Structures of Vac8-containing protein complexes reveal the underlying mechanism by which Vac8 regulates multiple cellular processes.

Vac8, a yeast vacuolar protein with armadillo repeats, mediates various cellular processes by changing its binding partners; however, the mechanism by which Vac8 differentially regulates these processes remains poorly understood. Vac8 interacts with Nvj1 to form the nuclear-vacuole junction (NVJ) and with Atg13 to mediate cytoplasm-to-vacuole targeting (Cvt), a selective autophagy-like pathway that delivers cytoplasmic aminopeptidase I directly to the vacuole. In addition, Vac8 associates with Myo2, a yeast class V myosin, through its interaction with Vac17 for vacuolar inheritance from the mother cell to the emerging daughter cell during cell divisions. Here, we determined the X-ray crystal structure of the Vac8-Vac17 complex and found that its interaction interfaces are bipartite, unlike those of the Vac8-Nvj1 and Vac8-Atg13 complexes. When the key amino acids present in the interface between Vac8 and Vac17 were mutated, vacuole inheritance was severely impaired in vivo. Furthermore, binding of Vac17 to Vac8 prevented dimerization of Vac8, which is required for its interactions with Nvj1 and Atg13, by clamping the H1 helix to the ARM1 domain of Vac8 and thereby preventing exposure of the binding interface for Vac8 dimerization. Consistently, the binding affinity of Vac17-bound Vac8 for Nvj1 or Atg13 was markedly lower than that of free Vac8. Likewise, free Vac17 had no affinity for the Vac8-Nvj1 and Vac8-Atg13 complexes. These results provide insights into how vacuole inheritance and other Vac8-mediated processes, such as NVJ formation and Cvt, occur independently of one another.

Human atlastins are sufficient to drive the fusion of liposomes with a physiological lipid composition.

The dynamin-like GTPase atlastin is believed to be the minimal machinery required for homotypic endoplasmic reticulum (ER) membrane fusion, mainly because Drosophila atlastin is sufficient to drive liposome fusion. However, it remains unclear whether mammalian atlastins, including the three human atlastins, are sufficient to induce liposome fusion, raising doubts about their major roles in mammalian cells. Here, we show that all human atlastins are sufficient to induce fusion when reconstituted into liposomes with a lipid composition mimicking that of the ER. Although the fusogenic activity of ATL1, which is predominantly expressed in neuronal cells, was weaker than that of ATL2 or ATL3, the addition of M1-spastin, a neuron-specific factor, markedly increased ATL1-mediated liposome fusion. Although we observed efficient fusion between ER microsomes isolated from cultured, non-neuronal cells that predominantly express ATL2-1, an autoinhibited isoform of ATL2, ATL2-1 failed to support liposome fusion by itself as reported previously, indicating that cellular factors enable ATL2-1 to mediate ER fusion in vivo.

Upregulation of duck interleukin-17A during Riemerella anatipestifer infection.

Although IL-17 cytokines play critical roles in host defense immunity, dysregulated expression of these cytokines is associated with inflammation and autoimmune diseases. Riemerella anatipestifer is the most important infectious bacterium in the duck industry. Interestingly, not all avian species are equally susceptible to R. anatipestifer infection. This paper reports the first description of mortality rate, bacterial burden, and expression profiles of immune-related genes between ducks and chickens infected with R. anatipestifer. Ducks exhibited increased susceptibility to R. anatipestifer infection compared to chickens, as determined by mortality rate and bacterial burden. Comparative expression analyses of immune-related genes in R. anatipestifer-infected tissues obtained from both species revealed that TLR3, TLR7, IL-2, IL-4, and IFN-γ transcript levels were higher in chickens, whereas TLR4 and IL-17A transcript levels were higher in ducks. Marked increases in expression of IL-17A and IL-6, but not TGF-ß, were associated with Th17 cell differentiation in duck splenic lymphocytes, but not in chicken splenic lymphocytes, stimulated with R. anatipestifer. Moreover, upregulation of IL-1ß, IL-6, and IL-17A mRNA expressions, but not TGF-ß, was confirmed in the liver and spleen of ducks infected with R. anatipestifer, indicating that IL-17A is strongly associated with Riemerella infection in ducks.

Identification and expression analysis of duck interleukin-17D in Riemerella anatipestifer infection.

Interleukin (IL)-17D is a proinflammatory cytokine with currently largely unknown biological functions. Here we provide the description of the sequence, bioactivity, and mRNA expression profile of duck IL-17D homologue. A full-length duck IL-17D (duIL-17D) cDNA with a 624-bp coding region was identified from the large intestine. duIL-17D shares approximately 94.7% identity with its chicken counterpart, which is also identified in this work. duIL-17D exhibits 62.6-68.4% and 52.1-53.1% identity with mammalian and piscine homologues. Recombinant duIL-17D promoted the expression of proinflammatory cytokines such as IL-6, IL-8, and IL-1ß in duck embryo fibroblast cells. Very low levels of duIL-17D transcript were observed in healthy lymphoid tissues, including bursa, thymus, and spleen, while duIL-17D expression was relatively high in the heart. The duIL-17D expression profiles were examined in mitogen-stimulated splenic lymphocytes, as well as tissues affected by Riemerella anatipestifer infection. The levels of duIL-17D were mostly upregulated in mitogen-activated splenic lymphocytes but downregulated in the liver and spleen of R. anatipestifer-infected ducks. These results provide new insights into the roles of IL-17D in host protective immune responses to Riemerella infection, which can therefore lead to further studies of its biological functions in different disease models of ducks and other avian species.

Molecular cloning, characterization and mRNA expression of duck interleukin-17F.

Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 510-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17. duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37-46% with corresponding mammalian homologues, and 53.5% with the previously described duck IL-17A (duIL-17A). The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes but not in poly I:C stimulated cells. duIL-17F forms heterodimers with duIL-17A. Recombinant duIL-17F, like duIL-17A, induced IL-1ß, IL-6, and IL-8 expression in duck embryonic fibroblasts (DEFs). duIL-17A, but not duIL-17F expression, was significantly up-regulated in the liver and spleen of Salmonella Typhimurium-infected ducks. Further analysis of the contributions of IL-17F to different Salmonella spp. or other disease models will be required to expand our understanding of its biological functions.