生物谷报道：端粒（线性染色体的端部）受各种结合蛋白的保护。纤毛虫中一个重要的端粒结合复合体是TBPalpa/beta。人类有一个TBPa同源结构POT1，但此前TBPb在纤毛虫以外的生物中没有被发现。现在，两个研究小组各自独立地识别出，神秘莫测的TBPb的人类同源结构为TPP1。这一研究结果发表在最新一期的《Nature》杂志上。令人吃惊的是，当POT1-TPP1复合体结合到端粒DNA上时，它并不抑制端粒酶的活性，而其他端粒结合蛋白却会抑制。相反，它会刺激端粒酶活性和核苷酸添加速率，即由核心端粒酶添加核苷酸的速率。
FIGURE 1. TPP1 is a homologue of ciliate TEBP- that interacts with POT1 to bind ssDNA.
a, TPP1 domain organization and TPP1-deletion mutants. RD, POT1-recruitment domain. S/T, Ser-rich region. TID, TIN2-interacting domain. b, Flag-tagged POT1 alone, or co-purified with wild-type TPP1 or TPP1 RD from 293T cells, were tested for their binding to the radiolabelled probe 49T3. Increasing amounts of non-radiolabelled 49T3 competitors (0, 30, 60, 120, 240 and 480 nM) were also added. c, POT1, TPP1 and telomere DNA form a ternary complex. Increasing amounts of insect-cell-purified POT1 (35, 70 and 140 nM), or TPP1 plus POT1 (17.5, 35 and 70 nM) were incubated with the 33P-labelled oligonucleotide 10 + 0 (TTACGGTTAGGGTTAG). The reactions were then treated with UV and glutaraldehyde to cross-link DNA to proteins, followed by SDS–PAGE and autoradiography. d, TPP1 enhances POT1 DNA-binding affinity. EMSA was performed using the radiolabelled oligonucleotide 10 + 0 (15 nM) and 2-fold dilutions (from 140 nM) of POT1 (open circles) or POT1 plus TPP1 (filled squares). e, Estimated Kd (nM) of POT1 and POT1–TPP1 to G-strand oligonucleotides with different 3'-end nucleotides.
原文出处:
TPP1 is a homologue of ciliate TEBP- and interacts with POT1 to recruit telomerase p559
Huawei Xin, Dan Liu, Ma Wan, Amin Safari, Hyeung Kim, Wen Sun, Matthew S. O'Connor and Zhou Songyang
doi:10.1038/nature05469
First paragraph | Full Text | PDF (841K) | Supplementary information
See also: Editor's summary
作者简介:
Zhou Songyang, Ph.D.
Assistant Professor, Departments of Biochemistry and Molecular and Cellular Biology
Ph.D., Molecular and Cellular Physiology, Tufts University
Postdoctoral, Biology, Massachusetts Institute of Technology,
Department of Biology, Harvard Medical School
Research Interests:
Our laboratory is interested in studying the molecular mechanisms of signaling pathways that regulate cell proliferation, survival, and aging. Understanding of these pathways is the key step in therapeutic remedy discovery for human diseases such as cancer and premature aging. We approach this issue in three aspects that include the study of protein-protein interaction domains, genome-wide genetic screens to identify factors important for these processes, and to develop new techniques that will further aid in our understanding of signal transduction.
(1) Proteomics and Protein-protein Interactions
Most signal proteins contain homology sequences (domains) that are necessary for specific protein-protein interactions. Hundreds of distinct families of protein domains are found in the human genome. These domains ensure proper targeting, assembly and firing of protein signal complexes. The identification of the targets of these domains therefore represents a critical step towards the understanding of signal networks. We have taken a unique approach to systematically study the binding specificities of protein domains. Using combinatorial peptide libraries, we have determined the specificities of a variety of important signaling domains, including SH2, SH3, PTB, BRCT, WW, PDZ, and protein kinase domains. These studies have not only provided structural basis for protein-ligand interactions through these domains, but also facilitated the identification of their in vivo targets. The lab is currently focusing on families of protein domains that regulate phosphorylation-dependent interactions. We are also interested in genome-wide predictions and identification of protein kinase targets, which utilizes techniques such as peptide libraries and mass spectrometry.
(2) Cell Senescence, Aging and Telomere Biology
The proper maintenance of telomeres is both essential for integrity of mammalian cells and tightly linked to cell senescence and aging. One major interest of the lab is to understand the molecular machinery that modulates telomere length and structure through proteomic approaches. Several telomeric protein complexes have been purified and identified in the lab. The molecular interactions between these proteins, how such interactions affect their function, and the role these protein complexes play in telomere maintenance in cell and animal will be investigated.
(3) Regulation of Cell Survival, Apoptosis, and Proliferation
Genetic screens play an important role in identifying central genes that regulate various signal pathways. My laboratory has developed a novel retrovirus-based technique (ERM) for genetic screens in mammalian cells. Such genetic screens do not require construction of cDNA expression libraries and allow genome-wide activation of cellular genes. We have already used this method to identify genome-wide mammalian genes that regulate cell growth and survival. Many genes that induce cell transformation and inhibit apoptosis have been cloned and their in vivo function will be studied in both cells and whole animals. In order to characterize the in vivo function of the identified genes, we are developing new technologies to establish cellular and animal models. In particular, we are improving techniques to activate and knock-out genes in mammalian somatic cells, and to generate chimera mice that express transgenes in their hematopoietic system.
Selected Publications:
Liu D, O'connor MS, Qin J, Songyang Z. (2004) Telosome, a Mammalian Telomere-associated Complex Formed by Multiple Telomeric Proteins. J Biol Chem. 279:51338-42.
Liu D, et al. (2004) PTOP interacts with POT1 and regulates its localization to telomeres. Nat Cell Biol. 6:673-80.
O'Connor MS, Safari A, Liu D, Qin J, Songyang Z. (2004) The human Rap1 protein complex and modulation of telomere length. J Biol Chem. 279:28585-91.
Songyang Z, Cantley LC. (2004) ZIP codes for delivering SH2 domains. Cell. 116:S41-3.
Rodriguez M, Li SS, Harper JW, Songyang Z. (2004) An oriented peptide array library (OPAL) strategy to study protein-protein interactions. J Biol Chem. 279:8802-7
Rodriguez M, Yu X, Chen J, Songyang Z. (2003) Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains. J Biol Chem. 2003 Dec 26;278(52):52914-8.
Xu J, Liu D, Songyang Z. (2002) The role of Asp-462 in regulating Akt activity. J Biol Chem. 277:35561-6.
Xu J, Liu D, Gill G, Songyang Z. (2001) Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides. J Cell Biol. 154:699-705.
Liu D, Yang XH, Songyang Z. (2000) Identification of CISK, a new member of the SGK kinase family that promotes IL-3 dependent survival. Curr. Biol. 10:1233-36.
For more publications, see listing on PubMed.
