Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate

Glycogen synthase (GYS1), in complex with glycogenin (GYG1), is the central enzyme of muscle glycogen biosynthesis, and its inhibition has been proposed as a therapeutic avenue for various glycogen storage diseases (GSDs). GYS1 activity is inhibited by phosphorylation of its N- and C- termini, which can be relieved by allosteric activation of glucose-6-phosphate. However, the structural basis of GYS1 regulation is unclear. Here, we present the first cryo-EM structures of phosphorylated human GYS1 complexed with a minimal interacting region of GYG1 in the inhibited, activated, and catalytically competent states at resolutions of 3.0-4.0 Å. These structures reveal how phosphorylations of specific N- and C- terminal residues are sensed by different arginine clusters that lock the GYS1 tetramer complex in an inhibited state via inter-subunit interactions. The allosteric activator, glucose-6-phopshate, promotes a conformational change by disrupting these interactions and increases flexibility of GYS1 allowing for a catalytically competent state to occur when bound to the sugar donor UDP-glucose. We also identify an inhibited-like conformation that has not transitioned into the activated state, whereby the locking interaction of phosphorylation with the arginine cluster impedes the subsequent conformational changes due to glucose-6-phosphate binding. Finally, we show that the PP1 phosphatase regulatory subunit PPP1R3C (PTG) is recruited to the GYS1:GYG1 complex through direct interaction with glycogen. Our data provide the first mechanistic insights into human glycogen synthase regulation.

ECS-based investigation of chloroplast ATP synthase regulation

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain which contains a cysteine couple and interacts with the torque-generating βDELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock. In vitro, γ-disulfide formation slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient . Here, we utilize in vivo absorption spectroscopy measurements for functional CF1Fo activity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure the present in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extra generated by an illumination, including its osmotic and electric components, and from there we estimate the lifetime of the light-generated ATP. In contrast with a previous report [Joliot and Joliot, Biochim. Biophys. Acta, 1777 (2008) 676-683], the CF1Fo γ-subunit exists mostly in an oxidized form in the dark-adapted state. To study the redox regulation of the CF1Fo, we used thiol agent infiltration in WT and a mutant that does not form the γ-disulfide. The obtained -dependent CF1Fo activity profile in the two γ-redox states in vivo reconciles with previous biochemical in vitro findings [Junesch and Gräber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two γ-redox state were similar at high . In the presence of the γ-dithiol, similar rates were obtained at a ~45 mV lower value compared to the oxidized state, which closely resembled the energetic gap of 0.7 ΔpH units reported in vitro.

Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.

CTP synthase regulation by miR-975 controls cell proliferation and differentiation in Drosophila melanogaster

CTP synthase (CTPsyn) is an essential metabolic enzyme. As a key regulator of the nucleotide pool, the protein has been found to be elevated in cancer models. In many organisms, CTPsyn compartmentalizes into filaments termed cytoophidia. For D. melanogaster, it is only its Isoform C i.e. CTPsynIsoC which forms the structure. The fruit fly’s testis is home to somatic and germline stem cells. Both micro and macro-cytoophidia are normally seen in the transit amplification regions close to its apical tip, where the stem-cell niche is located and development is at its most rapid. Here, we report that CTPsynIsoC overexpression causes the lengthening of cytoophidia throughout the entirety of the testicular body. A bulging apical tip is found in approximately one-third of like-genotyped males. Immunostaining shows that the cause of this tumour-like phenotype is most likely due to increased numbers of both germline cells and spermatocytes. We also report that under conditions whereby miR-975 is overexpressed, greater incidences of the same bulged-phenotype coincides with induced upregulation of CTPsynIsoC. However, RT-qPCR assays reveal that either overexpression genotype provokes a differential response in expression of a number of genes concurrently associated with CTPsyn and cancer, showing that the pathways CTPsynIsoC affect and miR-975 regulate may be completely independent of each other. This study presents the first instance of consequences of miRNA-asserted regulation upon CTPsyn in D. melanogaster, and further reaffirms the enzyme’s close ties to cancer and carcinogenesis.