Mitophagy reporter mouse analysis reveals increased mitophagy activity in disuse-induced muscle atrophy.

Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.

Angiopoietin Like Protein 2 (ANGPTL2) Promotes Adipose Tissue Macrophage and T lymphocyte Accumulation and Leads to Insulin Resistance.

OBJECTIVES: Angiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Adenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver. CONCLUSION: ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance.

Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein.

Four rhodopsins, bacteriorhodopsin (bR), halorhodopsin (hR), sensory rhodopsin (sR) and phoborhodopsin (pR) exist in archaeal membranes. bR and hR work as a light-driven ion pump. sR and pR work as a photo-sensor of phototaxis, and form signaling complexes in membranes with their respective cognate transducer proteins HtrI (with sR) and HtrII (with pR), through which light signals are transmitted to the cytoplasm. What is the determining factor(s) of the specific binding to form the complex? Binding of the wild-type or mutated rhodopsins with HtrII was measured by isothermal titration calorimetric analysis (ITC). bR and hR could not bind with HtrII. On the other hand, sR could bind to HtrII, although the dissociation constant (K(D)) was about 100 times larger than that of pR. An X-ray crystallographic structure of the pR/HtrII complex revealed formation of two specific hydrogen bonds whose pairs are Tyr199(pR)/Asn74(HtrII) and Thr189(pR)/Glu43(HtrII)/Ser62(HtrII). To investigate the importance of these hydrogen bonds, the K(D) value for the binding of various mutants of bR, hR, sR and pR with HtrII was estimated by ITC. The K(D) value of T189V(pR)/Y199F(pR), double mutant/HtrII complex, was about 100-fold larger than that of the wild-type pR, whose K(D) value was 0.16 microM. On the other hand, bR and hR double mutants, P200T(bR)/V210Y(bR) and P240T(hR)/F250Y(hR), were able to bind with HtrII. The K(D) value of these complexes was estimated to be 60.1(+/-10.7) microM for bR and to be 29.1(+/-6.1) microM for hR, while the wild-type bR and hR did not bind with HtrII. We concluded that these two specific hydrogen bonds play important roles in the binding between the rhodopsins and transducer protein.

Linker region of a halobacterial transducer protein interacts directly with its sensor retinal protein.

pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (K(D)) was estimated to be approximately 10 microM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrII(G83)(-)(Q149)) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppR(Gly224)(-)(His247)) and either the linker region (pHtrII(G83)(-)(Q149)) or the transmembrane region (pHtrII(M1)(-)(E114)) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction.

Interaction of Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II) with its cognate transducer probed by increase in the thermal stability.

Pharaonis phoborhodopsin (ppR, also called Natronobacterium pharaonis sensory rhodopsin II) and its transducer protein, pharaonis halobacterial transducer of ppR (pHtrII), form a signaling complex, and light signals are transmitted from the sensor to the transducer by the protein-protein interaction. A truncated pHtrII(1-159) consisting of intramembrane helices (expressing amino acid residues from the first to the 159th position) and ppR form the complex in a solution containing 0.1% n-dodecyl-beta-D-maltoside. At 75-85 degrees C, the time-dependent color loss of ppR was caused by denaturation. We found that pHtrII(1-159) retarded the denaturation rate of ppR. This increase in the thermal stability was used as a probe for the binding ability in the dark. Tyr199 of ppR and Asn74 of pHtrII(1-114) were proposed as amino acid residues interacting with each other through hydrogen bonding. Then,ppR and pHtrII(1-159) mutants at these positions were prepared to examine the effect on the binding in the dark. The wild-type and Y199F mutant can bind pHtrII(1-159), suggesting that the hydrogen bonding between these specific amino acid residues may not be the only cause of the binding, but the hydrophobic interaction via phenyl ring of ppR may contribute dominantly.