• ZAG and a potential role in systemic lipid homeostastis: examining the evidence from in vitro human studies and patients with chronic illness

      McDermott, Lindsay C.; Jadoon, Ayesha; Cunningham, Phil (Future Medicine, 2012-08-31)
      ZAG, a 42 kDa ubiquitously expressed soluble and secreted protein, consists of an MHC-like fold and binds fatty acids with an affinity similar to that of albumin. In human adipocytes, cytoplasmic and secreted ZAG appear inversely related to fat mass. Immunoblotting and biochemical measurements of human cachectic adipocytes and plasma suggest that ZAG’s lipolytic function may be mediated by βIAR. Plasma ZAG correlates with cholesterol in human populations, as does its single nucleotide polymorphism rs4215, which also associates with obesity. Biochemical and human genetic data, in vitro experiments and theoretical data imply that adipocyte ZAG’s expression is regulated by PPAR-γ and glucocorticoids. Cell biological experiments and data from human tissue indicate that ZAG may induce fatty-acid oxidation in skeletal muscle. Overall, these findings suggest ZAG’s participation in systemic lipid homeostasis. Understanding the molecular mechanisms behind ZAG’s in vivo behavior potentially allows for rational drug design to control body fat mass.
    • Zinc-induced oligomerisation of zinc α2 glycoprotein reveals multiple fatty acid binding sites

      Zahid, Henna; Miah, Layeque; Lau, Andy M.; Brochard, Lea; Hati, Debolina; Bui, Tam T. T.; Drake, Alex F.; Gor, Jayesh; Perkins, Stephen J.; McDermott, Lindsay C.; et al. (Portland Press, 2015-12-09)
      Zinc α2 glycoprotein (ZAG) is an adipokine with a class I major histocompatibility complex protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid, DAUDA, in the groove between the α1 and α2 domains. The surface of ZAG has about 15 weak zinc binding sites deemed responsible for precipitation from human plasma. Here the functional significance of these metal sites was investigated. Analytical ultracentrifugation and circular dichroism showed that zinc, but not other divalent metals, cause ZAG to oligomerise in solution. Thus ZAG dimers and trimers were observed in the presence of 1 mM and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY. Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid binding groove in ZAG contains at least two distinct fatty acid binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally-related immune protein Cd1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homeostasis occurs.
    • Zn-alpha2-glycoprotein, an MHC class I-related glycoprotein regulator of adipose tissues: modification or abrogation of ligand binding by site-directed mutagenesis

      McDermott, Lindsay C.; Freel, June A.; West, Anthony P.; Bjorkman, Pamela J.; Kennedy, Malcolm W.; University of Glasgow; California Institute of Technology (American Chemical Society, 2006-01-31)
      Zn-alpha(2)-glycoprotein (ZAG) is a soluble lipid-mobilizing factor associated with cancer cachexia and is a novel adipokine. Its X-ray crystal structure reveals a poly(ethylene glycol) molecule, presumably substituting for a higher affinity natural ligand, occupying an apolar groove between its alpha(1) and alpha(2) domain helices that corresponds to the peptide binding groove in class I MHC proteins. We previously provided evidence that the groove is a binding site for hydrophobic ligands that may relate to the protein's signaling function and that the natural ligands are probably (polyunsaturated) fatty acid-like. Using fluorescence-based binding assays and site-directed mutagenesis, we now demonstrate formally that the groove is indeed the binding site for hydrophobic ligands. We also identify amino acid positions that are involved in ligand binding and those that control the shape and exposure to solvent of the binding site itself. Some of the mutants showed minimal effects on their binding potential, one showed enhanced binding, and several were completely nonbinding. Particularly notable is Arg-73, which projects into one end of the binding groove and is the sole charged amino acid adjacent to the ligand. Replacing this amino acid with alanine abolished ligand binding and closed the groove to solvent. Arg-73 may therefore have an unexpected dual role in binding site access and anchor for an amphiphilic ligand. These data add weight to the distinctiveness of ZAG among MHC class I-like proteins in addition to providing defined binding-altered mutants for cellular signaling studies and potential medical applications.
    • β-alanine supplementation enhances human skeletal muscle relaxation speed but not force production capacity

      Hannah, Ricci; Jones, Rebecca Louise; Minshull, Claire; Artioli, Guilherme Giannini; Harris, Roger C.; Sale, Craig (American Physiological Society, 2015-03-01)
      PURPOSE: β-alanine (BA) supplementation improves human exercise performance. One possible explanation for this is an enhancement of muscle contractile properties, occurring via elevated intramuscular carnosine resulting in improved calcium sensitivity and handling. This study investigated the effect of BA supplementation on in vivo contractile properties and voluntary neuromuscular performance. METHODS: Twenty-three men completed two experimental sessions, pre- and post-28 days supplementation with 6.4 g·d-1 39 of BA (n = 12) or placebo (PLA; n = 11). During each session, force was recorded during a series of knee extensor contractions: resting and potentiated twitches and octet (8 pulses, 300 Hz) contractions elicited via femoral nerve stimulation; tetanic contractions (1 s, 1 – 100 Hz) via superficial muscle stimulation; and maximum and explosive voluntary contractions. RESULTS: BA supplementation had no effect on the force-frequency relationship, or the force responses (force at 25 ms and 50 ms from onset, peak force) of resting or potentiated twitches, and octet contractions (P > 0.05). Resting and potentiated twitch electromechanical delay and time-to-peak tension were unaffected by BA supplementation (P > 0.05), although half-relaxation time declined by 7-12% (P < 0.05). Maximum and explosive voluntary forces were unchanged after BA supplementation. CONCLUSION: BA supplementation had no effect on evoked force responses, implying that altered calcium sensitivity and/or release are not the mechanisms by which BA supplementation influences exercise performance. The reduced half-relaxation time with BA supplementation might, however, be explained by enhanced reuptake of calcium, which has implications for the efficiency of muscle contraction following BA supplementation.
    • β-alanine supplementation enhances human skeletal muscle relaxation speed but not force production capacity

      Jones, Rebecca Louise; Hannah, Ricci; Minshull, Claire; Artioli, Guilherme Giannini; Harris, Roger C.; Sale, Craig (2015-09-20)
    • β-alanine supplementation improves in-vivo fresh and fatigued muscle relaxation speed

      Jones, Rebecca Louise; Barnett, T.C.; Davidson, Joel; Maritza, Billy; Fraser, William D.; Harris, Roger C.; Sale, Craig (2017-09-20)
    • β-alanine supplementation improves in-vivo fresh and fatigued muscle relaxation speed

      Jones, Rebecca Louise; Barnett, Cleveland Thomas; Davidson, Joel; Maritza, Billy; Fraser, William D.; Harris, Roger C.; Sale, Craig; Nottingham Trent University; University of East Anglia; Norfolk and Norwich University Hospital; et al. (Springer International Publishing AG, 2017-03-27)
      PURPOSE: In fresh muscle, supplementation with the rate-limiting precursor of carnosine, β-alanine (BA), results in a decline in muscle half-relaxation time (HRT) potentially via alterations to calcium (Ca2+) handling. Accumulation of hydrogen cation (H+) has been shown to impact Ca2+ signalling during muscular contraction, carnosine has the potential to serve as a cytoplasmic regulator of Ca2+ and H+ coupling, since it binds to both ions. The present study examined the effect of BA supplementation on intrinsic in-vivo isometric knee extensor force production and muscle contractility in both fresh and fatigued human skeletal muscle assessed during voluntary and electrically evoked (nerve and superficial muscle stimulation) contractions. METHODS: Twenty-three males completed two experimental sessions, pre- and post- 28 day supplementation with 6.4 g.day-1 of BA (n = 12) or placebo (PLA; n = 11). Isometric force was recorded during a series of voluntary and electrically evoked knee extensor contractions. RESULTS: BA supplementation had no effect on voluntary or electrically evoked isometric force production, or twitch electromechanical delay and time-to-peak tension. There was a significant decline in muscle HRT in fresh and fatigued muscle conditions during both resting (3 ± 13%; 19 ± 26%) and potentiated (1 ± 15%; 2 ± 20%) twitch contractions. CONCLUSIONS: The mechanism for reduced HRT in fresh and fatigued skeletal muscle following BA supplementation is unclear. Due to the importance of muscle relaxation on total energy consumption, especially during short, repeated contractions, BA supplementation may prove to be beneficial in minimising contractile slowing induced by fatigue. TRIAL REGISTRATION: The trial is registered with Clinicaltrials.gov, ID number NCT02819505.
    • μECM process investigation considering pulse signal features and EDL capacitance

      Mortazavi, Mina; Ivanov, Atanas (Springer, 2019-05-22)
      Micro-electrochemical machining (μECM) is a controlled anodic dissolution process between electrodes. The anodic dissolution, which follows Faraday’s laws of electrolysis, depends on characteristics of the electrodes materials, electrolyte properties, and pulse signal features. μECM is a challenging multidisciplinary task in which quality of the process and features of the finished products depend on a complex relation between different machining parameters including, electrical features of pulse signal, chemical features of electrolyte, physical features of tools, and thermodynamic features of the process. In this paper, influential machining parameters will be reviewed briefly, and pulse signal features will be investigated and analyzed considering the behavior of the electrode-electrolyte interface. The interface has capacitive feature and plays an important role in micromachining performance. The proposed simulation work presents the requirement for the pulse on-time in order to provide the maximum possible charging-discharging time for the capacitive behavior of the electrode-electrolyte interface.