| 1. |
- Halim, Danny, et al.
(author)
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ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome
- 2016
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In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 25:3, s. 571-583
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Journal article (peer-reviewed)abstract
- Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a rare congenital disorder, in which heterozygous missense variants in the Enteric Smooth Muscle actin gamma-2 (ACTG2) gene have been recently identified. To investigate the mechanism by which ACTG2 variants lead to MMIHS, we screened a cohort of eleven MMIHS patients, eight sporadic and three familial cases, and performed immunohistochemistry, molecular modeling and molecular dynamics (MD) simulations, and in vitro assays. In all sporadic cases, a heterozygous missense variant in ACTG2 was identified. ACTG2 expression was detected in all intestinal layers where smooth muscle cells are present in different stages of human development. No histopathological abnormalities were found in the patients. Using molecular modeling and MD simulations, we predicted that ACTG2 variants lead to significant changes to the protein function. This was confirmed by in vitro studies, which showed that the identified variants not only impair ACTG2 polymerization, but also contribute to reduced cell contractility. Taken together, our results confirm the involvement of ACTG2 in sporadic MMIHS, and bring new insights to MMIHS pathogenesis.
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| 2. |
- Plaza-Menacho, Iván, et al.
(author)
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Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation.
- 2007
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:9, s. 6415-24
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Journal article (peer-reviewed)abstract
- The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.
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| 3. |
- Christaller, Wilhelm A A, et al.
(author)
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L1 syndrome diagnosis complemented with functional analysis of L1CAM variants located to the two N-terminal Ig-like domains.
- 2016
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In: Clinical Genetics. - : Wiley. - 0009-9163.
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Journal article (peer-reviewed)abstract
- L1CAM gene mutations cause neurodevelopmental disorders collectively termed L1 syndrome. Insufficient information about L1CAM variants complicates clinical prognosis, genetic diagnosis and genetic counseling. We combined clinical data, in silico effect predictions and functional analysis of four L1CAM variants, p.I37N, p.D202Y, p.M172I and p.T38M, located to the two N-terminal Ig-like domains present in five families with symptoms of L1 syndrome. Software tools predicted destabilizing effects of p.I37N and p.D202Y but results for p.T38M and p.M172I were inconsistent. Cell surface expression of mutant proteins L1-T38M, L1-M172I and L1-D202Y was normal. Conversely, L1-I37N accumulated in the endoplasmic reticulum and showed temperature-sensitive protein maturation suggesting that p.I37N induces protein misfolding. L1CAM-mediated cell-cell aggregation was severely impaired by L1CAM variants p.I37N, p.M172I and p.D202Y but was preserved by the variant p.T38M. Our experimental data indicate that protein misfolding and accumulation in the endoplasmic reticulum affect function of the L1CAM variant p.I37N whereas the variants p.M172I and p.D202Y impair homophilic interaction at the cell surface.
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