Human Nucleotide Research Articles

The human apoptosis inhibitor NAIP induces pyroptosis in macrophages infected with Legionella pneumophila

Human nucleotide oligomerization domain-like receptor family apoptosis inhibitory protein (NAIP) prevents apoptosis by inhibiting caspase-3, -7, and -9. Four functional Naip exist in the murine genome, each of which is equally similar to human NAIP. Among them, Naip5 induces pyroptosis by promoting caspase-1 activation in response to Legionella pneumophila infection in macrophages. However, the contribution of human NAIP to this response is unclear. To investigate the role of human NAIP in macrophage survival, we stably expressed human NAIP in RAW264.7 macrophages. Human NAIP inhibited camptothecin-induced apoptosis in macrophages; however, it promoted cytotoxicity in L. pneumophila-infected cells. This cytotoxicity was associated with caspase-1. In addition, human NAIP restricted the intracellular growth of L. pneumophila. L. pneumophila flagellin was required for cytotoxicity, caspase-1 activation, and restriction of intracellular bacterial growth. Expression of murine Naip5 produced comparable results. These data indicate that human NAIP regulates the host response to L. pneumophila infection in a manner similar to that of murine Naip5 and that human NAIP and murine Naip5 regulate cell survival by inhibiting apoptosis or by promoting pyroptosis in response to specific cellular signals.

Reply to Sousa et al.: Evaluation of competing J domain:Hsp70 complex models in light of methods used

We respond to Sousa et al. (1) who compare a crystal structure (2) of a disulfide-linked adduct of human Hsp70 nucleotide binding domain (NBD) and auxilin J domain, with the solution complex (3) of Escherichia coli Hsp70 NBD, substrate binding domain (SBD), substrate and DnaJ J domain. The molecular models are very different despite >60% sequence homology (the coordinates in ref. 3 are available on request).

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

The BDNF Val66Met Polymorphism Impairs Synaptic Transmission and Plasticity in the Infralimbic Medial Prefrontal Cortex

The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism is a common human single nucleotide polymorphism (SNP) that affects the regulated release of BDNF, and has been implicated in affective disorders and cognitive dysfunction. A decreased activation of the infralimbic medial prefrontal cortex (IL-mPFC), a brain region critical for the regulation of affective behaviors, has been described in BDNF(Met) carriers. However, it is unclear whether and how the Val66Met polymorphism affects the IL-mPFC synapses. Here, we report that spike timing-dependent plasticity (STDP) was absent in the IL-mPFC pyramidal neurons from BDNF(Met/Met) mice, a mouse that recapitulates the specific phenotypic properties of the human BDNF Val66Met polymorphism. Also, we observed a decrease in NMDA and GABA receptor-mediated synaptic transmission in the pyramidal neurons of BDNF(Met/Met) mice. While BDNF enhanced non-NMDA receptor transmission and depressed GABA receptor transmission in the wild-type mice, both effects were absent in BDNF(Met/Met) mice after BDNF treatment. Indeed, exogenous BDNF reversed the deficits in STDP and NMDA receptor transmission in BDNF(Met/Met) neurons. BDNF-mediated selective reversal of the deficit in plasticity and NMDA receptor transmission, but its lack of effect on GABA and non-NMDA receptor transmission in BDNF(Met/Met) mice, suggests separate mechanisms of Val66Met polymorphism upon synaptic transmission. The effect of the Val66Met polymorphism on synaptic transmission and plasticity in the IL-mPFC represents a mechanism to account for this impact of SNP on affective disorders and cognitive dysfunction.

An analysis of ERCC1, hENT1, RRM1, and RRM2 expression in resected pancreas adenocarcinoma: Implications for adjuvant treatment.

206 Background: Tumor overexpression of excision repair cross complementing gene 1 (ERCC1) was recently shown to be associated with decreased survival in patients with pancreas adenocarcinoma (PAC). Ribonucleotide reductase 1 and 2 (RRM) and human equilibrative nucleotide transporter 1 (hENT1) are integral to DNA synthesis and cellular transport and are implicated as poor prognostic factors in other malignancies. Their role in PAC is not well defined. Methods: A prospective database (n=220) was used to randomly select 95 pts who underwent pancreaticoduodenectomy for PAC between 1/00-10/08. IHC analysis was performed on tumor samples for RRM1 and 2, hENT1, and ERCC1. Main outcomes were recurrence-free (RFS) and overall survival (OS). Results: Median age was 63 yrs; median FU, RFS, and OS was 49, 10.6 and 15.5 mo. Median tumor size was 3cm, 26% had a positive margin, 34% were poorly differentiated, 61% had positive lymph nodes (LN), 88% had perineural invasion (PNI), and 45% had lymphovascular invasion (LVI). High expression of RRM1, RRM2, hENT1, and ERCC1 was present in 40%, 17%, 85%, and 16% of patients, respectively. High RRM2 was associated with reduced RFS (6.9 vs 16.0 mo; p<0.0001) and decreased OS (9.1 vs 18.4 mo; p<0.0001). High ERCC1 was associated with reduced RFS (6.1 vs 15 mo; p=0.04) and decreased OS (8.9 vs 18.1 mo; p=0.03). RRM1 and hENT1 were not associated with survival. After accounting for known adverse factors, high RRM2 and ERCC1 persisted as negative prognostic factors for RFS and OS (Table). A subset analysis of those who received adjuvant therapy (n=74) revealed the same negative effect of high RRM2 and ERCC1 on RFS and OS. Conclusions: High RRM2 and ERCC1 tumor expression are associated with reduced RFS and OS after resection of pancreas cancer. These biomarkers may help personalize adjuvant therapy. [Table: see text]

A New Look at Vitamin D Metabolism and “Idiopathic” Hypercalcemia

A New Look at Vitamin D Metabolism and “Idiopathic” Hypercalcemia

Serum paraoxonase-3 concentration in HIV-infected patients. Evidence for a protective role against oxidation

We investigated the influence of the HIV infection on serum paraoxonase-3 (PON3) concentration and assessed the relationships with lipoprotein-associated abnormalities, immunological response, and accelerated atherosclerosis. We studied 207 HIV-infected patients and 385 healthy volunteers. Serum PON3 was determined by in-house ELISA, and PON3 distribution in lipoproteins was investigated by fast-performance liquid chromatography (FPLC). Polymorphisms of the PON3 promoter were analyzed by the Iplex Gold MassArray(TM) method. PON3 concentrations were increased (about three times) in HIV-infected patients with respect to controls (P < 0.001) and were inversely correlated with oxidized LDL levels (P = 0.038). Long-term use of nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy was associated with a decrease of PON3 concentrations. In a multivariate linear regression analysis, these relationships were still strong when the main confounding covariates were considered. PON3 was mainly found in HDL in HIV-infected patients, but a substantial amount of the protein was detected in LDL particles. This study reports for the first time an important increase in serum PON3 concentrations in HIV-infected patients that is associated with their oxidative status and their treatment with NNRTI. Long-term, prospective studies are needed to confirm the possible influence of this enzyme on the course of this disease and its possible utility as an analytical biomarker.

Human sapovirus classification based on complete capsid nucleotide sequences

The genetically diverse sapoviruses (SaVs) are a significant cause of acute human gastroenteritis. Human SaV surveillance is becoming more critical, and a better understanding of the diversity and distribution of the viral genotypes is needed. In this study, we analyzed 106 complete human SaV capsid nucleotide sequences to provide a better understanding of their diversity. Based on those results, we propose a novel standardized classification scheme that meets the requirements of the International Calicivirus Scientific Committee. We believe the classification scheme and strains described here will be of value for the molecular characterization and classification of newly detected SaV genotypes and for comparing data worldwide.

Oxidative Damage Targets Complexes Containing DNA Methyltransferases, SIRT1, and Polycomb Members to Promoter CpG Islands

Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an invivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Synthesis and evaluation of potential inhibitors of human and Escherichia coli histidine triad nucleotide binding proteins

Based on recent substrate specificity studies, a series of ribonucleotide based esters and carbamates were synthesized and screened as inhibitors of the phosphoramidases and acyl-AMP hydrolases, Escherichia coli Histidine Triad Nucleotide Binding Protein (ecHinT) and human Histidine Triad Nucleotide Binding Protein 1 (hHint1). Using our established phosphoramidase assay, K(i) values were determined. All compounds exhibited non-competitive inhibition profiles. The carbamate based inhibitors were shown to successfully suppress the Hint1-associated phenotype in E. coli, suggesting that they are permeable intracellular inhibitors of ecHinT.

Polymorphic Diversity of UBX Domain D from cDNA Isolated from Pectoral Muscle of Korean Native Chicken

The objectives of this study are to identify specific functional genes which are related with growth and protein structure of the pectoral muscle of Korean native chicken. Pectoral muscle was isolated from three Korean native chickens (KNC, red brown, 12 months old, 2.41 <TEX>${\pm}$</TEX> 0.24 kg) and three Cornish chickens (16 month old, 2.76 <TEX>${\pm}$</TEX> 3.0 kg). The subtraction cDNA library was prepared in PCR4 Blunt-TOPO vector. The DNA sequence homology was compared with other breeds and species in GenBank. A clone NDS-81 was found to be unique for the DNA sequence homology with UBX family. Their partial sequence has high homology (98%) with chicken UBX domain D. Chicken UBX domain has chicken (93%), cattle (68%), dog (67%), mouse (64%) and, human (63%) nucleotide sequence homology. Several regions were mutated from T in chicken to C or G in the NDS-81 clone. The first site is LAD in chicken, but it was expressed as (L)RM in clone NDS-81. In this site, amino acids were changed from Ala to Arg, and from Asp to Met. The second site was changed from ER (Arg) in chicken to ED (Asp) in clone NDS-81. They are both containing functional side chains and play an important role in binding other proteins. Therefore, the clone NDS-81 could be a different candidate gene for the UBX family gene and could related with pectoral muscle structure of Korean native chicken.

Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated

Phosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC–MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.

Haplotype estimation from fuzzy genotypes using penalized likelihood.

The Composite Link Model is a generalization of the generalized linear model in which expected values of observed counts are constructed as a sum of generalized linear components. When combined with penalized likelihood, it provides a powerful and elegant way to estimate haplotype probabilities from observed genotypes. Uncertain (“fuzzy”) genotypes, like those resulting from AFLP scores, can be handled by adding an extra layer to the model. We describe the model and the estimation algorithm. We apply it to a data set of accurate human single nucleotide polymorphism (SNP) and to a data set of fuzzy tomato AFLP scores.

PAX3‐FOXO1 and FGFR4 in alveolar rhabdomyosarcoma

We and others have identified FGFR4 as a direct transcriptional target of the alveolar rhabdomyosarcoma (ARMS) specific fusion protein, PAX3-FOXO1. We hypothesized fibroblast growth factor receptor 4 (FGFR4) may act as an effector of PAX3-FOXO1, contributing to PAX3-FOXO1 tumorigenic phenotypes. However, we demonstrate that enhanced expression of FGFR4 does not contribute to inhibited differentiation, enhanced proliferation, or transformation downstream of PAX3-FOXO1 in primary mouse myoblasts. Therefore we were unable to identify any contribution of up regulation of wild type FGFR4 to PAX3-FOXO1 driven tumorigenesis. Conversely, a constitutively active mutant of FGFR4 can enhance primary myoblast proliferation and transformation, indicating activating mutations of FGFR4 could contribute to the development and progression of ARMS. We sequenced the FGFR4 mRNA from five ARMS cell lines and identified no somatic mutations, nor any association with any human single nucleotide polymorphism within the FGFR4 coding region.

TGF-β signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts

We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G. Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123–130]. In the present study, we show that TGF-β, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-β is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-β are prevented by inhibitors of TGF-β RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-β and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-β signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.

Challenges in the association of human single nucleotide polymorphism mentions with unique database identifiers

BackgroundMost information on genomic variations and their associations with phenotypes are covered exclusively in scientific publications rather than in structured databases. These texts commonly describe variations using natural language; database identifiers are seldom mentioned. This complicates the retrieval of variations, associated articles, as well as information extraction, e. g. the search for biological implications. To overcome these challenges, procedures to map textual mentions of variations to database identifiers need to be developed.ResultsThis article describes a workflow for normalization of variation mentions, i.e. the association of them to unique database identifiers. Common pitfalls in the interpretation of single nucleotide polymorphism (SNP) mentions are highlighted and discussed. The developed normalization procedure achieves a precision of 98.1 % and a recall of 67.5% for unambiguous association of variation mentions with dbSNP identifiers on a text corpus based on 296 MEDLINE abstracts containing 527 mentions of SNPs.The annotated corpus is freely available at http://www.scai.fraunhofer.de/snp-normalization-corpus.html.ConclusionsComparable approaches usually focus on variations mentioned on the protein sequence and neglect problems for other SNP mentions. The results presented here indicate that normalizing SNPs described on DNA level is more difficult than the normalization of SNPs described on protein level. The challenges associated with normalization are exemplified with ambiguities and errors, which occur in this corpus.

Identification of Novel Interaction Sites that Determine Specificity between Fibroblast Growth Factor Homologous Factors and Voltage-gated Sodium Channels

Fibroblast growth factor homologous factors (FHFs, FGF11-14) bind to the C termini (CTs) of specific voltage-gated sodium channels (VGSC) and thereby regulate their function. The effect of an individual FHF on a specific VGSC varies greatly depending upon the individual FHF isoform. How individual FHFs impart distinctive effects on specific VGSCs is not known and the specificity of these pairwise interactions is not understood. Using several biochemical approaches combined with functional analysis, we mapped the interaction site for FGF12B on the Na(V)1.5 C terminus and discovered previously unknown determinants necessary for FGF12 interaction. Also, we demonstrated that FGF12B binds to some, but not all Na(V)1 CTs, suggesting specificity of interaction. Exploiting a human single nucleotide polymorphism in the core domain of FGF12 (P149Q), we identified a surface proline that contributes a part of this pairwise specificity. This proline is conserved among all FHFs, and mutation of the homologous residue in FGF13 also leads to loss of interaction with a specific VGSC CT (Na(V)1.1) and loss of modulation of the resultant Na(+) channel function. We hypothesized that some of the specificity mediated by this proline may result from differences in the affinity of the binding partners. Consistent with this hypothesis, surface plasmon resonance data showed that the P149Q mutation decreased the binding affinity between FHFs and VGSC CTs. Moreover, immunocytochemistry revealed that the mutation prevented proper subcellular targeting of FGF12 to the axon initial segment in neurons. Together, these results give new insights into details of the interactions between FHFs and Na(V)1.x CTs, and the consequent regulation of Na(+) channels.

Probing for DNA damage with β-hairpins: Similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[ a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similar relative NER incision efficiencies for ∼65% of these substrates; the other cases deviate mostly by ∼30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER.

New words in human mutagenesis

BackgroundThe substitution rates within different nucleotide contexts are subject to varying levels of bias. The most well known example of such bias is the excess of C to T (C > T) mutations in CpG (CG) dinucleotides. The molecular mechanisms underlying this bias are important factors in human genome evolution and cancer development. The discovery of other nucleotide contexts that have profound effects on substitution rates can improve our understanding of how mutations are acquired, and why mutation hotspots exist.ResultsWe compared rates of inherited mutations in 1-4 bp nucleotide contexts using reconstructed ancestral states of human single nucleotide polymorphisms (SNPs) from intergenic regions. Chimp and orangutan genomic sequences were used as outgroups. We uncovered 3.5 and 3.3-fold excesses of T > C mutations in the second position of ATTG and ATAG words, respectively, and a 3.4-fold excess of A > C mutations in the first position of the ACAA word.ConclusionsAlthough all the observed biases are less pronounced than the 5.1-fold excess of C > T mutations in CG dinucleotides, the three 4 bp mutation contexts mentioned above (and their complementary contexts) are well distinguished from all other mutation contexts. This provides a challenge to discover the underlying mechanisms responsible for the observed excesses of mutations.

A Bioinformatics Workflow for Variant Peptide Detection in Shotgun Proteomics

Shotgun proteomics data analysis usually relies on database search. However, commonly used protein sequence databases do not contain information on protein variants and thus prevent variant peptides and proteins from been identified. Including known coding variations into protein sequence databases could help alleviate this problem. Based on our recently published human Cancer Proteome Variation Database, we have created a protein sequence database that comprehensively annotates thousands of cancer-related coding variants collected in the Cancer Proteome Variation Database as well as noncancer-specific ones from the Single Nucleotide Polymorphism Database (dbSNP). Using this database, we then developed a data analysis workflow for variant peptide identification in shotgun proteomics. The high risk of false positive variant identifications was addressed by a modified false discovery rate estimation method. Analysis of colorectal cancer cell lines SW480, RKO, and HCT-116 revealed a total of 81 peptides that contain either noncancer-specific or cancer-related variations. Twenty-three out of 26 variants randomly selected from the 81 were confirmed by genomic sequencing. We further applied the workflow on data sets from three individual colorectal tumor specimens. A total of 204 distinct variant peptides were detected, and five carried known cancer-related mutations. Each individual showed a specific pattern of cancer-related mutations, suggesting potential use of this type of information for personalized medicine. Compatibility of the workflow has been tested with four popular database search engines including Sequest, Mascot, X!Tandem, and MyriMatch. In summary, we have developed a workflow that effectively uses existing genomic data to enable variant peptide detection in proteomics.