No abstract

In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid-starved Mesh1 null mutant has highly downregulated DNA and protein synthesis-related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.

Non-motile primary cilium is an antenna-like structure whose defect is associated with a wide range of pathologies, including developmental disorders and cancer. Although mechanisms regulating cilia assembly have been extensively studied, how cilia disassembly is regulated remains poorly understood. Here, we report unexpected roles of Dishevelled 2 (Dvl2) and interphase polo-like kinase 1 (Plk1) in primary cilia disassembly. We demonstrated that Dvl2 is phosphorylated at S143 and T224 in a manner that requires both non-canonical Wnt5a ligand and casein kinase 1 epsilon (CK1e), and that this event is critical to interact with Plk1 in early stages of the cell cycle. The resulting Dvl2-Plk1 complex mediated Wnt5a-CK1e-Dvl2-dependent primary cilia disassembly by stabilizing the HEF1 scaffold and activating its associated Aurora-A (AurA), a kinase crucially required for primary cilia disassembly. Thus, via the formation of the Dvl2-Plk1 complex, Plk1 plays an unanticipated role in primary cilia disassembly by linking Wnt5a-induced biochemical steps to HEF1/AurA-dependent cilia disassembly. This study may provide new insights into the mechanism underlying ciliary disassembly processes and various cilia-related disorders.

Macroautophagy mediates the selective degradation of proteins and non-proteinaceous cellular constituents. Here, we show that the N-end rule pathway modulates macroautophagy. In this mechanism, the autophagic adapter p62/SQSTM1/Sequestosome-1 is an N-recognin that binds type-1 and type-2 N-terminal degrons (N-degrons), including arginine (Nt-Arg). Both types of N-degrons bind its ZZ domain. By employing three-dimensional modeling, we developed synthetic ligands to p62 ZZ domain. The binding of Nt-Arg and synthetic ligands to ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62 interaction with LC3, leading to the delivery of p62 and its cargoes to the autophagosome. Upon binding to its ligand, p62 acts as a modulator of macroautophagy, inducing autophagosome biogenesis. Through these dual functions, cells can activate p62 and induce selective autophagy upon the accumulation of autophagic cargoes. We also propose that p62 mediates the crosstalk between the ubiquitin-proteasome system and autophagy through its binding Nt-Arg and other N-degrons.