Antibacterial protein 3 homolog

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a complex of truncated mTOR and mammalian lethal with SEC13 protein 8 (mLST8) with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and a catalytic mechanism remarkably similar to canonical protein kinases. The active site is highly recessed owing to the FKBP12-rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR-activating mutations map to the structural framework that holds these elements in place, indicating that the kinase is controlled by restricted access. In vitro biochemistry shows that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. Rapamycin-FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting active-site access. The structures also reveal active-site residues and conformational changes that underlie inhibitor potency and specificity.

Macroautophagy/autophagy is known to be important for intracellular quality control in the lens. GJA8 is a major gap junction protein in vertebrate lenses. Mutations in GJA8 cause cataracts in humans. The well-known cataractogenesis mechanism is that mutated GJA8 leads to abnormal assembly of gap junctions, resulting in defects in intercellular communication among lens cells. In this study, we observed that ablation of Gja8b (a homolog of mammalian GJA8) in zebrafish led to severe defects in organelle degradation, an important cause of cataractogenesis in developing lens. The role of autophagy in organelle degradation in lens remains disputable. Intriguingly, we also observed that ablation of Gja8b induced deficient autophagy in the lens. More importantly, in vivo treatment of zebrafish with rapamycin, an autophagy activator that inhibits MAPK/JNK and MTORC1 signaling, stimulated autophagy in the lens and relieved the defects in organelle degradation, resulting in the mitigation of cataracts in gja8b mutant zebrafish. Conversely, inhibition of autophagy by treatment with the chemical reagent 3-MA blocked these recovery effects, suggesting the important roles of autophagy in organelle degradation in the lens in gja8b mutant zebrafish. Further studies in HLE cells revealed that GJA8 interacted with ATG proteins. Overexpression of GJA8 stimulated autophagy in HLE cells. These data suggest an unrecognized cataractogenesis mechanism caused by ablation of Gja8b and a potential treatment for cataracts by stimulating autophagy in the lens.

Defective mitophagy contributes to normal aging and various neurodegenerative and cardiovascular diseases. The newly developed methodologies to visualize and quantify mitophagy allow for additional progress in defining the pathophysiological significance of mitophagy in various model organisms. However, current knowledge regarding mitophagy relevant to human physiology is still limited. Model organisms such as mice might not be optimal models to recapitulate all the key aspects of human disease phenotypes. The development of the human-induced pluripotent stem cells (hiPSCs) may provide an exquisite approach to bridge the gap between animal mitophagy models and human physiology. To explore this premise, we take advantage of the pH-dependent fluorescent mitophagy reporter, mt-Keima, to assess mitophagy in hiPSCs and hiPSC-derived cardiomyocytes (hiPSC-CMs). We demonstrate that mt-Keima expression does not affect mitochondrial function or cardiomyocytes contractility. Comparison of hiPSCs and hiPSC-CMs during different stages of differentiation revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima hiPSC-CMs to analyze how mitophagy is altered under certain pathological conditions including treating the hiPSC-CMs with doxorubicin, a chemotherapeutic drug well known to cause life-threatening cardiotoxicity, and hypoxia that stimulates ischemia injury. We have further developed a chemical screening to identify compounds that modulate mitophagy in hiPSC-CMs. The ability to assess mitophagy in hiPSC-CMs suggests that the mt-Keima hiPSCs should be a valuable resource in determining the role mitophagy plays in human physiology and hiPSC-based disease models. The mt-Keima hiPSCs could prove a tremendous asset in the search for pharmacological interventions that promote mitophagy as a therapeutic target.