NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins.

Summary: NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways.

Background

NAD+ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD+-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD+ to nicotinamide ratio and the NAD+ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD+ metabolism requires analytical definition and quantification of the key metabolites.

Results

Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD+ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD+ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD+ or nicotinamide levels in ways that would increase Sir2 activity.

Conclusions

The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.

Despite the common occurrence of forkhead associated (FHA) phosphopeptide-binding domains and really interesting new gene (RING) E3 ubiquitin ligase domains, gene products containing both an N-terminal FHA domain and C-terminal RING domain constitute a highly distinctive intersection. Characterized FHA-RING ligases include the two vertebrate proteins, Checkpoint with FHA and RING (Chfr) and RING finger 8 (Rnf8), as well as three fungal proteins, Defective in mitosis (Dma1), Chf1 and Chf2. These FHA-RING ligases play roles in negative regulation of the cell division cycle, apparently by coupling protein phosphorylation events to specific ubiquitylation of target proteins. Here, the available data on upstream and downstream regulation of and by FHA-RING ligases are reviewed.

Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologs and FHIT homologs. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite no overall sequence similarity. Features of FHIT, a diadenosine polyphosphate hydrolase and candidate tumor suppressor, are predicted from HINT-nucleotide structures.

HIT (histidine triad)1 proteins, named for a motif related to the sequence HφHφHφφ, (φ a hydrophobic amino acid) are a superfamily of nucleotide hydrolases and transferases, which act on the α-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5′-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylylsulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding-site. The potential roles of ASW and Hint in avian sexual development are discussed in an accompanying manuscript. Here we review what is known about biological activities of HIT proteins, the structural and biochemical bases for their functions, and propose a new enzyme mechanism for Hint and Fhit that may account for the differences between HIT hydrolases and transferases.

Background & Aims:

Hints, Histidine triad nucleotide-binding proteins, are adenosine monophosphate–lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2.

Methods:

Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 over expressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flowcytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays.

Results:

Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; kcat:0.0223 s-1;Km:128 μmol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 ± 0 6% vs 57.7 ± 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 ± 2.35 vs 49.7 ± 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knock-down expression of Hint2. Subcutaneous injection of HepG2 cells over expressing Hint2 in SCID mice resulted in smaller tumors (0.32 ± 0.13 g vs 0.85 ± 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is down regulated in hepatocellular carcinomas (−0.42 ± 0.58 log2 vs −0.11 ± 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival.

Conclusion:

Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

The first committed step in the biosynthesis of l-ascorbate from d-glucose in plants requires conversion of GDP-l-galactose to l-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-l-galactose to l-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from Arabidopsis thaliana as a specific GDP-l-galactose/GDP-d-glucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.

Eukaryotic cells encode AMP-lysine hydrolases related to the rabbit histidine triad nucleotide-binding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative 31P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory Escherichia coli exhibited activity on the model substrate adenosine monophosphoramidate (AMP-NH2). The Escherichia coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, over-expressed, purified and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than mammalian Hint on three model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knockout strain of Escherichia coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT enzymatic activity results in failure to grow in media containing 0.75 KCl, 0.9 M NaCl, 0.5 M NaOAc and 10 mM MnCl2. Thus, bacteria may possess nucleotidylylated phosphoramidate substrates that must be hydrolyzed to support growth under certain high salt conditions.

Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5′ sulfamoyl (AdoOSO2NH2) or N-ethylsulfamoyl (AdoOSO2NHCH2CH3) groups. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors: titration of each inhibitor into AMP-pNA assays revealed their Ki values. The N-ethylsulfamoyl derivative has a 13-fold binding advantage over the sulfamoyl adenosine. The 1.8 Å co-crystal structure of rabbit Hint with N-ethylsulfamoyl adenosine revealed a binding site for the ethyl group against Trp123, a residue that reaches across the Hint dimer interface to interact with the alkyl portion of the inhibitor and, presumably, the alkyl portion of a lysyl substrate. Ser107 is positioned to donate a hydrogen bond to the leaving group nitrogen. Consistent with a role in acid-base catalysis, the Hint S107A mutant protein displayed depressed catalytic activity.

Hint is a universally conserved, dimeric AMP-lysine hydrolase encoded on the avian Z chromosome. Tandemly repeated on the female-specific W chromosome, Asw is a potentially sex-determining, dominant negative Hint dimerization partner whose substrate-interacting residues were specifically altered in evolution. To test the hypothesis that Gln127 of Asw is responsible for depression and/or alteration of Hint enzyme activity, a corresponding mutant was created in the chicken Hint homodimer and a novel substrate was developed that links reversal of lysine modification to aminomethylcoumarin release. Strikingly, the Hint-W123Q substitution reduced kcat/Km for AMP-lysine hydrolysis 17-fold, while it increased specificity for AMP-paranitroaniline hydrolysis by 160-fold. The resulting 2700-fold switch in enzyme specificity suggests that Gln127 could be the dominant component of Asw dominant negativity in avian feminization.

Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors. Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis. Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate bound state. Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog. Single crystals obtained by vapor diffusion against ammonium sulfate diffracted Xrays to beyond 2.75 Å resolution. High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form.

Fhit, a member of the Histidine Triad superfamily of nucleotide-binding proteins, binds and cleaves diadenosine polyphosphates and functions as a tumor suppressor in human epithelial cancers. Function of Fhit in tumor suppression does not require diadenosine polyphosphate cleavage but correlates with the ability to form substrate complexes. As diadenosine polyphosphates are at lower cellular concentrations than mononucleotides, we sought to quantify interactions between Fhit and competitive inhibitors with the use of diadenosine polyphosphate analogs containing fluorophores in place of one nucleoside. ApppAMC, ApppBODIPY and GpppBODIPY, synthesized in high yield, are effective Fhit substrates, producing AMP or GMP plus fluorophore diphosphates. GpppBODIPY cleavage is accompanied by a 5.4-fold increase in flourescence because BODIPY fluorescence is quenched by stacking with guanine. Titration of unlabeled diadenosine polyphosphates, inorganic pyrophosphate, mononucleotides, and inorganic phosphate into fluorescent assays provided values of Km and KI as competitive inhibitors. The data indicate that Fhit discriminates between good substrates via kcat and against cellular competitors in equilibrium binding terms. Surprisingly, pyrophosphate competes better than purine mononucleotides.

The design and synthesis of analogues of diadenosine 5′,5′″-P1,P3-triphosphate that are resistant to pyrophosphate hydrolysis is described in relation to their role in signaling and tumorigenesis involving the Fhit protein, the human fragile histidine triad protein, which is a novel Ap3A binding/cleaving protein.

Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein N-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations.

A novel human cytosolic flavin reductase, Nr1, was recently described that contains FMN, FAD, and NADPH cofactors. Though the targets of the related NADPH-dependent flavoprotein reductases, cytochrome P450 reductase, methionine synthase reductase, and nitric oxide synthase, are known, the cellular function of Nr1 is not clear. To explore expression and regulation of Nr1, we cloned fre-1, the Caenorhabditis elegans ortholog of Nr1, and discovered that it is transcribed as a bicistronic pre-mRNA together with dcs-1, the ortholog of the recently described scavenger mRNA decapping enzyme. We used the novel substrate, 7meGpppBODIPY, to demonstrate thatDCS-1 has low micromolar specificity for guanine ribonucleo-tides with the 7 memodification, whereas trimethylated G substrates are poor competitors. Contrary to earlier classification, DCS-1 is not a pyrophosphatase but a distant member of the Hint branch of the histidine triad superfamily of nucleotide hydrolases and transferases. These observations are consistent with the hypothesis that DCS-1 homologs may function in the metabolism of capped oligonucleotides generated following exosome-dependent degradation of short-lived mRNA transcripts. We find that fre-1 and dcs-1 are coordinately expressed through worm development, are induced by heat shock, and have an early identical expression profile inhuman tissues. Furthermore, immunocytochemical analysis of the endogenous proteins in COS cells indicates that both are present in the nucleus and concentrated in a distinct perinuclear structure. Though no connection between these enzymes had been anticipated, our data and data from global expression and protein association studies suggest that the two enzymes jointly participate in responses to DNA damage, heat shock, and other stresses.

The histidine superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of GalT-related transferases. While substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit and GalT are known, good substrates had not been reported for any member of the Hint branch and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily. Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5′-monophosphoramidate in an active-site dependent manner at second order rates exceeding 1,000,000 M-1 s-1. Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperature. Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3 and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH, and to mutations in Cak1, which phosphorylates Kin28. The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers. Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity.

Checkpoint with forkhead-associated and RING (Chfr) is a ubiquitin ligase (E3) that establishes an antephase or prometaphase checkpoint in response to mitotic stress. Though ubiquitination is essential for checkpoint function, the sites, linkages and ubiquitin conjugating enzyme (E2) specificity are controversial. Here we dissect the function of the two Chfr homologs in S. cerevisiae, Chf1 and Chf2, overexpression of which retard cell cycle at both G1 and G2. Using a genetic assay, we establish that Ubc4 is required for Chf2-dependent G1 cell cycle delay and Chf protein turnover. In contrast, Ubc13/Mms2 is required for G2 delay and does not contribute to Chf protein turnover. By reconstituting cis and trans-ubiquitination activities of Chf proteins in purified systems and characterizing sites modified and linkages formed by tandem mass spectrometry, we discovered that Ubc13/Mms2-dependent modifications are a distinct subset of those catalyzed by Ubc4. Mutagenesis of Lys residues identified in vitro indicates that site-specific Ubc4-dependent Chf protein autoubiquitination is responsible for Chf protein turnover. Thus, combined genetic and biochemical analyses indicate that Chf proteins have dual E2 specificity accounting for different functions in the cell cycle.

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

Author Summary

Biosynthesis of nicotinamide adenine dinucleotide (NAD+) is fundamental to cells, because NAD+ is an essential co-factor for metabolic and gene regulatory pathways that control life and death. Two vitamin precursors of NAD+ were discovered in 1938. We recently discovered nicotinamide riboside (NR) as a third vitamin precursor of NAD+ in eukaryotes, which extends yeast life span without caloric restriction and protects damaged dorsal root ganglion neurons from degeneration. Biosynthesis of NAD+ from NR requires enzyme activities in either of two pathways. In one pathway, specific NR kinases, including human Nrk1 and Nrk2, phosphorylate NR to nicotinamide mononucleotide. A second and Nrk-independent pathway is initiated by yeast nucleoside-splitting enzymes, Urh1 and Pnp1. We solved five crystal structures of human Nrk1 and, on the basis of co-crystal structures with substrates, suggested that the enzyme might be able to phosphorylate a novel compound, nicotinic acid riboside (NaR). We then demonstrated that human Nrk enzymes have dual specificity as NR/NaR kinases in vitro, and we established the ability of NaR to be used as a vitamin precursor of NAD+ via pathways initiated by Nrk1, Urh1, and Pnp1 in living yeast cells. Thus, starting from the structure of human Nrk1, we discovered a synthetic vitamin precursor of NAD+ and suggest the possibility that NaR is a normal NAD+ metabolite.

Eukaryotic nicotinamide riboside kinase (Nrk) converts nicotinamide riboside to NAD+ by phosphorylation and adenylylation. The structures of this enzyme bound to several substrates lead to identification of new pathways to NAD+

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.

Author Summary

Recent advances in sequence-based approaches to “knockdown” gene function have opened the door to an array of approaches to uncover functions for genes of interest. Vertebrate knockdown strategies—such as morpholinos (MOs) in zebrafish or RNA interference-based strategies in mammalian systems—have been demonstrated to be effective, rapid, and cost-efficient reverse-genetic approaches for studying gene function. However, their deployment has to date been limited by a number of technical (genomic, biological, and off-targeting) hurdles. One of the notable and unexpected findings from our work using MOs has been a series of observations surrounding unanticipated effects that are independent of the intended gene target. We have identified and characterized a recently described p53 induction pathway due to off-targeting that appears to be shared between knockdown technologies. This study reconciles a series of unexpected findings that show p53 upregulation at the transcriptional level in a subset of short inhibitory RNA- and MO-treated vertebrate systems. Moreover, concurrent p53 knockdown provides a new approach to facilitate the identification of previously hidden gene functions. This study provides both a new gene knockdown enhancement tool as well as additional insight into an important and conserved pathway implicated in cellular toxicity.

Four recent 'chemical genomic' studies, using genome-scale collections of yeast gene deletions, have presented complementary approaches to identifying gene-drug and pathway-drug interactions.

Many drugs have unknown, controversial or multiple mechanisms of action. Four recent 'chemical genomic' studies, using genome-scale collections of yeast gene deletions that were either arrayed or barcoded, have presented complementary approaches to identifying gene-drug and pathway-drug interactions.

In birds females are heterogametic with a ZW karyotype, while males are ZZ homogametes but the molecular basis for sexual differentiation in birds is unknown. Genomic and expression data suggest that Asw may feminize chicks, dominantly interfering with Hint function by heterodimerization.

Background

In birds and some lizards, females are heterogametic with a ZW karyotype, while males are ZZ homogametes. The molecular basis for sexual differentiation in birds is unknown: arguments exist for doses of Z masculinizing chicks and for W information feminizing. ASW was identified as a tandemly repeated gene conserved on avian W chromosomes that is expressed in early female development and appears to be an inactive form of avian Z-encoded HINT. Hint is a dimeric enzyme that hydrolyzes AMP linked to lysine, whose enzyme activity is required for regulation of the Cdk7 homologous Kin28 kinase in yeast. Of 16 residues most conserved across all life forms for AMP interactions, 15 are sexually dimorphic in birds, that is, altered in the female-specific Asw protein. Genomic and expression data suggest that Asw may feminize chicks, dominantly interfering with Hint function by heterodimerization.

Results

We consider whether positive cooperativity could explain how Hint heterodimerization with an inert enzyme might reduce specific activity by more than 50% and provide data sufficient to reject this model. Instead, we hypothesize that Asw carries a signal for mislocalization and/or proteolysis, and/or dominantly suppresses the remaining Hint active site to function as a dominant negative.

Conclusions

Molecular modeling suggests that Asw and Hint can heterodimerize and that Gln 127, an Asw-specific alteration for Trp123, dominantly interferes with the Hint active site. An extra dose of HINT in ZZW chicks, and thus more Hint homodimer, may partially overcome the feminizing influence of ASW and lead to the observed intersexual characteristics of ZZW triploids.

Background

The FHIT gene is lost early in the development of many tumors. Fhit possesses intrinsic ApppA hydrolase activity though ApppA cleavage is not required for tumor suppression. Because a mutant form of Fhit that is functional in tumor suppression and defective in catalysis binds ApppA well, it was hypothesized that Fhit-substrate complexes are the active, signaling form of Fhit. Which substrates are most important for Fhit signaling remain unknown.

Results

Here we demonstrate that dinucleoside polyphosphate levels increase 500-fold to hundreds of micromolar in strains devoid of the Saccharomyces cerevisiae homolog of Fhit, Hnt2. Accumulation of dinucleoside polyphosphates is reversed by re-expression of Hnt2 and is active site-dependent. Dinucleoside polyphosphate levels depend on an intact adenine biosynthetic pathway and time in liquid culture, and are induced by heat shock to greater than 0.1 millimolar even in Hnt2+ cells.

Conclusions

The data indicate that Hnt2 hydrolyzes both ApppN and AppppN in vivo and that, in heat-shocked, adenine prototrophic yeast strains, dinucleoside polyphosphates accumulate to levels in which they may saturate Hnt2.

Background

The human FHIT gene is inactivated early in the development of many human cancers and loss of Fhit in mouse predisposes to cancer while reintroduction of FHIT suppresses tumor formation via induction of apoptosis. Fhit protein, a diadenosine polyphosphate hydrolase, does not require hydrolase activity to function in tumor suppression and may signal for apoptosis as an enzyme-substrate complex. Thus, high affinity nonhydrolyzable substrate analogs may either promote or antagonize Fhit function, depending on their features, in Fhit + cells. Previously synthesized analogs with phosphorothioadenosyl substitutions and "supercharged" branches do not bind better than natural substrates and thus have limited potential as cellular probes.

Results

Here we link adenosine 5'-O-phosphates and phosphorothioates to short-chain polyols to generate a series of substrate analogs. We obtain structure-activity data in the form of in vitro Fhit inhibition for four types of analog substitutions and describe two compounds, inhibitory constants for which are 65 and 75-fold lower than natural substrates.

Conclusions

The best Fhit inhibitors obtained to date separate two or more 5'-O-phosphoromonothioadenosyl moieties with as many bond lengths as in AppppA, maintain oxygen at the location of the α-β bridging oxygen, and replace carbon for the β phosphorus.