A Genome-wide Linkage Analysis and Mutation Analysis of Hereditary Congenital Blepharoptosis in a Japanese Family

Hereditary congenital ptosis (PTOS) is defined as drooping of the upper eyelid without any other accompanying symptoms and distinguished from syndromic blepharoptosis.Two previous linkage analyses assigned a PTOS locus (PTOS1) to 1p32-p34.1 and another (PTOS2) to Xq24-q27.1. In addition, in a sporadic case with a balanced chromosomal translocation t(1;8)(p34.3;q21.12), the ZFHX4 (zinc finger homeodomain 4) gene was found to be disrupted at the 8q21.12 breakpoint, but there was no gene at the 1p34.3 breakpoint, suggesting the existence of the third PTOS locus (PTOS1) at 8q21.12. We carried out a genome-wide linkage analysis in a Japanese PTOS family and calculated two-point and multipoint LOD scores with reduced penetrance. Haplotype analysis gave three candidate disease-responsible regions, i.e., 8q21.11-q22.1, 12q24.32-q24.33 and 14q21.1-q23.2. Although the family size is too small to define one of them, 8q21.11-q22.1 is a likely candidate region, because it contains the previously reported translocation breakpoint above. We thus performed mutation, Southern-blot and methylation analyses of ZFHX4, but could not find any disease specific change in the family. Nevertheless, our data may support the localization of PTOS1.長崎大学学位論文 学位記番号:博（医歯薬）甲第153号 博士（医学）学位授与年月日:平成20年3月19

Structural Transformation of the Tandem Ubiquitin-Interacting Motifs in Ataxin-3 and Their Cooperative Interactions with Ubiquitin Chains

The ubiquitin-interacting motif (UIM) is a short peptide with dual function of binding ubiquitin (Ub) and promoting ubiquitination. We elucidated the structures and dynamics of the tandem UIMs of ataxin-3 (AT3-UIM12) both in free and Ub-bound forms. The solution structure of free AT3-UIM12 consists of two α-helices and a flexible linker, whereas that of the Ub-bound form is much more compact with hydrophobic contacts between the two helices. NMR dynamics indicates that the flexible linker becomes rigid when AT3-UIM12 binds with Ub. Isothermal titration calorimetry and NMR titration demonstrate that AT3-UIM12 binds diUb with two distinct affinities, and the linker plays a critical role in association of the two helices in diUb binding. These results provide an implication that the tandem UIM12 interacts with Ub or diUb in a cooperative manner through an allosteric effect and dynamics change of the linker region, which might be related to its recognitions with various Ub chains and ubiquitinated substrates

Ataxin-3 Plays a Role in Mouse Myogenic Differentiation through Regulation of Integrin Subunit Levels

BACKGROUND: During myogenesis several transcription factors and regulators of protein synthesis and assembly are rapidly degraded by the ubiquitin-proteasome system (UPS). Given the potential role of the deubiquitinating enzyme (DUB) ataxin-3 in the UPS, and the high expression of the murine ataxin-3 homolog in muscle during embryogenesis, we sought to define its role in muscle differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence analysis, we found murine ataxin-3 (mATX3) to be highly expressed in the differentiated myotome of E9.5 mouse embryos. C2C12 myoblasts depleted of mATX3 by RNA interference exhibited a round morphology, cell misalignment, and a delay in differentiation following myogenesis induction. Interestingly, these cells showed a down-regulation of alpha5 and alpha7 integrin subunit levels both by immunoblotting and immunofluorescence. Mouse ATX3 was found to interact with alpha5 integrin subunit and to stabilize this protein by repressing its degradation through the UPS. Proteomic analysis of mATX3-depleted C2C12 cells revealed alteration of the levels of several proteins related to integrin signaling. CONCLUSIONS: Ataxin-3 is important for myogenesis through regulation of integrin subunit levels.This work was financed by the Fundacao para a Ciencia e a Tecnologia (FCT) (POCI/SAU-MMO/60412/2002) and by National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) grant RO1 NS038712 to HLP. MCC, FB, AJR, and RJT were supported by the FCT fellowships (SFRH/BD/9759/2003 and SFRH/BPD/28560/2006), (SFRH/BPD/17368/2004), (SFRH/BD/17066/2004), (SFRH/BD/29947/2006), respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript