Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the 18 mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants, the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wild-type PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.
A single recurrent mutation in the regulatory subdomain of a bone morphogenetic protein type I receptor kinase has been linked to heterotopic ossification in classic fibrodysplasia ossificans progressiva (FOP). As a result of a substitution at 1 residue by only 1 other side chain (Arg206His) in just 1 of the 4 type I BMP receptors (ALK2/ACVR1), soft connective tissues progressively metamorphose through an endochondral process into cartilage that is replaced by bone. The substitution of arginine for histidine, also a basic residue yet with the singular property of ionization/protonation over the physiological pH range, led to the hypothesis of an aberrant, pH-sensitive switch mechanism for the ligand-independent activation of BMP signaling through the mutant receptor kinase in patients presenting with classic FOP. To test a potential aspect of the putative pH-dependent mechanism, i.e. loss of autoinhibition of the kinase mediated by the inhibitory protein FKBP12, in vitrointeraction analyses with purified wild-type and R206H ALK2 kinase and FKBP12 proteins were performed. Interactions between the kinases and inhibitory proteins were analyzed qualitatively and quantitatively by native gel electrophoresis and HPLC size exclusion chromatography and with an optical biosensor (Octet; ForteBio). Binding of inhibitory protein by the R206H mutant was diminished 3-fold relative to the wild type kinase at a physiological pH, yet below this value (<∼7.5) pronounced nonspecific interactions, particularly with the mutant, prevented comparative evaluations. In conclusion, substitution with histidine leads to partial loss of inhibition of the mutant type I receptor through diminished binding of FKBP12, which may act as a gradient reader in morphogenetic contexts.
Numerous epidemiological and experimental animal studies have indicated that chronic psychological stress may promote tumor development. However, the underlying molecular mechanisms by which chronic stress promotes tumorigenesis remain to be fully elucidated and animal models have not yet been well established. In the present study, an established mouse model of repeated social defeat stress (RSDS), was generated and used to investigate the effect of stress on tumor growth and metastasis. C57BL/6 mice were exposed to RSDS for 10 days, followed by subcutaneousl inoculation with Lewis lung carcinoma cells for seven days. The tumor weight and volume as well as the number of the lung metastatic nodules were then determined. Vascular endothelial growth factor (VEGF) serum levels were measured using ELISAs. In addition, expression levels of VEGF receptor (VEGFR) and L1 cell adhesion molecule (L1CAM) messenger (m)RNA were confirmed using reverse transcription quantitative polymerase chain reaction. Furthermore, protein expression levels of phosphorlyated extracellular signal-regulated kinase (pERK), matrix metalloproteinase (MMP)-2 and MMP-9 were examined using western blot analysis. The results showed that RSDS significantly increased the weight and the volume of the primary tumor as well as the number of the lung metastatic nodules. Serum VEGF levels were significantly higher in the tumor-stress group compared with those of the unstressed tumor mice. In addition, tumors in stressed animals demonstrated markedly enhanced expression of VEGFR-2 and L1CAM mRNA as well as pERK, MMP-2 and MMP-9 protein expression. In conclusion, these results suggested that RSDS contributed to lung cancer progression, angiogenesis and metastasis, which was partially associated with increased VEGF secretion and therefore the activation of the ERK signaling pathway, resulting in the induction of MMP-2 and MMP-9 protein expression.
The paired-domain transcription factor PAX9 plays a critical role in tooth development, as heterozygous mutations in PAX9 have been shown to be associated with human tooth agenesis. In this study, we report 2 novel missense mutations, gly6arg (G6R) and ser43lys (S43K), in the paired domain of PAX9 in Chinese patients with varying degrees of nonsyndromic tooth agenesis. Excluding third molars, the individual with the G6R mutation was missing 2 mandibular incisors and a maxillary premolar, while the phenotype of individuals with the S43K mutation consisted of peg-shaped upper lateral incisors and missing molars, premolars and canines. As these 2 mutations occur at highly conserved amino acids in the PAX gene family and between different species, we further analyzed the effects of the mutations on the function of the resulting proteins. Immunofluorescence and immunoblotting studies showed that the mutations did not alter nuclear localization in mammalian cells. Gel shift and super shift assays indicate that both mutant proteins bound DNA at a lower level than the normal protein, with G6R having a greater affinity for DNA than S43K. Likewise, the G6R protein was able to transcriptionally activate a Bmp4 promoter construct to a greater extent than S43K. Our finding that the severity of tooth agenesis in the patients was correlated to the DNA-binding capacity of the mutated PAX9 9proteins supports the hypothesis that DNA binding is responsible for the genetic defect.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.