比值降低(P＜0.05),LC3B与线粒体共标减少,线粒体膜电位下降(P＜0.01);与高糖组相比,保肾通络方组足细胞骨架改善,ROS生成减少(P＜0.01),nephrin、ATG5、ATG7表达显著增加(P＜0.05),LC3Ⅱ/LC3Ⅰ比值上升(P＜0.05),LC3B与线粒体荧光共标增多,线粒体膜电位升高(P＜0.01)。结论 保肾通络方含药血清能够减轻高糖诱导的足细胞氧化损伤,其作用可能与改善足细胞线粒体自噬有关。