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Z Ronai

Publications and source records attributed to Z Ronai.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of p53 localization and transcription by the HECT domain E3 ligase WWP1.

As a key cellular regulatory protein p53 is subject to tight regulation by several E3 ligases. Here, we demonstrate the role of HECT domain E3 ligase, WWP1, in regulating p53 localization and activity. WWP1 associates with p53 and induces p53 ubiquitylation. Unlike other E3 ligases, WWP1 increases p53 stability; inhibition of WWP1 expression or expression of a ligase-mutant form results in decreased p53 expression. WWP1-mediated stabilization of p53 is associated with increased accumulation of p53 in cytoplasm with a concomitant decrease in its transcriptional activities. WWP1 effects are independent of Mdm2 as they are seen in cells lacking Mdm2 expression. Whereas WWP1 limits p53 activity, p53 reduces expression of WWP1, pointing to a possible feedback loop mechanism. Taken together, these findings identify the first instance of a ubiquitin ligase that causes stabilization of p53 while inactivating its transcriptional activities.

Amino Acid Motifs↗
PubMed2005

The polymorphic nature of the human dopamine D4 receptor gene: a comparative analysis of known variants and a novel 27 bp deletion in the promoter region.

BACKGROUND: The human dopamine D4 receptor (DRD4) is a candidate gene of great interest in molecular studies of human personality and psychiatric disorders. This gene is unique in having an exceptionally high amount of polymorphic sites both in the coding and in the promoter region. RESULTS: We report the identification of a new 27 bp deletion starting 524 bp upstream of the initiation codon (27 bp del) of the dopamine D4 receptor (DRD4) gene, in the close vicinity of the -521C>T SNP. The presence of the 27 bp deletion leads to the misgenotyping of the -616C>G SNP by the Sau96 I RFLP method, thus the genotype determination of the mutation is of additional importance. The frequency of this novel sequence variation is considerably low (allele frequency is = 0.16%), as no homozygotes, and only 3 heterozygote carriers were found in a healthy, unrelated Caucasian sample (N = 955). CONCLUSION: Remarkably, the deleted region contains consensus sequences of binding sites for several known transcription factors, suggesting that the different alleles may affect the transcriptional regulation of the gene. A comparison of methods and results for the allelic variations of the DRD4 gene in various ethnic groups is also discussed, which has a high impact in psychiatric genetic studies.

Binding Sites↗
PubMed2004

A novel A/G SNP in the -615th position of the dopamine D4 receptor promoter region as a source of misgenotyping of the -616 C/G SNP.

The polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene containing several single nucleotide polymorphisms (SNPs) has recently become a focus of association studies in psychiatric genetics. Most SNP genotyping methods are based on the two-step procedure of restriction fragment length polymorphism (RFLP). An alternative technique is a single-step method of allele-specific amplification (ASA), previously introduced for genotyping the -521 C/T SNP of the DRD4 promoter region and applied here for the -616 C/G SNP. Parallel genotyping of individuals with the novel ASA method and the conventionally used Ava II RFLP showed a potential underestimation of the -616 GG genotype frequency by the conventional method. Sequencing the dubious samples clearly demonstrated a novel A/G SNP at the -615th position influencing the Ava II digestion and thus resulting in misgenotyping. To avoid this problem, we introduced the Sau96 I RFLP for the -616 C/G genotyping as this restriction enzyme is not sensitive for the -615 A/G sequence variation. Allele (-616 G = 0.48; -616 C = 0.52) and genotype (-616 GG = 0.25; -616 GC = 0.46; -616 CC = 0.29) frequencies were determined by both the novel ASA and the Sau96 I methods. The obtained genotype frequencies corresponded to the Hardy-Weinberg equilibrium in our healthy Caucasian sample (N = 534, P = 0.168). Using these methods, no association was found between the -616 C/G SNP and personality factors of Cloninger's temperament and character inventory (N = 153) in our population.

Gene Frequency↗
PubMed2004

Human personality dimensions of persistence and harm avoidance associated with DRD4 and 5-HTTLPR polymorphisms.

The associations of human personality traits as measured by the Temperament and Character Inventory (TCI) with two genetic polymorphisms, the dopamine D4 receptor (DRD4) gene exon III repeat polymorphism (VNTR) and the serotonin transporter-linked functional polymorphism (5-HTTLPR) are presented in a population of 157 ethnically homogeneous Caucasians. No association was found between Novelty Seeking and the DRD4 VNTR, but male individuals with a 7-repeat allele exhibited significantly lower Persistence scores. The 5-HTTLPR polymorphism itself had no significant effect on any of the temperament dimensions, but a significant DRD4 VNTR x 5-HTTLPR interaction was observed for Harm Avoidance, the subgroup with a s/s 5-HTTLPR, 7-repeat DRD4 genotype showed a higher mean Harm Avoidance score than the other groups. These results are discussed in relation to the recent findings on infant temperament. Association between the DRD4 7-repeat allele and Persistence can be theoretically linked to the 7-repeat allele as a risk factor for attention deficit hyperactivity disorder.

Adult↗
PubMed2004

Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene.

DNA-protein interaction in the 5' upstream polymorphic region of the dopamine D4 receptor (DRD4) gene was analyzed by capillary electrophoretic mobility shift assay (CEMSA). The sequence of interest was amplified using a fluorescent primer and applied as a probe in the binding assays with HeLa nuclear extract. Serial dilution of the probe resulted in a concentration dependent DNA-protein complex formation. Sp 1 specific oligonucleotide competitor significantly inhibited the DNA-protein complex formation. A non-specific competitor, differing only in three base pairs, showed weaker effect pointing to the contribution of the Sp 1 recognition sequence in the complex. Polymorphic competitors were also prepared from homozygous individuals possessing either duplicated (2 x 120 bp) or single copy (1 x 120 bp) of the 120 bp repeat sequence and were used against the Sp 1 specific probe in competition assays. Our data provide experimental evidence for the binding of Sp 1 to the 120 bp duplicated sequence of the DRD4 5' upstream region and suggest enhanced binding capacity of the duplicated form.

5' Flanking Region↗
PubMed2003

Association of D4 dopamine receptor gene and serotonin transporter promoter polymorphisms with infants' response to novelty.

Effects of DRD4 and 5-HTTLPR length polymorphisms have been reported on neonatal and infant temperament as well as adult personality traits. The 7-repeat form of the DRD4 III exon VNTR polymorphism has been associated with childhood ADHD, and recently we have reported its link with attachment disorganization in a nonclinical population of infants. Here, we report associations of these polymorphisms with infant temperament at 12 months of age. Maternal accounts of temperament and observed response to novelty were investigated for 90 infants, who were independently genotyped for the DRD4 III exon, and for 5-HTT-linked promoter region length polymorphisms. Maternal rating of temperament was not affected by these polymorphisms, but we found combined genotype effects for infants' observed responses to a novel, anxiety-provoking stimulus: the appearance of, and approach by, a stranger. Infants with at least one copy of both the 7-repeat DRD4 allele and the long variant of 5-HTTLPR (7(+), l/l&l/s) responded with significantly less anxiety than infants with other genotypes. However, infants with the 7-repeat DRD4 allele and homozygous for the short form of 5-HTTLPR (7(+), s/s) showed more anxiety and resistance to the stranger's initiation of interaction. These genotype effects were not redundant with the previously reported association between the 7-repeat DRD4 allele and disorganized attachment behavior. Although both temperament and attachment behavior were affected by the DRD4 repeat polymorphism, the effect on temperament measures was modified by the infants' 5-HTTLPR genotype.

Carrier Proteins↗
PubMed2002

Further evidence for the role of the dopamine D4 receptor (DRD4) gene in attachment disorganization: interaction of the exon III 48-bp repeat and the -521 C/T promoter polymorphisms.

In non-clinical low-risk populations 15% of infants show disorganized attachment behavior(1,2) with their caregivers in the Strange Situation,(3) a mildly stressful laboratory procedure testing infants' ability to cope with separation anxiety. Disorganization of early attachment has been primarily ascribed to inadequate parenting,(2,4,5) and has been associated with childhood behavior problems(6,7)and adolescent psychopathological tendencies.(5) We have recently reported an association between the DRD4 exon III 48 basepair repeat polymorphism and disorganization of infants' attachment behavior towards their mother in a low-social-risk group of 1-year-old infants:(8) the risk for disorganized attachment among infants carrying the 7-repeat allele was fourfold. Here we report further evidence for the involvement of the dopamine D4 receptor gene in attachment disorganization. The same group of infants was genotyped for the functional -521 C/T single nucleotide polymorphism (SNP) in the upstream regulatory region of the DRD4 gene(9) in order to test the association with attachment disorganization both alone and in interaction with the DRD4 exon III 7-repeat allele. While the -521 C/T genotype itself had no effect on attachment status (chi(2) = 0.41, df = 2, P = 0.82), there was an interaction between the structural 48-bp repeat polymorphism and the -521 C/T promoter polymorphism: the association between disorganized attachment and the 7-repeat allele was enhanced in the presence of the -521 T allele (chi(2) = 6.61 and 6.67, df = 1, P < 0.025 for CT and TT genotypes, respectively). In the presence of both risk alleles the odds ratio for disorganized attachment increased tenfold. This result supports our previous postulation that the DRD4 gene plays a role in the development of attachment behavior in low-risk, non-clinical populations.

Alleles↗
PubMed2001

Analysis of dopamine D4 receptor gene polymorphism using microchip electrophoresis.

A microfabricated electrophoresis device was used for rapid polymerase chain reaction product analysis in genotyping the dopamine D4 receptor gene (DRD4) 48 base pairs repeat polymorphism. An allelic ladder, prepared from homozygous individuals, was used as internal standard during the microchip electrophoresis based analysis. Comparison of this novel separation method with the conventional slab gel and previously reported ultra-thin-layer techniques confirmed the reliability of this new method. Genotyping of 332 healthy Hungarian individuals gave the following allele frequencies: two-repeat: 0.089; three-repeat: 0.026; four-repeat: 0.674; five-repeat: 0.011; six-repeat: 0.002; seven-repeat: 0.189; eight-repeat: 0.011. The genotype frequencies obtained showed no deviation from the Hardy-Weinberg equilibrium (p>0.903), further underlying the reliability of this new genotyping technique.

Base Sequence↗
PubMed2001

Distinct pattern of p53 phosphorylation in human tumors.

The protein product of the tumor suppressor gene p53 is phosphorylated on multiple residues by several protein kinases. Using a battery of 10 antibodies developed against different phosphorylated and acetylated residues of p53, we compared the pattern of p53 phosphorylation and acetylation in tumor-derived cell lines, tumor samples, and non-neoplastic cells. Irrespective of tumor types or the presence of p53 mutation, phosphorylation and acetylation of p53 was substantially higher in samples obtained from tumor tissues than those found in non-transformed samples. Among the 10 sites analysed, phosphorylation of residues 15, 81, 392, and acetylation were among the more frequent modifications. Analysis of two of the more abundant phosphorylation or acetylation sites on p53 is sufficient to detect 72% of tumor-derived p53 proteins. The distinct pattern of p53 phosphorylation and acetylation in human tumors may offer a new means to monitor the status and activity of p53 in the course of tumor development and progression.

Acetylation↗
PubMed2001

The Mdm-2 amino terminus is required for Mdm2 binding and SUMO-1 conjugation by the E2 SUMO-1 conjugating enzyme Ubc9.

Covalent attachment of SUMO-1 to Mdm2 requires the activation of a heterodimeric Aos1-Uba2 enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm2 by Ubc9, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Upon Sumo-1 conjugation, Mdm2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide ligase (E3) activity toward p53, thereby increasing its oncogenic potential. Because of the biological implication of Mdm2 sumoylation, we mapped Ubc9 binding on Mdm2. Here we demonstrate that Ubc9 can associate with Mdm2 only if amino acids 40-59 within the N terminus of Mdm2 are present. Mdm2 from which amino acids 40-59 have been deleted can no longer be sumoylated. Furthermore, addition of a peptide that corresponds to amino acids 40-59 on Mdm2 to a sumoylation reaction efficiently inhibits Mdm2 sumoylation in vitro and in vivo. In UV-treated cells Mdm2 exhibits reduced association with Ubc9, which coincides with decreased Mdm2 sumoylation. Our findings regarding the association of Ubc9 with Mdm2, and the effect of UV-irradiation on Ubc9 binding, point to an additional level in the regulation of Mdm2 sumoylation under normal growth conditions as well as in response to stress conditions.

3T3 Cells↗
PubMed2001

Induction of beta-transducin repeat-containing protein by JNK signaling and its role in the activation of NF-kappaB.

Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed.

3T3 Cells↗
PubMed2001

ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage.

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.

Amino Acid Substitution↗
PubMed2001

Expression of ring finger-deleted TRAF2 sensitizes metastatic melanoma cells to apoptosis via up-regulation of p38, TNFalpha and suppression of NF-kappaB activities.

Mechanisms underlying radiation and chemotherapy resistance, the hallmark of human melanoma, are not well understood. Here we demonstrate that expression levels of signal adaptor protein TRAF2 coincide with melanoma resistance to UV-irradiation. Altered TRAF2 signaling by a form of TRAF2, which lacks the ring finger domain (TRAF2DeltaN), increases activities of p38 MAPK, ATF2, and the level of TNFalpha expression. Forced expression of TRAF2DeltaN in HHMSX highly metastatic melanoma cells that lack Fas expression and thus utilize the TNFalpha-TNFR1 as the major apoptotic pathway sensitized cells to UV-induced apoptosis. An over twofold increase in degree of apoptosis was observed in TRAF2DeltaN expressing cells that were treated with actinomycin D, anisomycin or with the radiomimetic drug neocarzinostatin. Sensitization by TRAF2DeltaN is selective since it was not observed in response to either Taxol or cis-platinum treatment. TRAF2DeltaN effects are primarily mediated via p38 since inhibition of p38 reduces, whereas activation of p38 promotes the level of UV-induced apoptosis. Conversely, activation of IKK attenuates the sensitization of melanoma by TRAF2DeltaN, indicating that p38-mediated suppression of NF-kappaB activity is among TRAF2DeltaN effects. Our finding identifies p38, TNFalpha and NF-kappaB among key players that efficiently sensitizes melanoma cells to UV-, ribotoxic (anisomycin) and radiomimetic chemicals-induced programmed cell death in response to aberrant TRAF2 signaling.

Acetyltransferases↗
PubMed2001

Identification of new JNK substrate using ATP pocket mutant JNK and a corresponding ATP analogue.

Modification of the ATP pocket on protein kinases allows selective use of an ATP analogue that exhibits high affinity for the altered kinases. Using this approach, we altered the ATP-binding site on JNK and identified N(6)-(2-phenythyl)-ATP, a modified form of ATP that exhibits high specificity and affinity for the modified, but not the wild type form, of JNK. Using modified JNK and its ATP analogue enables the detection of novel JNK substrates. Among substrates identified using this approach is heterogeneous nuclear ribonucleoprotein K, which is involved in transcription and post-transcriptional mRNA metabolism. The newly identified substrate can be phosphorylated by JNK on amino acids 216 and 353, which contribute to heterogeneous nuclear ribonucleoprotein K mediated transcriptional activities.

Adenosine Triphosphate↗
PubMed2001

Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus.

c-Jun N-terminal kinase (JNK)-mediated cell signaling pathways are regulated endogenously in part by protein-protein interactions with glutathione S-transferase P1-1 (GSTP1-1) (). Using purified recombinant proteins, combined with fluorescence resonance energy transfer technology, we have found that the C terminus of JNK is critical to the interaction with GSTP1-1. The apparent K(d) for full-length JNK was 188 nm and for a C-terminal fragment (residues 200-424) 217 nm. An N-terminal fragment (residues 1-206) did not bind to GSTP1-1. Increased expression of the C-terminal JNK fragment in a tetracycline-inducible transfected NIH3T3 cell line produced a concentration-dependent increase in the kinase activity of JNK under normal, unstressed growth conditions indicating a dominant-negative effect. This suggests that the fragment can compete with endogenous full-length functional JNK resulting in dissociation of the GSTP1-1-JNK interaction and concomitant JNK enzyme activation. By using an antibody to hemagglutinin-tagged C-JNK, a concentration-dependent co-immunoprecipitation of GSTP1-1 was achieved. These data provide evidence for direct interactions between the C-terminal of JNK and GSTP1-1 and a rationale for considering GSTP1-1 as a critical ligand-binding protein with a role in regulating kinase pathways.

3T3 Cells↗
PubMed2001

Stabilization and activation of p53 by the coactivator protein TAFII31.

Regulation of the stability of p53 is key to its tumor-suppressing activities. mdm2 directly binds to the amino-terminal region of p53 and targets it for degradation through the ubiquitin-proteasome pathway. The coactivator protein TAF(II)31 binds to p53 at the amino-terminal region that is also required for interaction with mdm2. In this report, we demonstrate that expression of TAF(II)31 inhibits mdm2-mediated ubiquitination of p53 and increases p53 levels. TAF(II)31-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts. UV-induced stabilization of p53 coincides with an increase in p53-associated TAF(II)31 and a corresponding decrease in mdm2-p53 interaction. Non-p53 binding mutant of TAF(II)31 fails to stabilize p53. Our results suggest that direct interaction of TAF(II)31 and p53 not only mediates p53 transcriptional activation but also protects p53 from mdm2-mediated degradation, thereby resulting in activation of p53 functions.

3T3 Cells↗
PubMed2001

Genotyping the -521C/T functional polymorphism in the promoter region of dopamine D4 receptor (DRD4) gene.

The -521C/Tsingle nucleotide polymorphism (SNP) in the promoter region of the dopamine D4 receptor gene (DRD4) has recently been detected in oriental (Japanese) individuals and related to novelty seeking and schizophrenia. Here, we report the analysis of the -521C/T polymorphism in a Caucasian (Hungarian) population using two independent genotyping methods. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure utilized the Fspl restriction site around the -521 position. An additional, nonpolymorphic cleavage site was also included into the amplified region to serve as an internal standard for verifying the completion of the digestion. As another independent method, a tetraprimer system for single-tube allele-specific PCR (SAS-PCR) was developed to generate -521C and -521T specific PCR products with different fragment sizes. Consequently, genotyping with SAS-PCR is based on the gel-electrophoretic separation of the allele-specific double-stranded DNA (dsDNA) fragments. 119 healthy Hungarian individuals were genotyped for -521C/T polymorphism of the dopamine D4 promoter region, using both methods. Similar allele frequencies were found (-521C allele: 0.43; -521T allele: 0.57) as reported earlier for the Japanese population.

Alleles↗