Hydrogen sulfide (H 2 S), a messenger molecule generated by cystathionine γ-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H 2 S signals have been elusive. We now show that H 2 S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.

Summary Nuclear factor κB (NF-κB) is an anti-apoptotic transcription factor. We show that the anti-apoptotic actions of NF-κB are mediated by hydrogen sulfide (H2S) synthesized by cystathionine gamma-lyase (CSE). TNFα treatment triples H2S generation by stimulating binding of SP1 to the CSE promoter. H2S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H2S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the co-activator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early following TNFα treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. Anti-apoptotic influences of NF-κB, which are markedly diminished in CSE mutant mice. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its anti-apoptotic transcriptional activity.

Summary The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase which regulates glucose homeostasis and protein translation respectively via the GSK3β and mTOR pathways. Thus Akt, mTOR and GSK3β signaling are dramatically augmented in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 impacts this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby impacting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.

Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3sigma. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3sigma from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.

Intermediate filaments (IFs) are cytoskeletal polymers whose protein constituents are encoded by a large family of differentially expressed genes. Owing in part to their properties and intracellular organization, IFs provide crucial structural support in the cytoplasm and nucleus, the perturbation of which causes cell and tissue fragility and accounts for a large number of genetic diseases in humans. A number of additional roles, nonmechanical in nature, have been recently uncovered for IF proteins. These include the regulation of key signaling pathways that control cell survival, cell growth, and vectorial processes including protein targeting in polarized cellular settings. As this discovery process continues to unfold, a rationale for the large size of this family and the contextdependent regulation of its members is finally emerging.Intermediate filaments (IFs), first described by Holtzer and colleagues (Ishikawa et al. 1968) from studies of muscle in the late 1960s, serve as ubiquitous cytoskeletal scaffolds in both the nucleus and cytoplasm of higher metazoans (Erber et al. 1998). In human, mouse, and other mammalian genomes, ∼70 conserved genes encode proteins that can self-assemble into 10-to 12-nmwide IFs. Apart from three lamin-encoding genes, whose products localize to and function in the nucleus, the other ∼67 IF genes encode cytoplasmic proteins (Table 1; Hesse et al. 2001). Although heterogeneous in size, primary structure, and regulation, IF proteins share a common tripartite domain structure, with the defining feature being a centrally located, 310-residue-long ␣-helical domain (352 for lamins) containing long-range heptad repeats of hydrophobic/apolar residues (Fig. 1A). These conserved features were formalized with the cloning and sequencing of the first IF protein-encoding gene, keratin 14 (Hanukoglu and Fuchs 1982). The central "rod" domain mediates coiled-coil dimer formation and otherwise represents the major driving force sustaining selfassembly (for review, see Fuchs and Weber 1994;Herrmann and Aebi 2004;Parry 2005). The rod is flanked, at both ends, by nonhelical sequences that differ in length, sequence, substructure, and properties. Variations in the so-called "head" and "tail" domains account for the marked heterogeneity in IF protein size (M r ∼ 40-240 kDa) (Table 1) and other attributes. A "one gene/one protein" rule seems to prevail in the family, as relatively few IF mRNAs (lamin A/C, GFAP, peripherin, and synemin) (Table 1) yield distinct protein products via alternative splicing.Most biomedical researchers' understanding of fibrous cytoskeletal polymers is primarily influenced by the extraordinary properties of F-actin and microtubules, whose pleiotropic roles tend to be universal and can be investigated in cultured cell lines and simple model eukaryotes (Alberts et al. 2002). IFs are fundamentally different, as follows: Functionally, cytoplasmic IF proteins are not required for life at the single-cell level, as evidenced by their complete absence in yeast, in Drosophila (Erber ...

SUMMARY While the abuse of opiate drugs continues to rise, the neuroadaptations that occur with long-term drug exposure remain poorly understood. We describe here a series of chronic morphine-induced adaptations in ventral tegmental area (VTA) dopamine neurons, which are mediated via downregulation of AKT-mTORC2 (mammalian target of rapamycin complex-2). Chronic opiates decrease the size of VTA dopamine neurons in rodents, an effect seen in humans as well, and concomitantly increase the excitability of the cells but decrease dopamine output to target regions. Chronic morphine decreases mTORC2 activity, and overexpression of Rictor, a component of mTORC2, prevents morphine-induced changes in cell morphology and activity. Further, local knock-out of Rictor in VTA decreases DA soma size and reduces rewarding responses to morphine, consistent with the hypothesis that these adaptations represent a mechanism of reward tolerance. Together, these findings demonstrate a novel role for AKT-mTORC2 signaling in mediating neuroadaptations to opiate drugs of abuse.

Inositol pyrophosphates are highly energetic inositol polyphosphate molecules present in organisms from slime molds and yeast to mammals. Distinct classes of enzymes generate different forms of inositol pyrophosphates. The biosynthesis of these substances principally involves phosphorylation of inositol hexakisphosphate (IP6) to generate the pyrophosphate IP7. Initial insights into functions of these substances derived primarily from yeast, which contain a single isoform of IP6 kinase (yIP6K), as well as from the slime mold Dictyostelium. Mammalian functions for inositol pyrophosphates have been investigated by using cell lines to establish roles in various processes, including insulin secretion and apoptosis. More recently, mice with targeted deletion of IP6K isoforms as well as the related inositol polyphosphate multikinase (IPMK) have substantially enhanced our understanding of inositol polyphosphate physiology. Phenotypic alterations in mice lacking inositol hexakisphosphate kinase 1 (IP6K1) reveal signaling roles for these molecules in insulin homeostasis, obesity, and immunological functions. Inositol pyrophosphates regulate these processes at least in part by inhibiting activation of the serine-threonine kinase Akt. Similar studies of IP6K2 establish this enzyme as a cell death inducer acting by stimulating the proapoptotic protein p53. IPMK is responsible for generating the inositol phosphate IP5 but also has phosphatidylinositol 3-kinase activity—that participates in activation of Akt. Here, we discuss recent advances in understanding the physiological functions of the inositol pyrophosphates based in substantial part on studies in mice with deletion of IP6K isoforms. These findings highlight the interplay of IPMK and IP6K in regulating growth factor and nutrient-mediated cell signaling.

We fabricated CZTSSe thin films using optimized SLG-Mo/Zn/Cu/Sn (MZCT) as a stacked structure and described the phenomenon of Zn elemental volatilization using the MZCT stacked structure.