HARNESSING A SECONDARY DNA STRUCTURE AT THE TELOMERES USING A SMALL MOLECULE, TMPYP4: IMPLICATION IN CANCER THERAPY

Ph.DDOCTOR OF PHILOSOPH

Pathologic polyglutamine aggregation begins with a self-poisoning polymer crystal

A long-standing goal of amyloid research has been to characterize the structural basis of the rate-determining nucleating event. However, the ephemeral nature of nucleation has made this goal unachievable with existing biochemistry, structural biology, and computational approaches. Here, we addressed that limitation for polyglutamine (polyQ), a polypeptide sequence that causes Huntington’s and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. To identify essential features of the polyQ amyloid nucleus, we used a direct intracellular reporter of self-association to quantify frequencies of amyloid appearance as a function of concentration, conformational templates, and rational polyQ sequence permutations. We found that nucleation of pathologically expanded polyQ involves segments of three glutamine (Q) residues at every other position. We demonstrate using molecular simulations that this pattern encodes a four-stranded steric zipper with interdigitated Q side chains. Once formed, the zipper poisoned its own growth by engaging naive polypeptides on orthogonal faces, in a fashion characteristic of polymer crystals with intramolecular nuclei. We further show that self-poisoning can be exploited to block amyloid formation, by genetically oligomerizing polyQ prior to nucleation. By uncovering the physical nature of the rate-limiting event for polyQ aggregation in cells, our findings elucidate the molecular etiology of polyQ diseases

Inactivation of Chk2 and Mus81 Leads to Impaired Lymphocytes Development, Reduced Genomic Instability, and Suppression of Cancer

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81Δex3-4/Δex3-4 mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81Δex3-4/Δex3-4 lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81Δex3-4/Δex3-4 background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer

Telomere Biology—Insights into an Intriguing Phenomenon

Bacteria and viruses possess circular DNA, whereas eukaryotes with typically very large DNA molecules have had to evolve into linear chromosomes to circumvent the problem of supercoiling circular DNA of that size. Consequently, such organisms possess telomeres to cap chromosome ends. Telomeres are essentially tandem repeats of any DNA sequence that are present at the ends of chromosomes. Their biology has been an enigmatic one, involving various molecules interacting dynamically in an evolutionarily well-trimmed fashion. Telomeres range from canonical hexameric repeats in most eukaryotes to unimaginably random retrotransposons, which attach to chromosome ends and reverse-transcribe to DNA in some plants and insects. Telomeres invariably associate with specialised protein complexes that envelop it, also regulating access of the ends to legitimate enzymes involved in telomere metabolism. They also transcribe into repetitive RNA which also seems to be playing significant roles in telomere maintenance. Telomeres thus form the intersection of DNA, protein, and RNA molecules acting in concert to maintain chromosome integrity. Telomere biology is emerging to appear ever more complex than previously envisaged, with the continual discovery of more molecules and interplays at the telomeres. This review also includes a section dedicated to the history of telomere biology, and intends to target the scientific audience new to the field by rendering an understanding of the phenomenon of chromosome end protection at large, with more emphasis on the biology of human telomeres. The review provides an update on the field and mentions the questions that need to be addressed

Telomere Biology-Insights into an Intriguing Phenomenon

10.3390/cells6020015CELLS6

Molecular xenomonitoring of diurnally subperiodic Wuchereria bancrofti infection in Aedes (Downsiomyia) niveus (Ludlow, 1903) after nine rounds of Mass Drug Administration in Nancowry Islands, Andaman and Nicobar Islands, India.

A group of four human inhabited Nancowry Islands in Nicobar district in the Andaman and Nicobar Islands, India having a population of 7674 is the lone focus of diurnally sub-periodic Wuchereria bancrofti (DspWB) that is transmitted by Aedes niveus (Ludlow). Microfilaria (Mf) prevalence was above 1% even after nine rounds of Mass Drug Administration (MDA) with DEC and albendazole. Molecular xenomonitoring (MX) was conducted to identify appropriate vector sampling method and assess the impact. BioGents Sentinel traps, gravid traps and human baited double bed nettraps were used in three locations in each village to collect Aedes niveus female mosquitoes. Subsequently daytime man landing collections (MLC) were carried out in all the 25 villages in the islands. Collections were compared in terms of the number of vector mosquitoes captured per trap collection. Females of Ae. niveus were pooled, dried and processed for detecting filarial parasite DNA using RT-PCR assay. Vector infection rate was estimated using PoolScreen software. Only 393 female mosquitoes including 44 Ae. niveus (11.2%) were collected from 459 trap collections using three trapping devices. From 151 MLCs, 2170 Ae. niveus female mosquitoes were collected. The average prevalence of W. bancrofti DNA was 0.43%. Estimated upper 95% CI exceeded the provisional prevalence threshold of 0.1% in all the villages, indicating continued transmission as observed in Mf survey. MLCs could be the choice, for now, to sample Ae. niveus mosquitoes. The PCR assay used in MX for nocturnally periodic bancroftian filariasis could be adopted for DspWB. The vector-parasite MX, can be used to evaluate interventions in this area after further standardization of the protocol

Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis

<div><p>Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. <em>Rnf8^−/−</em> mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in <em>Rnf8^−/−</em> mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in <em>Rnf8^−/−</em> mice, we have generated <em>Rnf8^−/−p53^−/−</em> mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to <em>Rnf8^−/−</em> mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in <em>Rnf8^−/−</em> mice. Similarly, the senescence phenotype of <em>Rnf8^−/−</em> mouse embryonic fibroblasts was rescued in p53 null background. <em>Rnf8^−/−p53^−/−</em> cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, <em>Rnf8^−/−p53^−/−</em> mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either <em>Rnf8^−/−</em> or <em>p53^−/−</em> mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.</p> </div

Increased basal and residual γ-H2ax foci in <i>Rnf8^−/−p53^−/−</i> MEFs.

<p>(A) γ-H2ax staining for <i>Rnf8^−/−p53^−/−</i> MEFs and controls. <i>Rnf8^−/−p53^−/−</i>, <i>Rnf8^−/−</i>, <i>p53^−/−</i> and <i>WT</i> early passage primary MEFs were either mock-treated (UT) or irradiated (8 Gy) and allowed to recover for 0.5, 4 and 24 hours. Cells were then fixed, stained using anti-γ-H2ax antibody and counterstained with DAPI. (B) Percentage of untreated cells that contained 10 or more γ-H2ax foci. Data are presented as the means SD of at least 3 independent experiments. At least 100 cells were quantified per experiment. * denotes statistical significance (P<0.05; student t-test). (C, D) Percentage of cells that showed more than 10 γ-H2ax foci at 0.5 and 4 hours post-irradiation respectively. At least 100 cells were quantified for each experiment. (E) Percentage of cells showing 10 or more γ-H2ax foci 24 hours after irradiation. Data are presented as the means SD of at least 3 independent experiments. At least 100 cells were quantified per experiment. * indicates statistical significance (P<0.05; student t-test).</p