Galina Polekhina

AMPK β Subunit Targets Metabolic Stress Sensing to Glycogen

Abstract AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients [1], as well as hormones, including adiponectin and leptin [2, 3]. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK [4]. Mammalian AMPK is an αβγ heterotrimer with multiple isoforms of each subunit comprising α1, α2, β1, β2, γ1, γ2, and γ3, which have varying tissue and subcellular expression [5, 6]. Mutations in the AMPK γ subunit cause glycogen storage disease in humans [7], but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK β subunit contains a functional glycogen binding domain (β-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished β-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK β1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.

A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications

BACKGROUND Glutathione S-transferases (GSTs) are a multifunctional group of enzymes, widely distributed in aerobic organisms, that have a critical role in the cellular detoxification process. Unlike their mammalian counterparts, bacterial GSTs often catalyze quite specific reactions, suggesting that their roles in bacteria might be different. The GST from Proteus mirabilis (PmGST B1-1) is known to bind certain antibiotics tightly and reduce the antimicrobial activity of beta-lactam drugs. Hence, bacterial GSTs may play a part in bacterial resistance towards antibiotics and are the subject of intense interest. RESULTS Here we present the structure of a bacterial GST, PmGST B1-1, which has been determined from two different crystal forms. The enzyme adopts the canonical GST fold although it shares less than 20% sequence identity with GSTs from higher organisms. The most surprising aspect of the structure is the observation that the substrate, glutathione, is covalently bound to Cys 10 of the enzyme. In addition, the highly structurally conserved N-terminal domain is found to have an additional beta strand. CONCLUSIONS The crystal structure of PmGST B1-1 has highlighted the importance of a cysteine residue in the catalytic cycle. Sequence analyses suggest that a number of other GSTs share this property, leading us to propose a new class of GSTs - the beta class. The data suggest that the in vivo role of the beta class GSTs could be as metabolic or redox enzymes rather than conjugating enzymes. Compelling evidence is presented that the theta class of GSTs evolved from an ancestral member of the thioredoxin superfamily.

Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling.

Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 Å resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded β-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-α receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-α-mediated NF-κB activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.

Molecular basis of glutathione synthetase deficiency and a rare gene permutation event.

Glutathione synthetase (GS) catalyses the production of glutathione from γ‐glutamylcysteine and glycine in an ATP‐dependent manner. Malfunctioning of GS results in disorders including metabolic acidosis, 5‐oxoprolinuria, neurological dysfunction, haemolytic anaemia and in some cases is probably lethal. Here we report the crystal structure of human GS (hGS) at 2.1 Å resolution in complex with ADP, two magnesium ions, a sulfate ion and glutathione. The structure indicates that hGS belongs to the recently identified ATP‐grasp superfamily, although it displays no detectable sequence identity with other family members including its bacterial counterpart, Escherichia coli GS. The difficulty in identifying hGS as a member of the family is due in part to a rare gene permutation which has resulted in a circular shift of the conserved secondary structure elements in hGS with respect to the other known ATP‐grasp proteins. Nevertheless, it appears likely that the enzyme shares the same general catalytic mechanism as other ligases. The possibility of cyclic permutations provides an insight into the evolution of this family and will probably lead to the identification of new members. Mutations that lead to GS deficiency have been mapped onto the structure, providing a molecular basis for understanding their effects.

The identification of mitochondrial DNA variants in glioblastoma multiforme

BackgroundMitochondrial DNA (mtDNA) encodes key proteins of the electron transfer chain (ETC), which produces ATP through oxidative phosphorylation (OXPHOS) and is essential for cells to perform specialised functions. Tumor-initiating cells use aerobic glycolysis, a combination of glycolysis and low levels of OXPHOS, to promote rapid cell proliferation and tumor growth. Glioblastoma multiforme (GBM) is an aggressively malignant brain tumor and mitochondria have been proposed to play a vital role in GBM tumorigenesis.ResultsUsing next generation sequencing and high resolution melt analysis, we identified a large number of mtDNA variants within coding and non-coding regions of GBM cell lines and predicted their disease-causing potential through in silico modeling. The frequency of variants was greatest in the D-loop and origin of light strand replication in non-coding regions. ND6 was the most susceptible coding gene to mutation whilst ND4 had the highest frequency of mutation. Both genes encode subunits of complex I of the ETC. These variants were not detected in unaffected brain samples and many have not been previously reported. Depletion of HSR-GBM1 cells to varying degrees of their mtDNA followed by transplantation into immunedeficient mice resulted in the repopulation of the same variants during tumorigenesis. Likewise, de novo variants identified in other GBM cell lines were also incorporated. Nevertheless, ND4 and ND6 were still the most affected genes. We confirmed the presence of these variants in high grade gliomas.ConclusionsThese novel variants contribute to GBM by rendering the ETC. partially dysfunctional. This restricts metabolism to anaerobic glycolysis and promotes cell proliferation.

GSTZ1d: a new allele of glutathione transferase zeta and maleylacetoacetate isomerase.

The zeta class glutathione transferases (GSTs) are known to catalyse the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate (FAA), and the biotransformation of dichloroacetic acid to glyoxylate. A new allele of human GSTZ1, characterized by a Thr82Met substitution and termed GSTZ1d, has been identified by analysis of the expressed sequence tag (EST) database. In European Australians, GSTZ1d occurs with a frequency of 0.16. Like GSTZ1b-1b and GSTZ1c-1c, the new isoform has low activity with dichloroacetic acid compared with GSTZ1a-1a. The low activity appears to be due to a high sensitivity to substrate inhibition. The maleylacetoacetate isomerase (MAAI) activity of all known variants was compared using maleylacetone as a substrate. Significant differences in activity were noted, with GSTZ1a-1a having a notably lower catalytic efficiency. The unusual catalytic properties of GSTZ1a-1a in both reactions suggest that its characteristic arginine at position 42 plays a significant role in the regulation of substrate access and/or product release. The different amino acid substitutions have been mapped on to the recently determined crystal structure of GSTZ1-1 to evaluate and explain their influence on function.

Mapping the Intermedilysin-Human CD59 Receptor Interface Reveals a Deep Correspondence with the Binding Site on CD59 for Complement Binding Proteins C8α and C9

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.

Kinetic properties of missense mutations in patients with glutathione synthetase deficiency.

Patients with hereditary glutathione synthetase (GS) (EC 6.3.2.3) deficiency present with variable clinical pictures, presumably related to the nature of the mutations involved. In order to elucidate the relationship between genotype, enzyme function and clinical phenotype, we have characterized enzyme kinetic parameters of missense mutations R125C, R267W, R330C and G464V from patients with GS deficiency. One of the mutations predominantly affected the K(m) value, with decreased affinity for glycine, two mutations influenced both K(m) and V(max) values, and one mutation reduced the stability of the enzyme. This characterization agrees well with predictions based on the recently reported crystal structure of human GS. Thus our data indicate that different mutations can affect the catalytic capacity of GS by decreasing substrate affinity, maximal velocity or enzyme stability.

Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases.

Glutathione S-transferases (GSTs) normally use hydroxy-group-containing residues in the N-terminal domain of the enzyme for stabilizing the activated form of the co-substrate, glutathione. However, previous mutagenesis studies have shown that this is not true for Beta class GSTs and thus the origin of the stabilization remains a mystery. The recently determined crystal structure of Proteus mirabilis GST B1-1 (PmGST B1-1) suggested that the stabilizing role might be fulfilled in Beta class GSTs by one or more residues in the C-terminal domain of the enzyme. To test this hypothesis we mutated His(106) and Lys(107) of PmGST B1-1 to investigate their possible role in the enzymes catalytic activity. His(106) was mutated to Ala, Asn and Phe, and Lys(107) to Ala and Arg. The effects of the replacement on the activity, thermal stability and antibiotic-binding capacity of the enzyme were examined. The results are consistent with the involvement of His(106) and Lys(107) in interacting with glutathione at the active site but these residues do not contribute significantly to catalysis, folding or antibiotic binding.

Structure of the N-terminal domain of human thioredoxin-interacting protein.

Thioredoxin-interacting protein (TXNIP) is one of the six known α-arrestins and has recently received considerable attention owing to its involvement in redox signalling and metabolism. Various stress stimuli such as high glucose, heat shock, UV, H2O2 and mechanical stress among others robustly induce the expression of TXNIP, resulting in the sequestration and inactivation of thioredoxin, which in turn leads to cellular oxidative stress. While TXNIP is the only α-arrestin known to bind thioredoxin, TXNIP and two other α-arrestins, Arrdc4 and Arrdc3, have been implicated in metabolism. Furthermore, owing to its roles in the pathologies of diabetes and cardiovascular disease, TXNIP is considered to be a promising drug target. Based on their amino-acid sequences, TXNIP and the other α-arrestins are remotely related to β-arrestins. Here, the crystal structure of the N-terminal domain of TXNIP is reported. It provides the first structural information on any of the α-arrestins and reveals that although TXNIP adopts a β-arrestin fold as predicted, it is structurally more similar to Vps26 proteins than to β-arrestins, while sharing below 15% pairwise sequence identity with either.