Biochemical characterization,localization, and tissue distribution of the longer form of mouse SIRT3

SIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C‐terminus of human SIRT3 but lacks an N‐terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 5′ rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full‐length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD⁺. Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.     

Role of N-terminus in function and dynamics of sirtuin 7: an in silico study

Abstract

The sirtuin family comprises seven NAD⁺-dependent histone deacetylases named SIRT1 to SIRT7. The least investigated SIRT7 is currently considered as a promising therapeutic target for cardiovascular diseases, diabetes and different types of cancer. So far, its structure was not experimentally resolved, except of a fragment of its N-terminus. The aim of this study was to create in silico model of SIRT7 containing its core together with N-terminus, which is known to affect the enzyme’s catalytic activity and to find pockets that could be targeted by structure-based virtual screening. Homology model of SIRT7 was prepared using X-ray structures of other sirtuins and a resolved fragment of the N-terminus of SIRT7 as templates. All atom-unbiased molecular dynamics simulations were performed. It was found that N-terminus of SIRT7 remains in spatial proximity of the catalytic core for considerable fraction of time, and therefore, it may affect its catalytic activity by helping the enzyme to hold the substrate peptide. It may also participate in holding and release of the cofactor. Preferred orientations of NAD⁺?and acetyl-lysine inside SIRT7 were found, with all components forming a stable complex. Molecular dynamics provided an ensemble of conformations that will be targeted with virtual screening. Reliable in silico structure of SIRT7 will be a useful tool in searching for its inhibitors, which can be potential drugs in cancer treatment.

Communicated by Ramaswamy H. Sarma     

Characterization and possible redox regulation of the purified calmodulin‐dependent NAD+ kinase from Lycopersicon pimpinellifolium

The soluble and calmodulin (CaM)‐dependent NAD⁺ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short‐term NaCl stress (Delumeau, Morère‐Le Paven, Montrichard, Laval‐Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol‐modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD⁺ kinase. A three‐step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD⁺ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL^?1 bovine CaM. The activity of NAD⁺ kinase was strongly inhibited by thiol‐modifying reagents and oxidized glutathione. NAD⁺ kinase was also found to be air‐inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five‐fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD⁺ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP‐thioredoxin reductase system.     

NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor

Mycobacterium tuberculosis codes for an essential NAD+-dependent DNA ligase (MtuLigA) which is a novel, validated, and attractive drug target. We created mutants of the enzyme by systematically deleting domains from the C-terminal end of the enzyme to probe for their functional roles in the DNA nick joining reaction. Deletion of just the BRCT domain from MtuLigA resulted in total loss of activity in in vitro assays. However, the mutant could form an AMP-ligase intermediate that suggests that the defects caused by deletion of the BRCT domain occur primarily at steps after enzyme adenylation. Furthermore, genetic complementation experiments using a LigA deficient E. coli strain demonstrates that the BRCT domain of MtuLigA is necessary for bacterial survival in contrast to E. coli and T. filiformis LigA, respectively. We also report the identification, through virtual screening, of a novel N-substituted tetracyclic indole that competes with NAD+ and inhibits the enzyme with IC50 in the low muM range. It exhibits approximately 15-fold better affinity for MtuLigA compared to human DNA ligase I. In vivo assays using LigA deficient S. typhimurium and E. coli strains suggest that the observed antibacterial activity of the inhibitor arises from specific inhibition of LigA over ATP ligases in the bacteria. In silico ligand-docking studies suggest that the exquisite specificity of the inhibitor arises on account of its mimicking the interactions of NAD+ with MtuLigA. An analysis of conserved water in the binding site of the enzyme suggests strategies for synthesis of improved inhibitors with better specificity and potency.     

Discovery of TBC1D7 as a Potential Driver for Melanoma Cell Invasion

Metastasis is the leading cause for mortality in melanoma patients. Here, an unbiased mass spectrometry‐based quantitative proteomic method is utilized to assess differential protein expression in a matched pair of primary/metastatic melanoma cell lines (i.e., WM‐115/WM‐266‐4) derived from the same patient. It is found that TBC1D7 is overexpressed in metastatic over primary melanoma cells, and elevated expression of TBC1D7 promotes the invasion of these melanoma cells in vitro, partly through modulating the activities of secreted matrix metalloproteinases 2 and 9. Additionally, interrogation of publicly available data show that higher mRNA expression of TBC1D7 predicts poorer survival in melanoma patients. Together, the results suggest TBC1D7 as a driver for melanoma cell invasion, which is an important element in melanoma metastasis. The proteomic data generated from this study may also be useful for exploring the roles of other proteins in melanoma metastasis.     

The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae

Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.     

MCF-7/VD(R): a new vitamin D resistant cell line

Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (M?rk Hansen et al. [2000a]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF-7/VD(R) cell line, a stable subclone of the human MCF-7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1alpha,25(OH)(2)D(3). Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF-7/VD(R) cells contain fully functional VDRs, although in a lower number than seen with the parental MCF-7 cells. The regulation of the 24-hydroxylase enzyme appeared to be intact in the MCF-7/VD(R) cells and no differences with regard to growth rate and morphological appearance between the MCF-7/VD(R) cells and the parental MCF-7 cells were observed. Interestingly, however, the sensitivity of the MCF-7/VD(R) cells to the pure anti-estrogen ICI 182,780 was found to be increased. The MCF-7/VD(R) cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance.     

MicroRNA Let‐7 targets the ecdysone signaling pathway E75 gene to control larval–pupal development in Bactrocera dorsalis

MicroRNAs (miRNAs) regulate various biological processes during insect developme nt;however, their role in larval-pupal development in oriental fruit fly, Bactrocera dorsalis (Hendel) remains unknown. In the current study, we address the biological function of a conserved miRNA, Bdo-Let-7 in the regulation of BdE75 gene, which belongs to the ecdysone signaling pathway and participates in the larval-pupal development in B. dorsalis. Using dual luciferase reporter assay in HEK293T cells we show that Bdo-Let-7 miRNA interacts with the 3' untranslated region of BdE75 gene and suppresses its expression. The Bdo-Let?7 and BdE75 are also co-expressed in the larval-pupal stages and in different tissues of B. dorsalis .In in vivo experiments, the injectio n of Bdo-Let-7 agomir and antagomir in third instar larvae down- and up-regulated the expression of BdE75、 respectively. The 20-hydroxyecdysone (20E) injection assay shows that 20E up-regulated the expression of Bdo-Let-7 on the 5th day of the larvae. Moreover, abnormal pupation and eclosion were observed after larval Bdo-Let-7 antagomir injection. Based on these results, we show that Bdo-Let-7 regulates the ecdysone signaling pathway through the exact dose of BdE75 gene, and is indispensable for normal larval-pupal development in B. dorsalis.     

Knockdown of RBBP7 unveils a requirement of histone deacetylation for CPC function in mouse oocytes

During mouse oocyte maturation histones are deacetylated, and inhibiting this deacetylation leads to abnormal chromosome segregation and aneuploidy. RBBP7 is a component of several different complexes that contain histone deacetylases, and therefore could be implicated in histone deacetylation. We find that Rbbp7 is a dormant maternal mRNA that is recruited for translation during oocyte maturation to regulate the histone deacetylation. Importantly, we show that the maturation-associated decrease of histone acetylation is required for localization and function of the chromosomal passenger complex (CPC) during oocyte meiotic maturation. This finding can explain the phenotypes of oocytes where Rbbp7 is depleted by an siRNA/morpholino cocktail including severe chromosome misalignment, improper kinetochore–microtubule attachments, impaired SAC function, cytokinesis defects, and increased incidence of aneuploidy at metaphase II (Met II). These results implicate RBBP7 as a novel regulator of histone deacetylation during oocyte maturation and provide evidence that such deacetylation is required for proper chromosome segregation by regulating localized CPC function.     

Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate

The development of tolerant microorganisms is needed for the efficient fermentation of inhibitory lignocellulose hydrolysates. In the current work, the fermentation performance of six selected strains of Saccharomyces cerevisiae in dilute-acid spruce hydrolysate was compared using two different modes of fermentation; either single pulse addition of hydrolysate to exponentially growing cells or continuous feeding of the same amount of hydrolysate in a controlled fed-batch fermentation was made. All strains performed better in fed-batch mode than when all hydrolysate was added at once. However, the difference between strain performances varied significantly in the two fermentation modes. Large differences were observed between strains during the fed-batch experiments in the in vitro ability to reduce the furan compounds furfural and 5-hydroxymethyl furfural (HMF). A common feature among the strains was the induction of NADPH-coupled reduction of furfural and HMF, with the exception of strain CBS 8066. This strain also performed relatively poorly in both batch and fed-batch fermentations. Strain TMB3000--previously isolated from spent sulphite liquor fermentation--was by far the most efficient strain with respect to specific fermentation rate in both pulse addition and fed-batch mode. This strain was the only strain showing a significant constitutive NADH-coupled in vitro reduction of HMF. The ability to induce NADPH-coupled reduction together with the level of the apparently constitutive NADH-coupled reduction appeared to be key factors for selecting a suitable strain for fed-batch conversion of lignocellulose hydrolysate.     

Antibacterial activity of 7-aminocholesterol, a new sterol

Abstract A new sterol, 7-aminocholesterol, which inhibits growth of Saccharomyces cerevisiae , also displayed antibiotic activity against Gram-positive bacteria. The 50% growth inhibitory concentration against strains of Listeria innocua L. monocytogenes, Staphylococcus aureus, Enterococcus hirae and Bacillus cereus was 3 μM.     

CD157, the Janus of CD38 but with a unique personality

CD157 is a pleiotropic ectoenzyme which belongs to the CD38 family and to the growing number of leukocyte surface molecules known to act independently as both receptors and enzymes. A 45-kDa surface structure with a GPI anchor, the CD157 molecule displays two distinct domains in its extracellular component. The first is implicated in the enzymic activities of the molecule and the second features adhesion/signalling properties. CD157 shares several characteristics with CD38, including a similar amino acid sequence and enzymic functions. Both molecules are involved in the metabolism of NAD(+), and the CD157 gene is synthenic on 4p15 with CD38, with which it also shares a unique genomic organization. Their conservation in phylogeny is striking evidence for their relevance in the life and death cycle of the cell.     

Crystal structure of Lon protease: molecular architecture of gated entry to a sequestered degradation chamber

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine‐lysine catalytic dyad. We report the 2.0‐Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three‐tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight‐ and weak‐binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP‐driven protein unfolding and translocation. The bowl‐shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.     

Establishment of association of an Mg2+-dependent endonuclease with the rat liver nuclear matrix in cryonecrosis

Previously, we characterized the endonucleolytic activity of the nuclear matrix prepared from rat liver cryopreserved in liquid nitrogen. The enzymic activity was attributed to a 23 kDa, Mg(2+)-dependent and sequence non-specific endonuclease (p23) stably associated with the nuclear matrix. Here we show that p23 was absent from the nuclear matrix prepared from fresh liver. Instead, both ex vivo (cryopreservation), as well as in vivo-induced necrosis by repeated freezing/thawing of liver tissue in an anaesthetized rat, promoted the activation and translocation of p23 to the nuclear matrix. Considering that ex vivo and in vivo freezing/thawing of the liver were accompanied by morphological (nuclear compaction) and biochemical events (increased LDH activity, disorderly genomic DNA degradation, absence of lamin proteolysis, appearance of 62 and 50 kDa necrotic cleavage products of PARP-1) commonly observed during necrosis, and because the association of p23 with the nuclear matrix was saturable, reflecting the existence of a limited number of distinct high affinity sites on the nuclear matrix for p23, we concluded that the activation of the nuclear matrix-associated endonuclease p23 is a feature of liver cryonecrosis. Although cryonecrosis represents a typical example of acute cell damage, our results suggest that it is realized by ordered molecular events.     

Engineering the pentose phosphate pathway to improve hydrogen yield in recombinant Escherichia coli

Among various routes for the biological hydrogen production, the NAD(P)H-dependent pentose phosphate (PP) pathway is the most efficient for the dark fermentation. Few studies, however, have focused on the glucose-6-phosphate 1-dehydrogenase, encoded by zwf, as a key enzyme activating the PP pathway. Although the gluconeogenic activity is essential for activating the PP pathway, it is difficult to enhance the NADPH production by regulating only this activity because the gluconeogenesis is robust and highly sensitive to concentrations of glucose and AMP inside the cell. In this study, the FBPase II (encoded by glpX), a regulation-insensitive enzyme in the gluconeogenic pathway, was activated. Physiological studies of several recombinant, ferredoxin-dependent hydrogenase system-containing Escherichia coli BL21(DE3) strains showed that overexpression of glpX alone could increase the hydrogen yield by 1.48-fold compared to a strain with the ferredoxin-dependent hydrogenase system only; the co-overexpression of glpX with zwf increased the hydrogen yield further to 2.32-fold. These results indicate that activation of the PP pathway by glpX overexpression-enhanced gluconeogenic flux is crucial for the increase of NAD(P)H-dependent hydrogen production in E. coli BL21(DE3).     

Mammalian DIS3L2 exoribonuclease targets the uridylated precursors of let-7 miRNAs

The mechanisms of gene expression regulation by miRNAs have been extensively studied. However, the regulation of miRNA function and decay has long remained enigmatic. Only recently, 3′ uridylation via LIN28A-TUT4/7 has been recognized as an essential component controlling the biogenesis of let-7 miRNAs in stem cells. Although uridylation has been generally implicated in miRNA degradation, the nuclease responsible has remained unknown. Here, we identify the Perlman syndrome-associated protein DIS3L2 as an oligo(U)-binding and processing exoribonuclease that specifically targets uridylated pre-let-7 in vivo. This study establishes DIS3L2 as the missing component of the LIN28-TUT4/7-DIS3L2 pathway required for the repression of let-7 in pluripotent cells.