Neus Cols

Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis

A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.

RECOMBINANT SYNTHESIS OF MOUSE ZN3-BETA AND ZN4-ALPHA METALLOTHIONEIN 1 DOMAINS AND CHARACTERIZATION OF THEIR CADMIUM(II) BINDING CAPACITY

Abstract. Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn4-αMT and Zn3-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn3-βMT with CdCl2 results in the formation of Cd3-βMT. The addition of excess Cd(II) leads to Cd4-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd9-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd3-βMT species is stable in the presence of this strong metal-chelating agent.

A new insight into the Ag+ and Cu+ binding sites in the metallothionein β domain

Abstract The copper( I ) and silver( I ) binding properties of the β fragment of recombinant mouse metallothionein 1 have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn 3 -βMT at pH 7 or apo-βMT at pH 3 with Cu + leads to the formation of species having the same stoichiometry and structure: Cu 6 -βMT, Cu 7 -βMT and Cu 10 -βMT. In the first stage of the titration of Zn 3 -βMT with Cu + at pH 7 one additional species of formula Cu 4 Zn 1 -βMT was detected. In contrast, the titration of Zn 3 -βMT at pH 7.5 and of apo-βMT at pH 2.5 with Ag + proceeds through different reaction pathways, affording Zn x Ag 3 -βMT, Ag 6 -βMT and Ag 9 -βMT or Ag 3 -βMT, Ag 6 -βMT and Ag 9 -βMT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag 6 -βMT and Ag 9 -βMT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.

Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase

Tyr152 and Lys156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short‐chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr152 to Glu or Gin, Lys156 to Ile, Gly184 to Leu, and the double mutant Gly130 to Cys and Gly133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr152 and Lys156are involved in catalysis and that Gly130, Gly133 and Gly184 contribute substantially to the structure of the active form.

Zinc(II) is required for the in vivo and in vitro folding of mouse copper metallothionein in two domains

We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu,Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)α(Cu4Zn1)β-MT, (Cu3Zn2)α(Cu4Zn1)β-MT and (Cu4Zn1)α(Cu6)β-MT. The cooperative formation of (Cu3Zn2)α(Cu4Zn1)β-MT from (Zn4)α(Cu4Zn1)β-MT indicates that the preference of Cu(I) for binding to the β domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent α and β fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)α(Cu4Zn1)β-MT], while that of αMT has rendered a mixture of Cu4Zn1-αMT (40%), Cu5Zn1-αMT (20%) and Cu7-αMT (40%). In the case of βMT, a mixture of Cu6-βMT (25%) and Cu7-βMT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-βMT from native sources may not be adequate.

Drosophila alcohol dehydrogenase: evaluation of Ser139 site-directed mutants.

Drosophila alcohol dehydrogenase (DADH) belongs to the large and highly heterogeneous (15–30% residue identity) short‐chain dehydrogenase/reductase family (SDR). It is the only reported member that oxidizes mainly ethanol and 2‐propanol among other alcohols. To confirm the role of Ser139 we constructed two site‐directed mutants, Ser139Ala and Ser139Cys, which show no enzymatic activity. Molecular replacement and data from crystallographically refined 3D structures confirm the position of Ser139, whose hydroxyl group faces the cleft of the presumed catalytic pocket, very close to Tyr152 and Lys156. Thus, consistent with the constitution of the catalytic triad of other SDR, our results suggest that Ser139 of DADH is directly involved in the catalytic reaction.

An intact eight-membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity.

All drosophilid alcohol dehydrogenases contain an eight‐member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short‐chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X‐ray crystallography of the SlADHT114V mutant revealed a broken water chain, the overall 3D structure of the binary enzyme–NAD+ complex was almost identical to the wild‐type enzyme (SlADHwt). As for the SlADHwt, steady‐state kinetic studies revealed that catalysis by the SlADHT114V mutant was consistent with a compulsory ordered reaction mechanism where the co‐enzyme binds to the free enzyme. The mutation caused a reduction of the kon velocity for NAD+ and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme–NAD+–alcohol complex. Furthermore, it increased the pKa value of the group in the binary enzyme–NAD+ complex that regulates the kon velocity of alcohol and alcohol‐competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis.

The comparison of mouse full metallothionein‐1 versus α and β domains and metallothionein‐1‐to‐3 mutation following traumatic brain injury reveals different biological motifs

Traumatic injury to the brain is one of the leading causes of injury‐related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock‐out mice. We herewith report the results comparing full mouse MT‐1 with the independent α and β domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota‐rod tests; all the proteins showed a modest effect in the former test, while only full MT‐1 improved the performance of animals in the rota‐rod, and the α domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host‐response genes such as GFAP, Mac1 and ICAM, in some cases being the β domain more effective than the α domain or even the full MT‐1. A MT‐1‐to‐MT‐3 mutation blunted some but not all the effects caused by the normal MT‐1, and in some cases increased its potency. Thus, splitting the two MT‐1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

Genetic Analysis of High Bone Mass Cases from the BARCOS Cohort of Spanish Postmenopausal Women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM.

Recombinant synthesis and metal-binding abilities of mouse metallothionein 1 and its α- and β-domains

Heterologous expression in E.coli, coupled with spectroscopic analyses, has enabled the metal binding properties of the recombinant mouse Zn-MT, Zn-αMT and Zn-βMT to be characterized. Cd(II) binding properties of Zn7-MT and Zn4-αMT have shown that they behave like the native proteins and those of the Zn3-βMT fragment have been reported for the first time. In contrast, the behavior of these proteins towards Hg(II), Cu(I) and Ag(I), which is strongly influenced by the pH and the stabilization time, differs from that of the native forms. Furthermore, recombinant Cd-βMT has also been expressed and characterized.