The structure and function of the mitochondrial peroxiredoxin SP-22

Substrate protein-22 (SP-22) is a 22kDa bovine protein that functions as a thioredoxin-dependent peroxidase in the mitochondrial matrix. The exact physiological targets of SP-22 remain to be elucidated; however its antioxidant activity has been well documented in the protection of several free radical- sensitive enzymes. SP-22 has been assigned to a novel peroxidase family called the peroxiredoxins (Prxs). Prxs use electrons provided by free thiol groups to reduce hydrogen peroxide, a potent oxidising agent that can contribute to oxidative stress either directly or via the hydroxyl radical generated by the Fenton reaction. Prxs can be divided into two sub-groups (1-Cys and 2-Cys) depending on the number of conserved cysteine residues they possess. SP-22 is a 2-Cys Prx with three cysteines, not an uncommon feature, at positions 47, 66 and 168. Following sequence analysis C47 is proposed to be the catalytic residue, containing a sulphenic acid group, functioning as a 2-electron redox centre. The cloning and overexpression of recombinant SP-22 and three cysteine mutants (C47S, C66S and C168S) in bacterial cells as N-terminal His-tag proteins is reported. The overexpression conditions for optimal protein solubility and the subsequent purification by metal chelate chromatography were also determined. A comparison of reducing and non reducing SDS-PAGE of wild type and mutant SP-22s established that SP-22 contains a dimeric unit linked by two intermolecular disulphide bonds, in its oxidised state. These involve C47 of one monomer and C168 of the opposing subunit. This result is consistent with findings for other 2-Cys Prx members. Previous work in this laboratory involving the purification of the mitochondrial 2-oxoacid dehydrogenase member, pyruvate dehydrogenase (PDC) from bovine heart, and its subsequent separation into its three enzyme components by size exclusion chromatography, established that the dihydrolipoamide dehydrogenase component (E3) co-eluted with another contaminating protein. Following N-terminal sequencing this protein was identified to be SP-22. Preliminary results using the techniques of Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) have confirmed a physical association between SP-22 and E3, binding with an affinity in the micromolar range. The functional significance of this interaction is discussed with respect to the possible protection of PDC and the oxoglutarate dehydrogenase complex (OGDC) against oxidative modification of the catalytically-active thiol groups possessed by the individual components. (Abstract shortened by ProQuest.)

Insights into the Catalytic Mechanism of Glutathione S-Transferase: The Lesson from Schistosoma haematobium

SummaryGlutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS−; in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a π-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pKa of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison

The cyclic peptide G4CP2 enables the modulation of galactose metabolism in yeast by interfering with GAL4 transcriptional activity

Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed

Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063

The 1.8\u2005\uc5 resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using \u3b2-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography

Boosting of post-exposure human T-cell and B-cell recall responses in vivo by Burkholderia pseudomallei-related proteins.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped to prevent disease. Here, we used a humanized mouse model (hu-PBL-SCID), reconstituted with human peripheral blood mononuclear cells from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N-PilO2) for boosting both T-cell and B-cell immune responses. All three antigens boosted the production of specific antibodies in vivo, and increased the number of antibody and interferon-γ-secreting cells, and induced antibody affinity maturation. Moreover, antigen-specific antibodies isolated from either seropositive individuals or boosted mice, were found to enhance phagocytosis and oxidative burst activities from human polymorphonuclear cells. Our study demonstrates that FliC, OmpA and N-PilO2 can stimulate human memory T and B cells and highlight the potential of the hu-PBL-SCID system for screening and evaluation of novel protein antigens for inclusion in future vaccine trials against melioidosis

Inhibition of classical and alternative modes of respiration in Candida albicans leads to cell wall remodelling and increased macrophage recognition

The human fungal pathogen Candida albicans requires respiratory function for normal growth, morphogenesis and virulence. Mitochondria therefore represent an enticing target for the development of new antifungal strategies. This possibility is bolstered by the presence of characteristics specific to fungi. However, respiration in C. albicans, as in many fungal organisms, is facilitated by redundant electron transport mechanisms, making direct inhibition a challenge. In addition, many chemicals known to target the electron transport chain are highly toxic. Here we make use of chemicals with low toxicity to efficiently inhibit respiration in C. albicans. We find that use of the Nitric Oxide donor, Sodium Nitroprusside (SNP), and the alternative oxidase inhibitor, SHAM, prevents respiration, leads to a loss of viability and to cell wall rearrangements that increase the rate of uptake by macrophages in vitro and in vivo. We propose that SNP+SHAM treatment leads to transcriptional changes that drive cell wall re-arrangement but which also prime cells to activate transition to hyphal growth. In line with this we find that pre-treatment of C. albicans with SNP+SHAM leads to an increase in virulence. Our data reveals strong links between respiration, cell wall remodelling and activation of virulence factors. Our findings demonstrate that respiration in C. albicans can be efficiently inhibited with chemicals, which are not damaging to the mammalian host, but that we need to develop a deeper understanding of the roles of mitochondria in cellular signalling if they are to be developed successfully as a target for new antifungals

A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a “lid” domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG

A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a “lid” domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG

Durvalumab (MEDI 4736) in combination with extended neoadjuvant regimens in rectal cancer : a study protocol of a randomised phase II trial (PRIME-RT)

Acknowledgements We are grateful to Mr George Davidson and Ms Monica Jeffers for their input with writing the PRIME-RT protocol and patient information sheet. This study is co-sponsored by the University of Glasgow and NHS Greater Glasgow and Clyde. Funding PRIME-RT is funded by Astrazeneca and receives core funding from CRUK Clinical Trials Unit Glasgow for the purposes of trial set-up and data collection. The trial is co-sponsored by the University Of Glasgow and NHS Greater Glasgow and Clyde.Peer reviewedPublisher PD

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery