A Mammalian Mitophagy Receptor,Bcl2-L-13, Recruits the ULK1 Complex to Induce Mitophagy

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PEI-CS/siRNA复合颗粒对肝癌耐药细胞BEL7402/5-FU中MRE11表达的影响

目的:探讨聚乙烯亚胺-壳聚糖(PEI-CS)/si RNA复合颗粒对肝癌耐药细胞BEL7402/5-FU中MRE11表达的影响。方法:采用复凝聚法将PEI-CS(100μg/m L)与不同浓度的MRE11 si RNA-FAM形成PEI-CS/si RNA复合颗粒,并转染BEL7402/5-FU细胞,用荧光显微镜和Real-time PCR检测转染效率和沉默效率。结果:荧光显微镜观察结果显示:转染细胞48 h后,3.125、6.25、12.5、25、50μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为62.31%、76.09%、79.99%、86.49%、96.59%。转染细胞48、72、96 h后,12.5μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为78.22%、55.76%、42.85%,25μg/m L的si RNA与PEI-CS形成的复合颗粒的转染率分别为83.67%、74.23%、67.45%。Real-time PCR检测结果显示:25μg/m L的si RNA与PEI-CS形成的复合颗粒转染48小时后,对BEL7402/5-FU细胞中MRE11基因的沉默效率为35.4%。结论:聚乙烯亚胺-壳聚糖/si RAN复合颗粒能有效转染肝癌耐药细胞Bel7402/5-FU,并对BEL7402/5-FU细胞中MRE11基因表达有一定抑制作用。     

ACE、CYP11B2基因多态性与慢性心力衰竭患者醛固酮脱逸的关系

目的:研究CYP11B2-344C/T(醛固酮合成酶)及ACEI/D(血管紧张素转化酶)基因多态性与慢性心力衰竭(CHF)患者实施ACEI治疗后出现醛固酮脱逸表现的关系。方法:回顾分析2008年10月至2012年10月我科收治的252例CHF患者,全部患者应用ACEI治疗3月,醛固酮在基线以上为醛固酮脱逸,依据此标准将患者分为研究组(脱逸组,n=86)与对照组(非脱逸组,n=166),依据PCR(聚合酶链反应)及RFLP(片段长度限制多态性)等方法分别检测两组CYP11B2及ACE基因型,比较两组基因型频率的分布。结果:252例患者中,共86例出现醛固酮脱逸,发生率为34.1%。全部受试患者CYP11B2基因型及ACE基因型频率与Weinberg-Hardy平衡均相符(P均0.05)。研究组ACE I/D三种基因型的组间分布与对照组相较,无统计学差异(P0.05);CYP11B2基因TT型的频率与对照组相较,呈明显统计学差异(P0.05),等位基因C/T频率的组间分布同对照组相较,亦呈明显差异(P0.05)。研究组ACEI/D的基因多态性及CYP11B2-344C/T的多态性中,基因型联合组间分布与对照组相较,无统计学差异(P0.05)。结论:ACE基因多态性与CHF患者ACEI治疗后出现醛固酮脱逸无关,CYP11B2基因T等位基因及TT基因型多态性可能是CHF患者ACEI治疗后发生醛固酮脱逸的高危因素。醛固酮脱逸时,ACE、CYP11B2基因不具有协同效果。     

Uniparental mitochondrial DNA inheritance is not affected in Ustilago maydis Δatg11 mutants blocked in mitophagy

Background

Maternal or uniparental inheritance (UPI) of mitochondria is generally observed in sexual eukaryotes, however, the underlying mechanisms are diverse and largely unknown. Recently, based on the use of mutants blocked in autophagy, it has been demonstrated that autophagy is required for strict maternal inheritance in the nematode Caenorhabditis elegans. Uniparental mitochondrial DNA (mtDNA) inheritance has been well documented for numerous fungal species, and in particular, has been shown to be genetically governed by the mating-type loci in the isogamous species Cryptococcus neoformans, Phycomyces blakesleeanus and Ustilago maydis. Previously, we have shown that the a2 mating-type locus gene lga2 is decisive for UPI during sexual development of U. maydis. In axenic culture, conditional overexpression of lga2 triggers efficient loss of mtDNA as well as mitophagy. To assess a functional relationship, we have investigated UPI in U. maydis Δatg11 mutants, which are blocked in mitophagy.

Results

This study has revealed that Δatg11 mutants are not affected in pathogenic development and this has allowed us to analyse UPI under comparable developmental conditions between mating-compatible wild-type and mutant strain combinations. Explicitly, we have examined two independent strain combinations that gave rise to different efficiencies of UPI. We demonstrate that in both cases UPI is atg11-independent, providing evidence that mitophagy is not critical for UPI in U. maydis, even under conditions of strict UPI.

Conclusions

Until now, analysis of a role of mitophagy in UPI has not been reported for microbial species. Our study suggests that selective autophagy does not contribute to UPI in U. maydis, but is rather a consequence of selective mtDNA elimination in response to mitochondrial damage.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0358-z) contains supplementary material, which is available to authorized users.     

S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells

Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca²⁺ entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca²⁺ entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca²⁺-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member – S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers.     

Lipid droplets regulate autophagosome biogenesis

The source of the autophagic membrane and the regulation of autophagosome biogenesis are still elusive open issues in the field of autophagy. In our recent study of the role of lipid droplets (LDs) and their constituents in autophagy, we provided evidence that both the biogenesis of LDs and its lipolysis by specific lipases are important for autophagosome biogenesis. Our study sheds new light on the source of the autophagic membrane and suggests that a flow of membranes from the endoplasmic reticulum (ER) to LDs, and from LDs to the ER, is essential for autophagosome biogenesis.     

Structure of yeast Ape1 and its role in autophagic vesicle formation

In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy.     

Hrr25: An emerging major player in selective autophagy regulation in Saccharomyces cerevisiae

As with the case of the mechanism of autophagosome formation, studies in yeast have taken a leading role in elucidating the molecular basis of target recognition during selective autophagy. Degradation targets are recognized by receptor proteins, which also bind to Atg8 homologs on growing phagophore membranes, leading to the loading of the targets into autophagosomes. However, it remains to be elucidated how these processes are regulated. In yeast, receptors also interact with the scaffold/adaptor protein Atg11, which subsequently recruits core Atg proteins onto receptor-target complexes to initiate autophagosome formation. Recently, we found that Hrr25, a homolog of CSNK1D/casein kinase 1δ, regulates 3 of 4 selective autophagy-related pathways in the budding yeast Saccharomyces cerevisiae by a uniform mechanism: phosphoregulation of the receptor-scaffold interaction.     

甘露糖结合凝集素对小鼠脾脏CD11c+髓样树突状细胞表型和

目的 探讨甘露聚糖结合凝集素(Mannan-binding lectin,MBL)对CD11c+髓样树突状细胞(CD11c+mDC)表型和功能的影响.方法 应用磁珠分选技术获得BALB/c小鼠脾脏CD1 1c+ mDC和CD4+T淋巴细胞.在CD11c+mDC中加入不同浓度的MBL(2.5～20 μg/mL)刺激,以不加MBL的细胞作为对照,应用ELISA法检测细胞培养上清液中的IL-12水平,流式细胞仪检测细胞表面分子CD40、CD80、CD86及HLA-DR的表达.用MTT法测定CD11c+mDC刺激CD4+T淋巴细胞的增殖能力.ELISA法检测细胞培养液中IL-4和IFN-γ水平.结果 MBL显著增强CD11c+mDC表面分子CD40、CD80、CD86及HLA-DR的表达和IL-12的分泌,促进CD4+T淋巴细胞的增殖和抗原递呈能力,诱导CD4+T向TH1反应分化.结论 MBL能够有效刺激CD11c+mDC的活化,诱导CD4+T淋巴细胞向TH1反应分化.     

牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物,是一种重要的新型抗癌药物。然而,紫杉醇在植物中含量低且难提取,限制了高效应用。利用基因工程手段,借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径,构建含有牻牛儿基牻牛儿基焦磷酸(Geranylgeranyl diphosphate,GGPP)合酶和紫杉二烯合酶的融合基因表达载体p Bg GGTS和独立表达盒表达载体p Bg GGg TS,并分别转入灰盖鬼伞LT2菌株中,经过选择性筛选、PCR鉴定、Southern blotting杂交验证,分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株,分别提取菌丝体和发酵液分析。GC-MS分析表明,两种工程菌株与原出发菌株的菌丝提取物无明显差异峰,而与出发菌株的发酵液提取物相比,两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰,采用GC-MS特征质量离子分析方法判定为紫杉二烯,分别为44 ng/L(转化p Bg GGg TS)和30 ng/L(转化p Bg GGTS)。结果表明,通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因,可以生物合成紫杉二烯。