The p73 DNA binding domain displays enhanced stability relative to its homologue, the tumor suppressor p53, and exhibits cooperative DNA binding

The p53 protein family is involved in the control of an intricate network of genes implicated in cell cycle, through to germ line integrity and development. Although the role of p53 is well-established, the intrinsic nature of its homologue p73 has yet to be fully elucidated. Here, the biochemical characterization and homology-based modeling of the p73 protein is presented and the implications for its function(s) examined. The DNA binding domains (DBDs) of p53, p63, and p73 bind to the specific target site of a 30-mer gadd45 dsDNA, as tested by EMSA. The monomeric DBDs bind cooperatively forming tetrameric complexes. However, a larger construct consisting of p73 DBD plus TET domain (p73 CT) and the corresponding p53 DBD plus TET domain (p53 CT) bind gadd45 differently than the respective DBDs. Significantly, p73 DBD exhibited enhanced thermodynamic stability relative to the p53 DBD but not compared to p63 DBD as shown by DSC, CD, and equilibrium unfolding. The p73 CT is less stable than p73 DBD. The modeling data show distinct electrostatic surfaces of p73 and p53 dimers when bound to DNA. Specifically, the p73 surface is less complementary for DNA binding, which may account for the differences in affinity and specificity for p53 REs. These stability and DNA binding data for p73 in vitro enhance and complement our understanding of the role of the p73 protein in vivo and could be exploited in designing strategies for cancer therapy in places where p53 is mutated.     

Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface

Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.     

Potential Contribution of Monoamine Oxidase A Gene Variants in ADHD and Behavioral Co-Morbidities: Scenario in Eastern Indian Probands

Attention deficit hyperactivity disorder (ADHD) is the most frequently diagnosed behavioral disorder in children with a high frequency of co-morbid conditions like conduct disorder (CD) and oppositional defiant disorder (ODD). These traits are controlled by neurotransmitters like dopamine, serotonin and norepinephrine. Monoamine oxidase A (MAOA), a mitochondrial enzyme involved in the degradation of amines, has been reported to be associated with aggression, impulsivity, depression, and mood changes. We hypothesized that MAOA can have a potential role in ADHD associated CD/ODD and analyzed 24 markers in a group of Indo-Caucasoid subjects. ADHD probands and controls (N = 150 each) matched for ethnicity and gender were recruited following the Diagnostic and Statistical Manual for Mental Disorders-IV. Appropriate scales were used for measuring CD and ODD traits. Markers were genotyped by PCR-based methods and data obtained analyzed using the Cocaphase program under UNPHASED. Only eight markers were found to be polymorphic. rs6323 “G” allele showed higher frequencies in ADHD (P = 0.0023), ADHD + CD (P = 0.03) and ADHD + ODD (P = 0.01) as compared to controls. Haplotype analysis revealed statistically significant difference for three haplotypes in ADHD cases (P < 0.02). Statistically significant differences were also noticed for haplotypes in ADHD + CD and ADHD + ODD cases (P < 0.01). LD analysis showed significant variation in different groups. Multidimensionality reduction analysis showed independent as well as interactive effects of markers. Genotypes showed correlation with behavioral problems in ADHD and ADHD + CD. We interpret that MAOA gene variants may contribute to the etiology of ADHD as well as associated co-morbid CD and ODD in this ethnic group.     

Cortisol responses in children and adults with attention deficit hyperactivity disorder (ADHD): a possible marker of inhibition deficits

Attention deficit hyperactivity disorder (ADHD) is a heterogeneous disease whose neurobiological background is not completely understood. It has been proposed that deficits of the inhibitory function with an underactive behavioral inhibition system (BIS) may be in the core of ADHD. In this regard, this review summarizes all studies that examine the involvement of cortisol in ADHD. Differences in cortisol responses from different ADHD subtypes, hyperactive/impulsive, inattentive, and combined, are analyzed. In addition, we examine the role of comorbidities as confounding factors in the study of cortisol in ADHD, including comorbid disruptive behavioral disorder (DBD), as well as anxiety and depressive disorders. Because ADHD is a neurodevelopmental condition and approximately half of the children enter adulthood with the disorder, we review cortisol studies in adults and children separately. Two diverse patterns of cortisol have been reported both in children and adults with ADHD. Blunted cortisol responses to stress are associated with comorbid DBD, whereas high cortisol responses are associated to comorbid anxiety disorders. Nevertheless, the inhibitory deficits in ADHD do not appear to be related directly to cortisol deficits in either children or adults. This review increases our understanding of the heterogeneity of ADHD and could help in determining new strategies for the treatment of these patients. Future studies including gender and a more systematic methodology to study the cortisol response are needed.     

Low-resolution structures of thyroid hormone receptor dimers and tetramers in solution

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.     

Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.

We have previously reported that the binding site repertoires of heterodimers formed between retinoid X receptor (RXR) and either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) bound to response elements consisting of directly repeated PuG(G/T)TCA motifs spaced by 1-5 bp [direct repeat (DR) elements 1-5] are highly similar to those of their corresponding DNA binding domains (DBDs). We have now mapped the dimerization surfaces located in the DBDs of RXR, RAR and TR, which are responsible for cooperative interaction on DR4 (RXR and TR) and DR5 (RXR and RAR). The D-box of the C-terminal CII finger of RXR provides one of the surfaces which is specifically required for the formation of the heterodimerization interfaces on both DR4 and DR5. Heterodimerization with the RXR DBD on DR5 specifically requires the tip of the RAR CI finger as the complementary surface, while a 7 amino acid sequence encompassing the 'prefinger region', but not the TR CI finger, is specifically required for efficient dimerization of TR and RXR DBDs on DR4. Importantly, DBD swapping experiments demonstrate not only that the binding site repertoires of the full-length receptors are dictated by those of their DBDs, but also that the formation of distinct dimerization interfaces between the DBDs are the critical determinants for cooperative DNA binding of these receptors to specific DRs.     

Facial Mimicry in 6–7 Year Old Children with Disruptive Behavior Disorder and ADHD

Background

Impairments in facial mimicry are considered a proxy for deficits in affective empathy and have been demonstrated in 10 year old children and in adolescents with disruptive behavior disorder (DBD). However, it is not known whether these impairments are already present at an earlier age. Emotional deficits have also been shown in children with attention-deficit/hyperactivity disorder (ADHD).

Aims

To examine facial mimicry in younger, 6–7 year old children with DBD and with ADHD.

Methods

Electromyographic (EMG) activity in response to emotional facial expressions was recorded in 47 children with DBD, 18 children with ADHD and 35 healthy developing children.

Results

All groups displayed significant facial mimicry to the emotional expressions of other children. No group differences between children with DBD, children with ADHD and healthy developing children were found. In addition, no differences in facial mimicry were found between the clinical group (i.e., all children with a diagnosis) and the typically developing group in an analysis with ADHD symptoms as a covariate, and no differences were found between the clinical children and the typically developing children with DBD symptoms as a covariate.

Conclusion

Facial mimicry in children with DBD and ADHD throughout the first primary school years was unimpaired, in line with studies on empathy using other paradigms.     

Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3

The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.9 kJ/mol. Fluorescence anisotropy titration experiments showed that at this temperature the association constants with the PRDI binding site are 1.1 x 10(6) M(-)(1) for DBD1 and 3.6 x 10(6) M(-)(1) for DBD3, corresponding to Gibbs energies of association of -34 and -37 kJ/mol, respectively. However, the larger binding energy of DBD3 is due to its larger electrostatic component, while its nonelectrostatic component is smaller than that of DBD1. Therefore, DBD1 appears to have more sequence specificity than DBD3. Binding of DBD1 to target DNA is characterized by a substantially larger negative enthalpy than binding of DBD3, implying that the more flexible structure of DBD1 forms tighter contacts with DNA than the more rigid structure of DBD3. Thus, the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs.     

HPA-axis activity in alcoholism: examples for a gene-environment interaction.

Genetic and environmental influences are both known to be causal factors in the development and maintenance of substance abuse disorders. This review aims to focus on the contributions of genetic and environmental research to the understanding of alcoholism and how gene-environment interactions result in a variety of addiction phenotypes. Gene-environment interactions have been reviewed by focusing on one of the most relevant environmental risk factors for alcoholism, stress. This is examined in more detail by reviewing the functioning of the hypothalamic-pituitary-adrenal (HPA) axis and its genetic and molecular components in this disorder. Recent evidence from animal and human studies have shown that the effects of stress on alcohol drinking are mediated by core HPA axis genes and are associated with genetic variations in those genes. The findings of the studies discussed here suggest that the collaborations of neuroscience, psychobiology and molecular genetics provide a promising framework to elucidate the exact mechanisms of gene-environment interactions as seen to convene upon the HPA axis and effect phenotypes of addiction.