Lucia Pellizzari

Redox Potential Controls the Structure and DNA Binding Activity of the Paired Domain

Pax proteins are transcriptional regulators controlling a variety of cell fates during animal development. This role depends on the intact function of the paired (Prd) domain that is able to recognize specific DNA sequences. The Prd domain is composed of two distinct helix-turn-helix subdomains, PAI and RED. Molecular functions of Pax proteins are subjected to different levels of regulation involving both pre-translational and post-translational mechanisms. By using Pax-5 and Pax-8 recombinant proteins, we demonstrate that the binding activity of the Prd domain is regulated through the oxidation/reduction of conserved cysteine residues. Mass spectrometry analysis and mutagenesis experiments demonstrate that the redox regulation is accomplished through the reversible formation of an intramolecular disulfide bridge involving the cysteines present in the PAI subdomain, whereas the RED subdomain appears quite insensitive to redox potential. Circular dichroism experiments indicate that only the reduced form of the Prd domain is able to undergo the proper conformational change necessary for sequence-specific DNA binding. Nuclear extracts from different cell lines contain an activity that is able to reduce the Paired domain and, therefore, to control the DNA binding activity of this protein. Immunodepletion of nuclear extracts demonstrate that the protein Ref-1 contributes to the redox regulation of the Prd DNA binding activity. Given the modular nature of the Prd domain and the independent DNA binding specificity of the PAI and RED subdomains, we propose that this control mechanism should be involved in “switching” among different DNA sequences and therefore different target genes.

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5′‐TAAT‐3′. The homeodomain of thyroid transcription factor 1 (TTF‐1HD) binds to sequences containing a 5′‐CAAG‐3′ core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF‐1HD by both structural and functional approaches. As already suggested by the three‐dimensional structure of TTF‐1HD, the DNA binding specificities of the TTF‐1, Antennapedia and Engrailed homeodomains, either wild‐type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3′ end of the core motif 5′‐NAAN‐3′. The nucleotide at the 5′ position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF‐1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

Correlations between individual clinical manifestations and CTG repeat amplification in myotonic dystrophy.

Myotonic dystrophy (DM) is a multisystemic disease caused by the expansion of a CTG repeat, located in the 3′‐untranslated region of the DMPK gene. The number of CTG repeats broadly correlates with the overall severity of the disease. However, correlations between CTG repeat number and presence/absence or severity of individual clinical manifestations in the same patients are yet scarce. In this study the number of CTG repeats detected in blood cells of 24 DM subjects was correlated with the severity of single clinical manifestations. The presence/absence of muscular atrophy, respiratory insufficiency, cardiac abnormalities, diabetes, cataract, sleep disorders, sterility or hypogonadism is not related to the number of CTG repeats. Muscular atrophy and respiratory insufficiency are present with the highest frequency, occurring in 96 and 92% of the cases, respectively. A significant correlation was found with age of onset (r=−0.57, p<0.01), muscular disability (r=0.46, p<0.05), intellective quotient (r=−0.58, p<0.01) and short‐term memory (r=−0.59, p<0.01). Therefore, the CTG repeat number has a predictive value only in the case of some clinical manifestations, this suggesting that pathogenetic mechanisms of DM may differ depending on the tissue.

HEX expression and localization in normal mammary gland and breast carcinoma

BackgroundThe homeobox gene HEX is expressed in several cell types during different phases of animal development. It encodes for a protein localized in both the nucleus and the cytoplasm. During early mouse development, HEX is expressed in the primitive endoderm of blastocyst. Later, HEX is expressed in developing thyroid, liver, lung, as well as in haematopoietic progenitors and endothelial cells. Absence of nuclear expression has been observed during neoplastic transformation of the thyroid follicular cells. Aim of the present study was to evaluate the localization and the function of the protein HEX in normal and tumoral breast tissues and in breast cancer cell lines.MethodsHEX expression and nuclear localization were investigated by immunohistochemistry in normal and cancerous breast tissue, as well as in breast cancer cell lines. HEX mRNA levels were evaluated by real-time PCR. Effects of HEX expression on Sodium Iodide Symporter (NIS) gene promoter activity was investigated by HeLa cell transfection.ResultsIn normal breast HEX was detected both in the nucleus and in the cytoplasm. In both ductal and lobular breast carcinomas, a great reduction of nuclear HEX was observed. In several cells from normal breast tissue as well as in MCF-7 and T47D cell line, HEX was observed in the nucleolus. MCF-7 treatment with all-trans retinoic acid enhanced HEX expression and induced a diffuse nuclear localization. Enhanced HEX expression and diffuse nuclear localization were also obtained when MCF-7 cells were treated with inhibitors of histone deacetylases such as sodium butyrate and trichostatin A. With respect to normal non-lactating breast, the amount of nuclear HEX was greatly increased in lactating tissue. Transfection experiments demonstrated that HEX is able to up-regulate the activity of NIS promoter.ConclusionOur data indicate that localization of HEX is regulated in epithelial breast cells. Since modification of localization occurs during lactation and tumorigenesis, we suggest that HEX may play a role in differentiation of the epithelial breast cell.

Ape1/Ref-1 expression and cellular localization in human thyroid carcinoma cell lines

For its DNA repair, transcription factor regulation and anti-apoptotic activity, the apurinic/apirimidinic Ape1/Ref-1 endonuclease is thought to play a relevant role in human tumorigenesis. In human thyroid tumors, we demonstrated an altered nuclear/cytoplasmic ratio in all the carcinomas examined but not in follicular adenomas. In this study, Ref-1 expression and cellular localization were analyzed in a series of human thyroid carcinoma cell lines. We found a reduced nuclear/cytoplasmic ratio in BCPAP, TPC1 and ARO cells and not in WRO cells. Such a pattern of expression corresponds to that observed in thyroid tumoral tissues except for the WRO cells which behave as the follicular adenomas rather than carcinomas. Thus, these cell lines represent an excellent in vitro model to analyze the molecular mechanisms involved in Ref-1 regulation and activity and clarify its role in thyroid tumorigenesis.

Co-operation between the PAI and RED subdomains of Pax-8 in the interaction with the thyroglobulin promoter.

Pax proteins are transcription factors that play an important role in the differentiation of several cell types. These proteins bind to specific DNA sequences through the paired domain. This evolutionarily conserved element is composed of two subdomains (PAI and RED), located at the N- and C-terminals, respectively. Due to the presence of these two subdomains, Pax proteins may recognize DNA in different modes, a possibility that has not been exhaustively explored yet. The C site of the thyroglobulin promoter is bound by the thyroid-specific transcription factor Pax-8. In this study we have characterized the mode by which the Pax-8 paired domain interacts with the C site. Results allow the identification of the respective positions of the PAI and RED subdomains when the full-length protein is bound to the C site. The binding of the isolated PAI and RED subdomains to the C site and to several related mutants was also evaluated. Both subdomains interact with DNA as a monomer and display a lower binding affinity than the full-length protein. Therefore, the Pax-8 paired domain-C site interaction occurs through a co-operation between the two subdomains. The binding properties of the PAI subdomain suggest that the co-operation between PAI and RED subdomains does not merely consist of the sum of contacts established by the single subdomain: the presence of the RED subdomain is necessary for correct DNA recognition by the PAI subdomain, thus accounting for a sort of chronology of events during DNA binding. Since the RED subdomain is much more variable than the PAI subdomain among Pax proteins, these results could explain how distinct Pax proteins may select different target genes.

Functional interference between contacting amino acids of homeodomains

In a protein, the function of an amino acid at some position depends on the amino acids at other positions. Here we demonstrate a functional interference between base‐contacting amino acids (at positions 50 and 54) of homeodomains. When, in the context of Antennapedia or Goosecoid homeodomains, Lys50 is paired to Tyr54 or Ala54 and Gln50 is paired to Met54, the resulting proteins efficiently discriminate among different DNA sequences. In contrast, in the presence of the pair Lys50‐Met54, both homeodomains show a reduced capability to discriminate among different DNA sequences. Sequence selection experiments performed in the context of the Goosecoid homeodomain suggest that the presence of Met54 precludes the base‐discriminating function of Lys50. These results may explain why the pair Lys50‐Met54 is never found in natural homeodomains.

Functional analysis of a novel RUNX2 missense mutation found in a family with cleidocranial dysplasia.

AbstractMutations of the RUNX2 gene result in dominantly inherited cleidocranial dysplasia (CCD). RUNX2 encodes for an osteoblast-specific transcription factor, which recognizes specific DNA sequences by the runt domain. DNA binding is stabilized by the interaction with the protein CBFβ, which induces structural modifications of the runt domain. A novel 574G>A RUNX2 missense mutation has been found in members of a family clinically diagnosed with CCD. This mutation causes the glycine at position 192 to change to arginine (G192R), in loop 9 of the runt domain. Unlike other residues of loop 9, G192 does not establish DNA contacts. Accordingly, the G192R mutant showed a 50% reduction in binding activity compared to the wild-type runt domain. However, the mutation completely abolished the activating properties of the protein on osteocalcin promoter. Moreover, the G192R mutant exerts a dominant-negative effect when overexpressed. Computer modeling indicated that the G192R mutation perturbs not only loop 9, but also other parts of the runt domain, suggesting impairment of the interaction with CBFβ.

Combined yolk sac tumor and adenocarcinoma in a gastric stump

Extragonadal yolk sac tumors of the gastrointestinal tract are extremely rare neoplasms. Their greater rarity compared with other extragonadal yolk sac tumors suggests that different pathogenetic mechanisms could be involved according to the site of origin. This report describes a case of a combined yolk sac tumor and adenocarcinoma that arose in a gastric stump in a man age 61 years 43 years after he underwent distal gastric resection and gastrojejunostomy (Billroth II operation) for a benign duodenal ulcer. The coexistence of an adenocarcinomatous component with the yolk sac component suggests that the two histologic patterns may represent distinct phenotypes arising from a common mucosal epithelial cell.

Molecular analysis of a human PAX6 homeobox mutant

Pax6 controls eye, pancreas and brain morphogenesis. In humans, heterozygous PAX6 mutations cause aniridia and various other congenital eye abnormalities. Most frequent PAX6 missense mutations are located in the paired domain (PD), while very few missense mutations have been identified in the homeodomain (HD). In the present report, we describe a molecular analysis of the human PAX6 R242T missense mutation, which is located in the second helix of the HD. It was identified in a male child with partial aniridia in the left eye, presenting as a pseudo-coloboma. Gel-retardation assays revealed that the mutant HD binds DNA as well as the wild-type HD. In addition, the mutation does not modify the DNA-binding properties of the PD. Cell transfection assays indicated that the steady-state levels of the full length mutant protein are higher than those of the wild-type one. In cotransfection assays a PAX6 responsive promoter is activated to a higher extent by the mutant protein than by the wild-type protein. In vitro limited proteolysis assays indicated that the presence of the mutation reduces the sensitivity to trypsin digestion. Thus, we suggest that the R242T human phenotype could be due to abnormal increase of PAX6 protein, in keeping with the reported sensitivity of the eye phenotype to increased PAX6 dosage.