Characterization Complex Involved In Rothmund-Thomson

Rothmund Thomson综合征(RTS)是一种罕见的疾病，与基因组不稳定、癌症易感性、皮肤和骨骼异常以及过早衰老的某些特征有关。这种疾病是由RECQL4突变引起的，RECQL4是RecQ家族的一种解旋酶，包括Werner和Bloom综合征中分别存在缺陷的蛋白质WRN和BLM。为了了解这种疾病的发病机制，我们纯化了一个人RECQL4复合体，并鉴定了它的相关成分。我们发现，RECQL4的大部分存在于HeLa细胞的细胞质提取物中，而不是主要有核的BLM和WRN解旋酶。然而，在未转化的Wi-38成纤维细胞中，RECQL4被发现主要是有核的，并且在转化的HeLa细胞中的总水平显著低于转化的HeLa细胞。从HeLa细胞中分离到RECQL4，它与UBR1和UBR2形成稳定的复合体。这些200kD的蛋白质是N-末端规则通路的泛素连接酶，其底物包括具有不稳定N-末端残基的蛋白质。这种蛋白分解途径的功能包括调节多肽输入、染色体稳定性、减数分裂、细胞凋亡和心血管发育。虽然UBR1和UBR2的已知作用是介导其底物的多泛素化(和随后的降解)，但UBR1/2结合的RECQL4在体内不是泛素化的，在HeLa细胞中是一个长期存在的蛋白质。分离的RECQL4-UBR1/2复合体具有DNA刺激的ATPase活性，但在基于DNA的解旋酶和转位酶检测中无效，在BLM解旋酶活性的检测中。我们的数据表明，N端规则通路的RECQL4和泛素连接酶可能在维持基因组稳定性方面发挥作用。这些研究的第一份报告现已发表在《人类分子遗传学》上。

Rothmund Thomson Syndrome (RTS) is a rare disease associated with genome instability, predisposition to cancer, skin and skeletal abnormalities, and some features of premature aging. The disease is caused by mutation in RECQL4, a putative helicase of the RecQ family which includes WRN and BLM, proteins defective in Werner and Bloom syndromes, respectively. To understand the mechanism of this disease, we have purified a human RECQL4 complex and identified its associated components. We found that the bulk of RECQL4 was present in a cytoplasmic extract of HeLa cells, in contrast to the largely nuclear BLM and WRN helicases. However, in untransformed WI-38 fibroblasts, RECQL4 was found to be largely nuclear, and was present at significantly lower total levels than in transformed HeLa cells. RECQL4 from HeLa cells was isolated as a stable complex with UBR1 and UBR2. These 200 kD proteins are ubiquitin ligases of the N-end rule pathway, whose substrates include proteins with destabilizing N-terminal residues. The functions of this proteolytic pathway include the regulation of peptide import, chromosome stability, meiosis, apoptosis, and cardiovascular development. Although the known role of UBR1 and UBR2 is to mediate polyubiquitylation (and subsequent degradation) of their substrates, the UBR1/2-bound RECQL4 was not ubiquitylated in vivo, and was a long-lived protein in HeLa cells. The isolated RECQL4-UBR1/2 complex had a DNA-stimulated ATPase activity but was inactive in DNA-based assays for helicases and translocases, the assays in which the BLM helicase was active. Our data suggest that RECQL4 and ubiquitin ligases of the N-end rule pathway may play a role in maintaining genomic stability. The first report of these studies is now published in Human Molecular Genetics.