Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children, and structural characterization of EV71 during its life cycle can aid in the development of therapeutics against HFMD. Here, we present the atomic structures of the full virion and an uncoating intermediate of a clinical EV71 C4 strain to illustrate the structural changes in the full virion that lead to the formation of the uncoating intermediate prepared for RNA release. Although the VP1 N-terminal regions observed to penetrate through the junction channel at the quasi-3-fold axis in the uncoating intermediate of coxsackievirus A16 were not observed in the EV71 uncoating intermediate, drastic conformational changes occur in this region, as has been observed in all capsid proteins. Additionally, the RNA genome interacts with the N-terminal extensions of VP1 and residues 32 to 36 of VP3, both of which are situated at the bottom of the junction. These observations highlight the importance of the junction for genome release. Furthermore, EV71 uncoating is associated with apparent rearrangements and expansion around the 2-and 5-fold axes without obvious changes around the 3-fold axes. Therefore, these structures enabled the identification of hot spots for capsid rearrangements, which led to the hypothesis that the protomer interface near the junction and the 2-fold axis permits the opening of large channels for the exit of polypeptides and viral RNA, which is an uncoating mechanism that is likely conserved in enteroviruses. IMPORTANCE Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD Human enterovirus 71 (EV71) is the leading causative agent for severe hand-foot-and-mouth diseases (HFMD) in infants and young children (1), and EV71 infection has caused significant morbidity and mortality in the Asia-Pacific regions. During 2008 to 2012, numerous outbreaks of HFMD occurred in mainland China, with over 2,000 fatal cases reported (www.chinacdc.cn). However, vaccines and effective drugs remain unavailable. EV71 belongs to Enterovirus species A within the Enterovirus genus of the Picornaviridae (2). The EV71 virion contains a singlestranded positive-sense RNA genome of 7.5 kb. The viral capsid, which has a diameter of approximately 300 Å, is composed of 60 copies each of VP1 to VP4 (3, 4) that are organized onto a quasi-Tϭ3 icosahedral lattice. The capsid proteins VP1, VP2, and VP3 each possess a ␤-sandwich jelly roll fold and form the outer surface of the capsid shell, whereas VP4 is situated inside the shell. The capsid surface is characterized by a depression encircling each 5-fold symmetry axis, which is referred to as the "canyon" and often contains the receptor-binding site in picornaviruses (5, 6). The hydrophobic pocket of VP1, which is located at the bottom of the canyon, contains a lipid moiety termed the "pocket factor," which likely stabilizes the mature virion. As observed in poliovirus and certain picornaviruses, receptor bindin...

The Na؉ /H ؉ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H ؉ for one extracellular Na ؉ . It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251-273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na ؉ /H ؉ exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly ␣-helical, with a break in the helix at the functionally critical residues Gly 261 -Glu 262 . The relative positions and orientations of the N-and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition. The mammalian Naϩ /H ϩ exchanger isoform 1 (NHE1) 6 is a ubiquitous integral membrane protein mediating removal of a single intracellular proton in exchange for one extracellular sodium ion (1). NHE1 has several cellular and physiological functions, including protecting cells from intracellular acidification (2, 3), promoting cell growth and differentiation (2), and regulating sodium fluxes and cell volume after osmotic shrinkage (4). The Na ϩ /H ϩ exchanger also plays a critical role in the damage that occurs during ischemia and reperfusion and may play a key role in mediating heart hypertrophy. Inhibition of the exchanger protects the myocardium in these two forms of heart disease (5, 6). Amiloride and its derivatives are inhibitors of the NHE1 isoform of the Na ϩ /H ϩ exchanger, and a new generation of Na ϩ /H ϩ exchanger inhibitors is being developed for clinical treatment of heart disease (7,8). In addition to these many physiological roles, more recently, the Na ϩ /H ϩ exchanger has been demonstrated to be involved in modulating cell motility and invasiveness of neoplastic breast cancer cells (9) and has been shown to be critical to cell motility in some cell types (10).NHE1 is composed of two domains as follows: an N-terminal membrane domain of ϳ500 amino acids and a C-terminal regulatory domain of ϳ315 amino acids (1, 6). The N-terminal membrane domain is responsible for ion movement and has 12 transmembrane segments ...

The AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by sensing the metabolic status of the cell. AMPK is regulated by phosphorylation and dephosphorylation as a result of changing AMP/ATP levels and by removal of inhibitory ubiquitin residues by USP10. In this context, we identified the GID-complex, an evolutionarily conserved ubiquitin-ligase-complex (E3), as a negative regulator of AMPK activity. Our data show that the GID-complex targets AMPK for ubiquitination thereby altering its activity. Cells depleted of GID-subunits mimic a state of starvation as shown by increased AMPK activity and macroautophagic/autophagic flux as well as reduced MTOR activation. Consistently, gid-genes knockdown in C. elegans results in increased organismal lifespan. This study may contribute to understand metabolic disorders such as type 2 diabetes mellitus and morbid obesity and implements alternative therapeutic approaches to alter AMPK activity.

The Na؉ /H ؉ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H ؉ in exchange for one extracellular Na ؉ . It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339 -363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 The mammalian Na ϩ /H ϩ exchanger isoform 1 (NHE1) 6 is a ubiquitous integral membrane protein that regulates intracellular pH by mediating removal of one intracellular proton in exchange for one extracellular sodium ion (1). NHE1 has many cellular and physiological functions. It promotes cell growth and differentiation (2), protects cells from intracellular acidification (2, 3), and also regulates sodium fluxes and cell volume after challenge by osmotic shrinkage (4). In addition, Na ϩ /H ϩ exchanger activity has been demonstrated to be involved in modulating cell motility and invasiveness of neoplastic breast cancer cells (5), and it has been shown to be critical to cell motility in some cell types (6). The Na ϩ /H ϩ exchanger also plays a critical causal role in heart hypertrophy and in the damage that occurs during ischemia and reperfusion. Inhibition of the exchanger with a new generation of Na ϩ /H ϩ exchanger inhibitors protects the myocardium during these disease states (7-10).NHE1 has an N-terminal membrane ion translocation domain of ϳ500 amino acids and a C-terminal regulatory domain of ϳ315 amino acids (1, 8). Wakabayashi et al. (11) used a combination of hydrophobicity analysis and cysteine-scanning mutagenesis to predict a topology with 3 membrane-associated segments and 12 integral transmembrane (TM) segments (Fig. 1A). A recent homology model, based on the Escherichia coli antiporter NhaA crystal structure (12), also predicts 12 TM segments, but assignments of transmembrane segments are different in comparison with the Wakabayashi topology (13).The exact details of cation coordination and translocation through the central ion pore of NHE1 have only recently begun to emerge. In this respect, we have identified TM IV (14) and TM VII (15) (with topology assignments according to Ref. 11) as functionally critical. More recently, we solved the structures of these TM segments and identified specific essential residues (16,17). Prolines 167 and 168 of TM IV were essential to NHE1 function (14) and Phe 161 was shown to be a porelining residue involved in transport using cysteine-scanning mutagenesis (16). The peptide was composed of an N-terminal ␤-turn region, followed by an extended (nonhelical) structure * This work was supported in part by gr...