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.     

Mitochondrial Stress-Initiated Aberrant Activation of the NLRP3 Inflammasome Regulates the Functional Deterioration of Hematopoietic Stem Cell Aging

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Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle

Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.     

Homeostatic Control of Sebaceous Glands by Innate Lymphoid Cells Regulates Commensal Bacteria Equilibrium

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Interaction of an amphipathic peptide with phosphatidycholine/phosphatidylethanolamine mixed membranes

The effect of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in mixed membranes with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on interaction with a class A amphipathic peptide, Ac-DWLKAFYDKVAEKLKEAF-NH₂ (Ac-18A-NH₂), was investigated. The fluorescence lifetime of 2-(9-anthroyloxy)stearic acid and ²H NMR spectra were used to evaluate the penetration of water molecules into the membrane interface and the order of lipid acyl chains, respectively. The results demonstrated that DOPE in the mixed membranes decreased the fluorescence lifetime and increased the acyl-chain order, and that Ac-18A-NH₂ affected them more for membranes with higher DOPE fractions. The partition coefficient (K_p) of the peptide to the mixed membranes was increased with the increase in the DOPE mole fractions. From the temperature dependence of the K_p values, the binding of Ac-18A-NH₂ to POPC/DOPE mixed membranes was found to be entropy-driven. The formation of an α-helix at the membrane’s surface is supposed to induce positive curvature strain, which decreases the headgroup hydration and acyl-chain order of lipids. Thus, the binding of Ac-18A-NH₂ to membranes is entropically more favorable at higher DOPE fractions since the peptide’s insertion into the membrane can decrease the order parameter and unfavorable headgroup hydration, which explains the enhanced peptide binding.     

The permeability to water and cryoprotectants of immature and mature oocytes in the zebrafish (Danio rerio)

To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.     

Quality control of Photosystem II: Cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 °C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

Structural analysis of obscurin gene in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiac disease characterized by left ventricular hypertrophy with diastolic dysfunction. Molecular genetic studies have revealed that HCM is caused by mutations in genes for sarcomere/Z-band components including titin/connectin and its associate proteins. However, disease-causing mutations can be found in about half of the patients, suggesting that other disease-causing genes remain to be identified. To explore a novel disease gene, we searched for obscurin gene (OBSCN) mutations in HCM patients, because obscurin interacts with titin/connectin. Two linked variants, Arg4344Gln and Ala4484Thr, were identified in a patient and functional analyses demonstrated that Arg4344Gln affected binding of obscurin to Z9-Z10 domains of titin/connectin, whereas Ala4484Thr did not. Myc-tagged obscurin showed that Arg4344Gln impaired obscurin localization to Z-band. These observations suggest that the obscurin abnormality may be involved in the pathogenesis of HCM.