The influenza virus M 2 proton-selective ion channel is known to be essential for acidifying the interior of virions during virus uncoating in the lumen of endosomes. The M 2 protein is a homotetramer that contains four 19-residue transmembrane (TM) domains. These TM domains are multifunctional, because they contain the channel pore and also anchor the protein in membranes. The M 2 protein is gated by pH, and thus we have measured pH-gated currents, the accessibility of the pore to Cu 2؉ , and the effect of a protein-modifying reagent for a series of TM domain mutant M 2 proteins. The results indicate that gating of the M 2 ion channel is governed by a single side chain at residue 41 of the TM domain and that this property is mediated by an indole moiety. Unlike many ion channels where the gate is formed by a whole segment of a protein, our data suggest a model of striking simplicity for the M 2 ion channel protein, with the side chain of Trp 41 blocking the pore of the M 2 channel when pH out is high and with this side chain leaving the pore when pH out is low. Thus, the Trp 41 side chain acts as the gate that opens and closes the pore.The prediction that the influenza A virus M 2 protein has a proton-selective ion channel activity (Refs. 1 and 2 and reviewed in Ref.3) arose from a coupling of various observations on the life cycle of influenza virus. The M 2 protein is an integral membrane protein that is expressed at the plasma membrane of influenza virus-infected cells and is incorporated in small amounts into budding virions (4, 5). Studies on the mechanism of action of the anti-viral drug, amantadine (1-aminoadamantine hydrochloride), indicated that viral escape mutants resistant to the drug mapped to the transmembrane (TM) 1 domain of the M 2 protein (6) and that amantadine affected two steps in the life cycle, virus uncoating and virus maturation. The effect of amantadine on inhibition of uncoating is general to all strains of influenza A virus (7, 8) (reviewed in Refs. 3, 9 -11). When a virion has entered the cell by receptor-mediated endocytosis and the virus particle is in the acidic environment of the endosomal lumen, the M 2 ion channel is activated and conducts protons across the viral membrane. The lowered internal virion pH is thought to weaken protein-protein interactions between the viral matrix protein (M1) and the ribonucleoprotein (RNP) core (7,(12)(13)(14)(15)). In the presence of amantadine, influenza virus uncoating is incomplete, because the M1 protein is not released from the RNPs and the RNPs fail to enter the nucleus. Normally, influenza virus RNPs are transcribed and replicated in the nucleus (reviewed in Ref. 10). For some influenza virus subtypes, amantadine inhibits a "late" step in virus replication. The M 2 ion channel activity is activated during transport of the M 2 protein through the exocytic pathway; this ion channel activity raises the lumenal pH of the trans Golgi network (TGN), equilibrating pH with that of the cytoplasm (1, 17-22). Thus, the intralumenal pH of the TGN i...

Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 (FUNDC1) was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with microtubule-associated protein light chain 3 beta (LC3B) through its LC3 interaction region (LIR). Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDC1 LIR peptide phosphorylated at Ser17 (pS17), demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS17. Alternatively, phosphorylated Tyr18 (pY18) and Ser13 (pS13) in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry (ITC) assays. Therefore, our structural and biochemical results reveal a working model for the specific recognition of FUNDC1 by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0328-8) contains supplementary material, which is available to authorized users.

The human PPIL1 (peptidyl prolyl isomerase-like protein 1) is a specific component of human 35 S U5 small nuclear ribonucleoprotein particle and 45 S activated spliceosome. It is recruited by SKIP, another essential component of 45 S activated spliceosome, into spliceosome just before the catalytic step 1. It stably associates with SKIP, which also exists in 35 S and activated spliceosome as a nuclear matrix protein. We report here the solution structure of PPIL1 determined by NMR spectroscopy. The structure of PPIL1 resembles other members of the cyclophilin family and exhibits PPIase activity. To investigate its interaction with SKIP in vitro, we identified the SKIP contact region by GST pulldown experiments and surface plasmon resonance. We provide direct evidence of PPIL1 stably associated with SKIP. The dissociation constant is 1.25 ؋ 10 ؊7 M for the N-terminal peptide of SKIP-(59 -129) with PPIL1. We also used chemical shift perturbation experiments to show the possible SKIP binding interface on PPIL1. These results illustrated that a novel cyclophilin-protein contact mode exists in the PPIL1-SKIP complex during activation of the spliceosome. The biological implication of this binding with spliceosome rearrangement during activation is discussed.Pre-mRNA splicing, the removal of introns from mRNA precursors, is indispensable to the expression of most eukaryotic genes. The splicing of mRNA is catalyzed by spliceosome, a large machine formed by an ordered interaction of several small nuclear ribonucleoproteins (snRNPs), 3 U1, U2, U5, U4/U6, and numerous other less stably associated non-snRNP splicing factors (1, 2). The formation of spliceosome goes through many intermediate stages. The stable intermediate complexes are the A, B, and C complexes. During the spliceosome maturation process, the most decisive step is the conversion from non-active complex B to the catalytically active spliceosome B*. The activated complex B* undergoes the first catalytic step of splicing and then forms complex C. Prior to the activation of the spliceosome and during the splicing process, a number of conformational rearrangements take place. Recently, human 45 S activated spliceosome (complex B*) and 35 S U5 snRNP have been isolated by immunoaffinity purification and characterized by mass spectrometry (3). Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition. More than 100 proteins were identified in the 45 S activated spliceosome, 80 of which are known splicing factors. The rest are non-snRNP proteins, including protein SKIP and one peptidyl prolyl isomerase-like protein 1 (PPIL1) (2-4).PPIL1 is a component in 45 S U5 snRNP in activated spliceosome (complex B*) and 35 S snRNP. It was believed to participate in the activation of spliceosome (5). The cDNA of PPIL1 was first cloned from human fetal brain, which encodes 166 amino acid residues. PPIL1 has 41.6% identity to human cyclophilin A (6). It belongs to a novel subfamily of cyclophilins, to...

Calorie restriction (CR) and fasting are common approaches to weight reduction, but the maintenance is difficult after resuming food consumption. Meanwhile, the gut microbiome associated with energy harvest alters dramatically in response to nutrient deprivation. Here, we reported that CR and high-fat diet (HFD) both remodeled the gut microbiota with similar microbial composition, Parabacteroides distasonis was most significantly decreased after CR or HFD. CR altered microbiota and reprogramed metabolism, resulting in a distinct serum bile acid profile characterized by depleting the proportion of non-12α-hydroxylated bile acids, ursodeoxycholic acid and lithocholic acid. Downregulation of UCP1 expression in brown adipose tissue and decreased serum GLP-1 were observed in the weight-rebound mice. Moreover, treatment with Parabacteroides distasonis or non-12α-hydroxylated bile acids ameliorated weight regain via increased thermogenesis. Our results highlighted the gut microbiota-bile acid crosstalk in rebound weight gain and Parabacteroides distasonis as a potential probiotic to prevent rapid post-CR weight gain.