Redox sulfur chemistry of the copper chaperone Atox1 is regulated by the enzyme glutaredoxin 1, the reduction potential of the glutathione couple GSSG/2GSH and the availability of Cu(I)

Glutaredoxins have been characterised as enzymes regulating the redox status of protein thiols via cofactors GSSG/GSH. However, such a function has not been demonstrated with physiologically relevant protein substrates in in vitro experiments. Their active sites frequently feature a Cys-xx-Cys motif that is predicted not to bind metal ions. Such motifs are also present in copper-transporting proteins such as Atox1, a human cytosolic copper metallo-chaperone. In this work, we present the first demonstration that: (i) human glutaredoxin 1 (hGrx1) efficiently catalyses interchange of the dithiol and disulfide forms of the Cys(12)-xx-Cys(15) fragment in Atox1 but does not act upon the isolated single residue Cys(41); (ii) the direction of catalysis is regulated by the GSSG/2GSH ratio and the availability of Cu(I); (iii) the active site Cys(23)-xx-Cys(26) in hGrx1 can bind Cu(I) tightly with femtomolar affinity (K(D) = 10(-15.5) M) and possesses a reduction potential of E(o)\u27 = -118 mV at pH 7.0. In contrast, the Cys(12)-xx-Cys(15) motif in Atox1 has a higher affinity for Cu(I) (K(D) = 10(-17.4) M) and a more negative potential (E(o)\u27 = -188 mV). These differences may be attributed primarily to the very low pKa of Cys23 in hGrx1 and allow rationalisation of conclusion (ii) above: hGrx1 may catalyse the oxidation of Atox1(dithiol) by GSSG, but not the complementary reduction of the oxidised Atox1(disulfide) by GSH unless Cu(aq)(+) is present at a concentration that allows binding of Cu(I) to reduced Atox1 but not to hGrx1. In fact, in the latter case, the catalytic preferences are reversed. Both Cys residues in the active site of hGrx1 are essential for the high affinity Cu(I) binding but the single Cys(23) residue only is required for the redox catalytic function. The molecular properties of both Atox1 and hGrx1 are consistent with a correlation between copper homeostasis and redox sulfur chemistry, as suggested by recent cell experiments. These proteins appear to have evolved the features necessary to fill multiple roles in redox regulation, Cu(I) buffering and Cu(I) transport

CAPS facilitates filling of the rapidly releasable pool of large dense-core vesicles

Calcium-activator protein for secretion (CAPS) is a cytosolic protein that associates with large dense-core vesicles and is involved in their secretion. Mammals express two CAPS isoforms, which share a similar domain structure including a Munc13 homology domain that is believed to be involved in the priming of secretory vesicles. A variety of studies designed to perturb CAPS function indicate that CAPS is involved in the secretion of large dense-core vesicles, but where in the secretory pathway CAPS acts is still under debate. Mice in which one allele of the CAPS-1 gene is deleted exhibit a deficit in catecholamine secretion from chromaffin cells. We have examined catecholamine secretion from chromaffin cells in which both CAPS genes were deleted and show that the deletion of both CAPS isoforms causes a strong reduction in the pool of rapidly releasable chromaffin granules and of sustained release during ongoing stimulation. We conclude that CAPS is required for the adequate refilling and/or maintenance of a rapidly releasable granule pool

CAPS1 Regulates Catecholamine Loading of Large Dense-Core Vesicles

SummaryCAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs

Two distinct secretory vesicle–priming steps in adrenal chromaffin cells

The calcium-dependent activator proteins for secretion, CAPS1 and CAPS2, facilitate syntaxin opening during synaptic vesicle priming

The cross-sectional GRAS sample: A comprehensive phenotypical data collection of schizophrenic patients

<p>Abstract</p> <p>Background</p> <p>Schizophrenia is the collective term for an exclusively clinically diagnosed, heterogeneous group of mental disorders with still obscure biological roots. Based on the assumption that valuable information about relevant genetic and environmental disease mechanisms can be obtained by association studies on patient cohorts of ≥ 1000 patients, if performed on detailed clinical datasets and quantifiable biological readouts, we generated a new schizophrenia data base, the GRAS (Göttingen Research Association for Schizophrenia) data collection. GRAS is the necessary ground to study genetic causes of the schizophrenic phenotype in a 'phenotype-based genetic association study' (PGAS). This approach is different from and complementary to the genome-wide association studies (GWAS) on schizophrenia.</p> <p>Methods</p> <p>For this purpose, 1085 patients were recruited between 2005 and 2010 by an invariable team of traveling investigators in a cross-sectional field study that comprised 23 German psychiatric hospitals. Additionally, chart records and discharge letters of all patients were collected.</p> <p>Results</p> <p>The corresponding dataset extracted and presented in form of an overview here, comprises biographic information, disease history, medication including side effects, and results of comprehensive cross-sectional psychopathological, neuropsychological, and neurological examinations. With >3000 data points per schizophrenic subject, this data base of living patients, who are also accessible for follow-up studies, provides a wide-ranging and standardized phenotype characterization of as yet unprecedented detail.</p> <p>Conclusions</p> <p>The GRAS data base will serve as prerequisite for PGAS, a novel approach to better understanding 'the schizophrenias' through exploring the contribution of genetic variation to the schizophrenic phenotypes.</p

Molecular aspects of copper transport in human cells: do Atox1 and hGrx1 have complementary roles?

© 2014 Dr. Jens BroseCopper is one of a minimum of thirteen known essential metals in eukaryotic biology, there are some prokaryotic and archae for which copper is likely not essential. This essentiality derives from the one-electron redox couple provided by the change between its di- and mono-valent forms and is utilised by a number of key redox enzymes. However, the same redox couple is responsible for the toxicity of copper, if its transport is uncontrolled within the cell. Imbalance between intake, storage, and excretion of copper can harm the cell and ultimately lead to the death of the organism. General mechanisms of copper homeostasis are understood but many details remain unclear. This work examines the chemistry and interaction of key proteins of copper’s pathway through mammalian cells to the trans-Golgi network. Glutaredoxins (Grxs) have been characterised as enzymes regulating the redox status of protein thiols via the cofactors glutathione (GSH) and glutathione disulphide (GS-SG). However, such a function has been demonstrated in model systems only using low molar mass compounds as substrates. It has not been demonstated with physiologically relevant protein substrates. The active sites of Grxs frequently feature Cys-xx-Cys motifs. Such motifs are also present as high affinity copper binding sites in copper-transporting proteins such as Atox1, a human cytosolic copper metallo-chaperone and in the metal binding domains of the copper transporting ATPases ATP7A and ATP7B. However, it has been predicted that this motif in hGrx1 does not bind copper. The aim of this study was to determine crucial thermodynamic properties of the proteins involved and to highlight their differences and similarities. These properties are essential to an understanding of functions of these proteins. In this work, it is demonstrated that: - (i) The active site Cys23-Pro-Tyr-Cys26 in human glutaredoxin 1 (hGrx1) can bind Cu(I) tightly with femtomolar affinity (KD = 10-15.5 M) and possesses a reduction potential of E°’ = -118 mV at pH 7.0. In contrast, the Cys12-Gly-Gly-Cys15 motif in Atox1 has a higher affinity for Cu(I) (KD = 10-17.4 M) and a more negative potential (E°’ = -188 mV). (ii) hGrx1 efficiently catalyses interchange of the dithiol and disulfide forms of the Cys12-Gly-Gly-Cys15 fragment in Atox1 but does not act upon the isolated single residue Cys41; (iii) The direction of this catalysis is regulated by the reduction potential imposed by the redox couple GS-SG/2GSH ratio and the availability of Cu(I). The final chapter describes the optimization of cell-free expression of the human copper transporter (hCtr1) and some initial characterization of its oligomeric state in detergent-solubilised form. Additionally, initial results for the incorporation of hCtr1 into lipid bilayer systems are presented. This step will be crucial for future copper binding and translocation assays

Molecular mechanisms of active zone function

In response to incoming action potentials the presynapse exocytoses neurotransmitter filled vesicles. Proper information encoding requires that the release occurs exactly at the junction to the postsynapse, that it is temporally tightly coupled to the electrical signal and that it proceeds efficiently. This review discusses the structure and function of the active zone, which is defined as the region of the presynapse that is specialized for vesicle release. Particular emphasis is put on the molecular players that control spatial restriction, efficiency and timing of exocytosis in the mammalian nervous system

A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1

ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane