Homocysteinylation and sulfhydration in diseases

: Homocysteine (Hcy) is an important intermediate in methionine metabolism and generation of one-carbon unit, and its dysfunction is associated with many pathological states. Although Hcy is a non-protein amino acid, many studies have demonstrated protein-related homocysteine metabolism and possible mechanisms underlying homocysteinylation. Homocysteinylated proteins lose their original biological function and have a negative effect on the various disease phenotypes. Hydrogen sulfide (H2S) has been recognized as an important gaseous signaling molecule with mounting physiological properties. H2S modifies small molecules and proteins via sulfhydration, which is supposed to be essential in the regulation of biological functions and signal transduction in human health and disorders. This review briefly introduces Hcy and H2S, further discusses pathophysiological consequences of homocysteine modification and sulfhydryl modification, and ultimately makes a prediction that H2S might exert a protective effect on the toxicity of homocysteinylation of target protein via sulfhydration. The highlighted information here yields new insights for the role of protein modification by Hcy and H2S in diseases.

Allosteric activation of an ion channel triggered by modification of mechanosensitive nano-pockets

Lipid availability within transmembrane nano-pockets of ion channels is linked with mechanosensation. However, the effect of hindering lipid-chain penetration into nano-pockets on channel structure has not been demonstrated. Here we identify nano-pockets on the large conductance mechanosensitive channel MscL, the high-pressure threshold channel. We restrict lipid-chain access to the nano-pockets by mutagenesis and sulfhydryl modification, and monitor channel conformation by PELDOR/DEER spectroscopy. For a single site located at the entrance of the nano-pockets and distal to the channel pore we generate an allosteric response in the absence of tension. Single-channel recordings reveal a significant decrease in the pressure activation threshold of the modified channel and a sub-conducting state in the absence of applied tension. Threshold is restored to wild-type levels upon reduction of the sulfhydryl modification. The modification associated with the conformational change restricts lipid access to the nano-pocket, interrupting the contact between the membrane and the channel that mediates mechanosensitivity.

Alphaxalone Binds in Inner Transmembrane β+–α− Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors

Background Neurosteroids like alphaxalone are potent anxiolytics, anticonvulsants, amnestics, and sedative-hypnotics, with effects linked to enhancement of γ-aminobutyric acid type A (GABAA) receptor gating in the central nervous system. Data locating neurosteroid binding sites on synaptic αβγ GABAA receptors are sparse and inconsistent. Some evidence points to outer transmembrane β+–α− interfacial pockets, near sites that bind the anesthetics etomidate and propofol. Other evidence suggests that steroids bind more intracellularly in β+–α− interfaces. Methods The authors created 12 single-residue β3 cysteine mutations: β3T262C and β3T266C in β3-M2; and β3M283C, β3Y284C, β3M286C, β3G287C, β3F289C, β3V290C, β3F293C, β3L297C, β3E298C, and β3F301C in β3-M3 helices. The authors coexpressed α1 and γ2L with each mutant β3 subunit in Xenopus oocytes and electrophysiologically tested each mutant for covalent sulfhydryl modification by the water-soluble reagent para-chloromercuribenzenesulfonate. Then, the authors assessed whether receptor-bound alphaxalone, etomidate, or propofol blocked cysteine modification, implying steric hindrance. Results Eleven mutant β3 subunits, when coexpressed with α1 and γ2L, formed functional channels that displayed varied sensitivities to the three anesthetics. Exposure to para-chloromercuribenzenesulfonate produced irreversible functional changes in ten mutant receptors. Protection by alphaxalone was observed in receptors with β3V290C, β3F293C, β3L297C, or β3F301C mutations. Both etomidate and propofol protected receptors with β3M286C or β3V290C mutations. Etomidate also protected β3F289C. In α1β3γ2L structural homology models, all these protected residues are located in transmembrane β+–α− interfaces. Conclusions Alphaxalone binds in transmembrane β+–α− pockets of synaptic GABAA receptors that are adjacent and intracellular to sites for the potent anesthetics etomidate and propofol.