Structural insights into Atg10-mediated formation of the autophagy-essential Atg12-Atg5 conjugate

The Atg12-Atg5 conjugate, which is formed by an ubiquitin-like conjugation system, is essential to autophagosome formation, a central event in autophagy. Despite its importance, the molecular mechanism of the Atg12-Atg5 conjugate formation has not been elucidated. Here, we report the solution and crystal structures of Atg10 and Atg5 homologs from Kluyveromyces marxianus (Km), a thermotolerant yeast. KmAtg10 comprises an E2-core fold with characteristic accessories, including two β strands, whereas KmAtg5 has two ubiquitin-like domains and a helical domain. The nuclear magnetic resonance experiments, mutational analyses, and crosslinking experiments showed that KmAtg10 directly recognizes KmAtg5, especially its C-terminal ubiquitin-like domain, by its characteristic two β strands. Kinetic analysis suggests that Tyr56 and Asn114 of?KmAtg10 may place the side chain of KmAtg5 Lys145 into the optimal orientation for its conjugation reaction with Atg12. These structural features enable Atg10 to mediate the formation of the Atg12-Atg5 conjugate without a specific E3 enzyme.     

Influence of APOE Genotype on Whole-Brain Functional Networks in Cognitively Normal Elderly

This study aimed to investigate the influence of apolipoprotein E (APOE) ε4 allele on whole-brain functional networks in cognitively normal (CN) elderly by applying graph theoretical analysis to brain glucose metabolism. Eighty-six CN elderly [28 APOE ε4 carriers (ε4+) and 58 non-carriers (ε4-)] underwent clinical evaluation and resting [¹⁸F] fluorodeoxyglucose positron emission tomography scan. Whole-brain functional networks were constructed from correlations of the 90 regions of interest using the automated anatomical labeling template, and analyzed using graph theoretical approaches. The overall small-world property seen in ε4- was preserved in ε4+. However, both local clustering and path length were lower in ε4+ compared to ε4-. In terms of the hubs of functional networks, ε4+ showed decreased centrality of the right hippocampus but increased centrality of several brain regions associated with the default mode network compared to ε4-. Our results indicate that genetic vulnerability to Alzheimer’s disease may alter whole-brain functional networks even before clinical symptoms appear.     

IL-10对哮喘小鼠血清IL-5和IgE的影响

目的:探讨IL-10治疗对哮喘鼠血清IL-5及IgE水平的影响.方法:制备20只小鼠哮喘模型,随机分为治疗组、哮喘组,前者使用IL-10治疗,后者未予治疗,用ELISA双抗体夹心法测定治疗组和哮喘组血清IL-5和IgE的水平.以10只健康小鼠为对照组.结果:与对照组比较,哮喘组IL-5和IgE水平较高,差异均有统计学意义(P＜0.05);经IL-10治疗后,治疗组的血清IL-5、IgE的水平明显低于哮喘组水平,其差异有统计学意义(P＜0.05);治疗组治疗后IL-5和和IgE检测值与正常组水平差异无统计学意义(p＞0.05).结论:IL-10能够降低哮喘小鼠血清的IL-5和IgE水平.     

湖南汉族人群IL-1O启动子和IL-1ra基因多态性与SLE相关性研究

目的:研究湖南汉族人群IL-10启动子和IL-1受体拮抗剂(IL-1ra)的基因多态性,探讨IL-10启动子和IL-1ra基因多态性与SLE疾病的关系。方法:PCR和限制性内切酶酶切分析SLE患者(n=83)和正常对照人群(n=125)IL-10启动子和IL-1ra基因多态性,对基因频率进行分析。结果:湖南汉族人群IL-1ra及IL-10启动子基因具有多态性;SLE患者IL-1RN*1等位基因的频率显著高于正常对照组(P<0.05,RR=5);SLE患者IL-10启动子区-597位*A、-824位*T和ACC亚型的基因频率高于正常对照组(P<0.001)。结论:SLE患者IL-1RN*1的基因频率、IL-10启动子区-597位和-824位的基因多态性与正常人比较有显著差异,提示以上基因可能与SLE的发病有一定相关性。     

Functional identification of Bombyx mori Atg13 in autophagy

The autophagy process involves a series of autophagy-related (Atg) proteins, which are conserved in eukaryotes. ULK1/Atg1-ATG13/Atg13 is the core protein complex for autophagy initiation in response to nutrient and hormone signaling. However, how Atg13 is regulated to participate in autophagy is unclear in insects. Here in Bombyx mori, the variation of BmAtg13 was correlated with autophagy induced by steroid hormone 20-hydroxyecdysone (20E) or starvation. Developmental profiles from feeding to prepupal stage revealed that there were two bands of BmAtg13 protein detected by western blot analysis, therein the upper band was intensively decreased, while the lower band was significantly increased which was in accordance with its mRNA variation; and immunofluorescent staining indicated that BmAtg13 was nucleocytoplasmic translocated during larval-pupal metamorphosis when autophagy was dramatically induced. BmAtg13 knockdown and overexpression both inhibits autophagy. Besides, 20E treatment-induced BmAtg13 gene expression, while blocking 20E signaling transduction by knockdown of BmUsp reduced both gene expression and protein level of BmAtg13. These results reveal that BmAtg13 is required for 20E- and starvation-induced autophagy in B. mori, which provides the foundation for further related studies.     

Increased Retinal Ganglion Cell Survival by Exogenous IL-2 Depends on IL-10, Dopamine D1 Receptors,and Classical IL-2/IL-2R Signaling Pathways

Neurochemical Research - Interleukin-2 (IL-2) is a classical pro-inflammatory cytokine known to display neuroprotective roles in the central nervous system including the retina. In the present...     

Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c

The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (Cycs ^G41S/G41S) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway.     

Structure of Atg5.Atg16, a complex essential for autophagy

Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5.Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97A resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy.     

Atg5-Independent Sequestration of Ubiquitinated Mycobacteria

Like several other intracellular pathogens, Mycobacterium marinum (Mm) escapes from phagosomes into the host cytosol where it can polymerize actin, leading to motility that promotes spread to neighboring cells. However, only ∼25% of internalized Mm form actin tails, and the fate of the remaining bacteria has been unknown. Here we show that cytosolic access results in a new and intricate host pathogen interaction: host macrophages ubiquitinate Mm, while Mm shed their ubiquitinated cell walls. Phagosomal escape and ubiquitination of Mm occured rapidly, prior to 3.5 hours post infection; at the same time, ubiquitinated Mm cell wall material mixed with host-derived dense membrane networks appeared in close proximity to cytosolic bacteria, suggesting cell wall shedding and association with remnants of the lysed phagosome. At 24 hours post-infection, Mm that polymerized actin were not ubiquitinated, whereas ubiquitinated Mm were found within LAMP-1–positive vacuoles resembling lysosomes. Though double membranes were observed which sequestered Mm away from the cytosol, targeting of Mm to the LAMP-1–positive vacuoles was independent of classical autophagy, as demonstrated by absence of LC3 association and by Atg5-independence of their formation. Further, ubiquitination and LAMP-1 association did not occur with mutant avirulent Mm lacking ESX-1 (type VII) secretion, which fail to escape the primary phagosome; apart from its function in phagosome escape, ESX-1 was not directly required for Mm ubiquitination in macrophages or in vitro. These data suggest that virulent Mm follow two distinct paths in the cytosol of infected host cells: bacterial ubiquitination is followed by sequestration into lysosome-like organelles via an autophagy-independent pathway, while cell wall shedding may allow escape from this fate to permit continued residence in the cytosol and formation of actin tails.     

Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity

The autophagy-related proteins ATG12 and ATG5 form a covalent complex essential for autophagy. Here, we demonstrate that ATG12 has distinct functions from ATG5 in pro-opiomelanocortin (POMC)-expressing neurons. Upon high-fat diet (HFD) consumption, mice lacking Atg12 in POMC-positive neurons exhibit accelerated weight gain, adiposity, and glucose intolerance, which is associated with increased food intake, reduced ambulation, and decreased LEP/leptin sensitivity. Importantly, although genetic deletion of either Atg12 or Atg5 renders POMC neurons autophagy-deficient, mice lacking Atg5 in POMC neurons do not exhibit these phenotypes. Hence, we propose nonautophagic functions for ATG12 in POMC neurons that counteract excessive weight gain in response to HFD consumption.     

Influence of acute alcohol intake and alcohol withdrawal on circulating levels of IL-6, IL-8, IL-10 and IL-12

Cytokine balance alterations are responsible for some of the systemic and hepatic manifestations of alcoholism. The present study was aimed to evaluate the influence of both acute alcohol abstinence (in alcoholics) and acute alcohol intake (in healthy subjects) on serum IL-6, IL-8, IL-10, and IL-12 levels. Serum cytokine concentrations were determined on admission and after a median of 6 days of ethanol abstinence in 29 patients with alcohol withdrawal syndrome. The same determinations were made in five healthy volunteers at baseline and after 36 h of a single 60 g-dose alcohol intake. Increased serum levels of IL-6, IL-10 and, to a lesser extent IL-8, declined in the few days after alcohol abstinence in patients with alcohol withdrawal syndrome. Serum IL-8 values increased after alcohol intake in healthy subjects. Rapid variation of serum cytokine levels along with alcohol intake or abstinence should be taken into account in cytokine studies in alcohol abusers.     

环境对沼泽红假单胞菌基因型的影响

<正>紫色光合细菌是不产氧光合细菌的一个重要分支,包括紫色非硫细菌和紫色硫细菌。其中紫色非硫细菌具有极其丰富的代谢模式,可进行光能异养、光能自养和化能异养生长,其代谢的多样性使得它们广泛存活于不同的生态系统中,如土壤、湖泊、海洋及底泥等[1-2]。沼泽红假单胞菌(Rhodopseudomonas palustris)是紫     

Functional Cardiac Lipolysis in Mice Critically Depends on Comparative Gene Identification-58

Efficient catabolism of cellular triacylglycerol (TG) stores requires the TG hydrolytic activity of adipose triglyceride lipase (ATGL). The presence of comparative gene identification-58 (CGI-58) strongly increased ATGL-mediated TG catabolism in cell culture experiments. Mutations in the genes coding for ATGL or CGI-58 in humans cause neutral lipid storage disease characterized by TG accumulation in multiple tissues. ATGL gene mutations cause a severe phenotype especially in cardiac muscle leading to cardiomyopathy that can be lethal. In contrast, CGI-58 gene mutations provoke severe ichthyosis and hepatosteatosis in humans and mice, whereas the role of CGI-58 in muscle energy metabolism is less understood. Here we show that mice lacking CGI-58 exclusively in muscle (CGI-58KOM) developed severe cardiac steatosis and cardiomyopathy linked to impaired TG catabolism and mitochondrial fatty acid oxidation. The marked increase in ATGL protein levels in cardiac muscle of CGI-58KOM mice was unable to compensate the lack of CGI-58. The addition of recombinant CGI-58 to cardiac lysates of CGI-58KOM mice completely reconstituted TG hydrolytic activities. In skeletal muscle, the lack of CGI-58 similarly provoked TG accumulation. The addition of recombinant CGI-58 increased TG hydrolytic activities in control and CGI-58KOM tissue lysates, elucidating the limiting role of CGI-58 in skeletal muscle TG catabolism. Finally, muscle CGI-58 deficiency affected whole body energy homeostasis, which is caused by impaired muscle TG catabolism and increased cardiac glucose uptake. In summary, this study demonstrates that functional muscle lipolysis depends on both CGI-58 and ATGL.     

Decrease in circulating DNA, IL-10 and BAFF levels in newly-diagnosed SLE patients after corticosteroid and chloroquine treatment

Arsenal of pattern-recognition receptors alongside antibody production machinery make B cells vulnerable to autoimmune response if an autoantigen elicits both pathways in a self-sustained fashion. Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies to DNA, RNA and related structures. Murine studies demonstrated autoreactive B cell activation upon TLR9 stimulation with DNA-containing immune complexes. This activation could be abolished with chloroquine, a drug used in SLE treatment that also blocks TLR9 signaling. We investigated whether chloroquine modulates TLR9 expression, circulating DNA levels and B cell-related cytokines in newly discovered, untreated SLE patients. TLR9 was measured in peripheral blood B cells by flow cytometry, serum DNA by real-time PCR, and IL-10 and BAFF by ELISA before treatment, after 3weeks on corticosteroids, and 3months after introduction of chloroquine. We found that circulating DNA is higher in SLE patients than in controls in every time-point and decreases significantly after chloroquine treatment. Untreated patients had higher serum IL-10 than controls or patients on corticosteroids. Also, corticosteroids decreased and chloroquine completely abolished CpG-mediated CD86 upregulation on B cells and IL-10 secretion in PBMC culture. Providing the TLR9 pathway activation demonstrates its importance in pathogenesis of human SLE, this data supports continuation of chloroquine in SLE treatment protocol. In addition, observed modulation of cytokine and DNA levels after immunomodulatory treatment prompts for inclusion of untreated patients in studies of human immune disorders.     

Arrhythmogenic Biophysical Phenotype for SCN5A Mutation S1787N Depends upon Splice Variant Background and Intracellular Acidosis

Background

SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. I _Na dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity.

Methods and Results

We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated I _Na was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like I _Na including peak I _Na density, activation and inactivation parameters, and late I _Na amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of I _Na late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.

Conclusion

The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Na_v1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.     

Down-regulated NOD2 by immunosuppressants in peripheral blood cells in patients with SLE reduces the muramyl dipeptide-induced IL-10 production

Background

Pattern recognition receptors (PRRs) such as Toll-like receptors are aberrantly expressed of peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients, for playing immunopathological roles.

Methodology/Principal Findings

We investigated the expression and function of the PRR nucleotide-binding oligomerization domain (NOD2) in SLE. NOD2 expression in T, B lymphocytes, monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) was assessed in SLE patients and healthy controls (HCs) using flow cytometric analysis. Ex vivo production of cytokines from PBMCs upon NOD2 agonist muramyl dipeptide (MDP) stimulation was assessed using Cytometric Bead Array. Over-expression of NOD2 in monocytes was observed in immunosuppressant naïve SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8+ T, monocytes, mDCs and pDCs. Ex vivo basal productions of cytokines (IL-6, IL-8 and IL-10) were significantly increased in immunosuppressant naïve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1β) from PBMC was significantly increased in immunosuppressant naïve patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with HCs. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naïve patients and HCs.

Conclusions/Significance

Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant naïve SLE patients which can subsequently lead to aberrant activation of PBMCs to produce proinflammatory cytokines, implicating the innate immune response for extracellular pathogens in the immunopathological mechanisms in SLE. Immunosuppressant therapy may downregulate NOD2 expression in CD8+ T lymphocytes, monocytes, and DCs in SLE patients which subsequently IL-10 reduction, contributing towards the regulation of immunopathological mechanisms of SLE, at the expense of increasing risk of bacterial infection.