Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

Site-Directed Mutagenesis on Human Cystathionine-γ-Lyase Reveals Insights into the Modulation of H2S Production

In recent years, increased interest has been directed towards hydrogen sulfide (H₂S) as the third gasotransmitter and its role in various diseases. Cystathionine-γ-lyase (CSE) is one of the enzymes responsible for the endogenous production of H₂S in mammals. With the aid of the crystal structures of human CSE and site-directed mutagenesis studies, we have identified several amino acid residues in CSE that are actively involved in the catalysis of H₂S production. Contrary to reports suggesting that Tyr114 is required for substrate binding, our results reveal a significant increase in the production of H₂S upon mutation of Tyr114 to phenylalanine. This is attributed to an increased rate of pyridoxal 5′-phosphate (PLP) regeneration due to weakened π-stacking interactions between Phe114 and PLP. Thr189 is also identified as a crucial residue where hydrogen bonding to Asp187 keeps the latter in an optimal position for hydrogen bonding to the pyridoxal nitrogen of PLP. Furthermore, mutation of Glu339 to lysine, alanine or tyrosine reveals the importance of the hydrophobicity of the 339th amino acid in determining the specificity of the enzyme for the catalysis of α,γ-elimination or α,β-elimination reaction. Our study also shows that the rate of H₂S production is increased with increasing exogenous PLP concentration, hence supporting our hypothesis that apo-CSE is formed during the catalysis of H₂S production. Taken together, these findings suggest novel routes towards the design of activators or inhibitors that modulate the production of H₂S; these modulators may also serve as lead compounds in the development of drugs or mechanistic probes in the study of various H₂S-related diseases.     

TIP47 protects mitochondrial membrane integrity and inhibits oxidative-stress-induced cell death

We found that overexpression of tail interacting protein of 47 kDa (TIP47), but not its truncated form (t-TIP47) protected NIH3T3 cells from hydrogen-peroxide-induced cell death, prevented the hydrogen-peroxide-induced mitochondrial depolarization determined by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC1), while suppression of TIP47 in HeLa cells facilitated oxidative-stress-induced cell death. TIP47 was located to the cytoplasm of untreated cells, but some was associated to mitochondria in oxidative stress. Recombinant TIP47, but not t-TIP47 increased the mitochondrial membrane potential (Δψ), and partially prevented Ca²⁺ induced depolarization. It is assumed that TIP47 can bind to mitochondria in oxidative stress, and inhibit mitochondria mediated cell death by protecting mitochondrial membrane integrity.     

The quest for molecular quasi-species in ligand-activity space and its application to directed enzyme evolution

We propose that the proper evolving unit in enzyme evolution is not a single “fittest molecule”, but a cluster of related variants denoted a “quasi-species”. A distribution of variants provides genetic variability and thereby reduces the risk of inbreeding and evolutionary dead-ends. Different matrices of substrates or activity modulators will lead to different selection criteria and divergent evolutionary trajectories. We provide examples from our directed evolution of glutathione transferases illustrating the interplay between libraries of enzyme variants and ligand matrices in the identification of quasi-species. The ligand matrix is shown to be crucial to the outcome of the search for novel activities. In this sense the experimental system resembles the biological environment in governing the evolution of enzymes.     

A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses

A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO₃ stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO₃), drought (PEG) and metals (CuCl_2, ZnCl₂, CdCl₂). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g⁻¹ dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu²⁺, Zn²⁺ and Cd²⁺ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl₂, ZnCl₂ or CdCl₂, respectively, compared with control after 6 h culture.     

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bgamma), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bgamma class of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass=20 kDa; pI=5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean+/-standard deviation, n=3) of 3.13+/-0.27 and 3.43+/-1.0 micromol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus.     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

The p53-induced Wig-1 protein binds double-stranded RNAs with structural characteristics of siRNAs and miRNAs

Wig-1 is a p53-induced zinc finger protein. Here we show that human Wig-1 binds long (>or=23 bp) dsRNAs with 5'-overhangs. The first zinc finger domain is necessary but not sufficient for this dsRNA-binding in vitro. Wig-1 also binds dsRNA in living cells via zinc fingers 1 and 2. Both zinc fingers 1 and 2 are important for Wig-1-mediated growth suppression. Moreover, Wig-1 binds 21 bp dsRNAs with 3'-protruding ends. These findings demonstrate that human Wig-1 can bind different types of dsRNAs, including dsRNAs resembling small interfering RNAs (siRNAs) and microRNAs (miRNAs), and indicate that dsRNA binding has a role in Wig-1-mediated regulation of cell growth.