Song (2015) Insights into autophagosome maturation revealed by the structures of ATG5 with its interacting partners, Autophagy, 11:1, 75-87, DOI: 10.4161/15548627.2014 Autophagy is a bulky catabolic process that responds to nutrient homeostasis and extracellular stress signals and is a conserved mechanism in all eukaryotes. When autophagy is induced, cellular components are sequestered within an autophagosome and finally degraded by subsequent fusion with a lysosome. During this process, the ATG12-ATG5 conjugate requires 2 different binding partners, ATG16L1 for autophagosome elongation and TECPR1 for lysosomal fusion. In our current study, we describe the crystal structures of human ATG5 in complex with an N-terminal domain of ATG16L1 as well as an internal AIR domain of TECPR1. Both binding partners exhibit a similar a-helical structure containing a conserved binding motif termed AFIM. Furthermore, we characterize the critical role of the C-terminal unstructured region of the AIR domain of TECPR1. These findings are further confirmed by biochemical and cell biological analyses. These results provide new insights into the molecular details of the autophagosome maturation process, from its elongation to its fusion with a lysosome.
Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N-and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.
The nuclear factor (NF)-kB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-kB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor a (TNFa) receptor 1 signaling complex (TNF-RSC) and attenuates TNFa-induced NF-kB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFa-induced increase in IkB kinase (IKK) activity, as well as the expression of NF-kB target genes. TNFa induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFa stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKg. Together, our findings suggest that MST1 negatively regulates TNFa-induced NF-kB signaling by targeting LUBAC.
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