Quantifying Nucleation In Vivo Reveals the Physical Basis of Prion-like Phase Behavior.

Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein's self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.

Synergistic interaction of Rnf8 and p53 in the protection against genomic instability and tumorigenesis.

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. Rnf8(-/-) 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 Rnf8(-/-) mice in a tissue- and cell type-specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8(-/-) mice, we have generated Rnf8(-/-)p53(-/-) mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8(-/-) mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8(-/-) mice. Similarly, the senescence phenotype of Rnf8(-/-) mouse embryonic fibroblasts was rescued in p53 null background. Rnf8(-/-)p53(-/-) cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8(-/-)p53(-/-) mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8(-/-) or p53(-/-) 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.

Targeting DNA-PKcs and telomerase in brain tumour cells.

BACKGROUND: Patients suffering from brain tumours such as glioblastoma and medulloblastoma have poor prognosis with a median survival of less than a year. Identifying alternative molecular targets would enable us to develop different therapeutic strategies for better management of these tumours. METHODS: Glioblastoma (MO59K and KNS60) and medulloblastoma cells (ONS76) were used in this study. Telomerase inhibitory effects of MST-312, a chemically modified-derivative of epigallocatechin gallate, in the cells were assessed using telomere repeat amplification protocol. Gene expression analysis following MST-312 treatment was done by microarray. Telomere length was measured by telomere restriction fragments analysis. Effects of MST-312 on DNA integrity were evaluated by single cell gel electrophoresis, immunofluorescence assay and cytogenetic analysis. Phosphorylation status of DNA-PKcs was measured with immunoblotting and effects on cell proliferation were monitored with cell titre glow and trypan blue exclusion following dual inhibition. RESULTS: MST-312 showed strong binding affinity to DNA and displayed reversible telomerase inhibitory effects in brain tumour cells. In addition to the disruption of telomere length maintenance, MST-312 treatment decreased brain tumour cell viability, induced cell cycle arrest and double strand breaks (DSBs). DNA-PKcs activation was observed in telomerase-inhibited cells presumably as a response to DNA damage. Impaired DNA-PKcs in MO59J cells or in MO59K cells treated with DNA-PKcs inhibitor, NU7026, caused a delay in the repair of DSBs. In contrast, MST-312 did not induce DSBs in telomerase negative osteosarcoma cells (U2OS). Combined inhibition of DNA-PKcs and telomerase resulted in an increase in telomere signal-free chromosomal ends in brain tumour cells as well. Interestingly, continual exposure of brain tumour cells to telomerase inhibitor led to population of cells, which displayed resistance to telomerase inhibition-mediated cell arrest. DNA-PKcs ablation in these cells, however, confers higher cell sensitivity to telomerase inhibition, inducing cell death. CONCLUSIONS: Efficient telomerase inhibition was achieved with acute exposure to MST-312 and this resulted in subtle but significant increase in DSBs. Activation of DNA-PKcs might indicate the requirement of NHEJ pathway in the repair telomerase inhibitor induced DNA damage. Therefore, our results suggest a potential strategy in combating brain tumour cells with dual inhibition of telomerase and NHEJ pathway.

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. Mus81(Δex3-4/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 Mus81(Δex3-4/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 Mus81(Δex3-4/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.

Protein supersaturation powers innate immune signaling.

Innate immunity protects us in youth but turns against us as we age. The reason for this tradeoff is unclear. Seeking a thermodynamic basis, we focused on death fold domains (DFDs), whose ordered polymerization has been stoichiometrically linked to innate immune signal amplification. We hypothesized that soluble ensembles of DFDs function as phase change batteries that store energy via supersaturation and subsequently release it through nucleated polymerization. Using imaging and FRET-based cytometry to characterize the phase behaviors of all 109 human DFDs, we found that the hubs of innate immune signaling networks encode large nucleation barriers that are intrinsically insulated from cross-pathway activation. We showed via optogenetics that supersaturation drives signal amplification and that the inflammasome is constitutively supersaturated in vivo. Our findings reveal that the soluble "inactive" states of adaptor DFDs function as essential, yet impermanent, kinetic barriers to inflammatory cell death, suggesting a thermodynamic driving force for aging.

DAmFRET measures saturating concentrations and toxicities of protein phase transitions in vivo.

Protein phase transitions broadly govern protein function and dysfunction. However, analyzing the consequences of specific phase transitions in cells is hindered by the low throughput and limited resolution of fluorescence microscopy, and this problem is compounded for proteins with complex phase behavior such as those implicated in age-associated neurodegenerative diseases. As one solution to this problem, we incorporated an orthogonally fluorescence proxy of total protein expression to adjust for effective cell volume differences in a flow cytometric assay for protein self-association-Distributed Amphifluoric FRET (DAmFRET)-thereby allowing the intracellular saturating concentrations of different proteins to be precisely compared in single experiments. We further found that the effective cell volume decreased in cells experiencing proteotoxicity, which provided a simple way to assign toxicity to specific phases of ectopically expressed proteins.

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.

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.

Diseases that typically occur later in life, such as Alzheimer's, are often caused by specific proteins clumping together into structures known as amyloids. Once the process starts, amyloids will continue to form, leading to worse symptoms that cannot be cured. The best way to treat these diseases is therefore to stop amyloids from arising in the first place. Amyloids initially develop by proteins coming together to create an unstable structure referred to as the nucleus. The instability of the nucleus means it cannot be observed directly, making it hard to study this nucleation process. To overcome this, Kandola, Venkatesan et al. investigated the simplest protein known to form an amyloid ­ polyglutamine, which is made up of a chain of repeating building blocks known as amino acids. Polyglutamine forms only one type of amyloid which is associated with nine neurodegenerative diseases, including Huntington's disease. However, it only does this when its chain of amino acids exceeds a certain length, suggesting that a specific structure may be required for nucleation to begin. Kandola, Venkatesan et al. made alternative versions of the polyglutamine protein which each contained slightly different sequences of amino acids that will alter the way the protein folds. They then tested how well these different variants could form amyloids in yeast cells. This revealed that in order to join together into a nucleus, polyglutamine needs to be able to fold into a zipper shape made up of four interlocking strands. The length of the protein required to form this shape is also the same length that causes the amyloid associated with neurodegenerative diseases. Kandola, Venkatesan et al. also found that polyglutamine tends to bind to nuclei that have already formed in a way that hinders their growth. This 'self-poisoning' affect could potentially be exploited as a way to pre-emptively stop amyloids from initially arising. These findings have uncovered a potential therapeutic strategy for blocking amyloid formation that could eventually benefit people with or at risk of developing neurodegenerative diseases linked to polyglutamine. Additionally, this approach provides a blueprint for understanding how other proteins undergo amyloid nucleation, including those responsible for Alzheimer's, Parkinson's, and other diseases.

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

Effects of rapamycin on the mechanistic target of rapamycin (mTOR) pathway and telomerase in breast cancer cells.

The mTOR pathway and the enzyme telomerase are two key players commonly upregulated in cancers. They render survival and proliferative advantage to cancer cells, and are regarded as attractive anticancer targets. Rapamycin, a macrolide antibiotic and mTOR inhibitor, has recently also been implicated in telomerase inhibition and telomere attrition, although the mechanisms remain poorly understood. Using breast cancer cells (MCF-7 and MDA-MB-231) wherein telomerase activity and mTOR pathway are concurrently overexpressed, this study sought to unravel novel mechanisms by which rapamycin may affect these pathways. Short term treatment with an acute dose of rapamycin inhibited the mTOR pathway and telomerase activity and induced G1 arrest. This arrest was independent of cyclin D1 and p21 levels and was not mediated by DNA damage in both cell types. While long term treatment with a clinically relevant dose of rapamycin resulted in compromised population doubling capacity and mTOR pathway inhibition, there was no effect on telomere functionality and telomerase activity as evidenced by our assessments of hTERT protein levels, in vitro telomerase activity, telomere length and telomere FISH analyses. We also found that sustained rapamycin treatment leading to Akt activation may play a role in resistance in the more invasive MDA-MB-231 cells. In summary, rapamycin specifically inhibits the activation of mTOR pathway. Moreover, we show for the first time that while acute short-term treatment with rapamycin induces telomerase inhibition, it does not affect telomerase activity nor does it inflict telomere dysfunction in breast cancer cells upon chronic long-term treatment with a clinically relevant dose. These findings may be useful while designing combinatorial treatment strategies with rapamycin inhibition in the clinic.

Assessment of genomic instability and proliferation index in cultured lymphocytes of patients with Down syndrome, congenital anomalies and aplastic anaemia.

One hundred and fifteen cases [Down Syndrome (DS) n = 75, Multiple Congenital Anomalies (MCA) n = 15 and Aplastic Anaemia (AA) n = 25], with respect to their nature of predisposition to cancer, were selected for clinical, cytogenetic and cyto-molecular studies to understand the severity of genomic instability according to the nature of the different diseases. Cytogenetic studies included chromosomal aberration (CA) assays and cytokinesis block micronucleus cytome (CBMN-Cyt) assays. In DS, MCA and AA, average frequencies of nuclear anomalies (NA) were 0.015 ±â€¯0.0006, 0.021 ±â€¯0.00123, 0.031 ±â€¯0.00098, respectively and CA were 0.107 ±â€¯0.003, 0.105 ±â€¯0.008, 0.158 ±â€¯0.006, respectively per metaphase. The extent of genomic instability in patients analysed by CBMN-Cyt assays and CA assays was statistically significant in all groups. Comparatively decreased cytokinesis block proliferation index (CBPI) observed in AA patients of 1.59 ±â€¯0.05, support the assumption that decreased levels of CBPI indicate increased genomic damage. Furthermore, we performed peptide nucleic acid fluorescence in situ hybridisation (PNA FISH) analysis to understand the mechanisms behind genomic instability and telomere dysfunction. PNA FISH showed increased frequencies of telomere signal free ends (0.98 ±â€¯0.13) in individuals with higher genomic instability. Therefore, the results demonstrate that increased chromosomal instability along with higher telomere attrition or loss may initiate gross DNA damage and leads to chromosomal instability, which is an important mechanism for triggering genomic instability - an important hallmark of cancer cells.

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.

Biomarkers of Ionizing Radiation Exposure: A Multiparametric Approach.

Humans are exposed to ionizing radiation not only through background radiation but also through the ubiquitous presence of devices and sources that generate radiation. With the expanded use of radiation in day-to-day life, the chances of accidents or misuse only increase. Therefore, a thorough understanding of the dynamic effects of radiation exposure on biological entities is necessary. The biological effects of radiation exposure on human cells depend on much variability such as level of exposure, dose rate, and the physiological state of the cells. During potential scenarios of a large-scale radiological event which results in mass casualties, dose estimates are essential to assign medical attention according to individual needs. Many attempts have been made to identify biomarkers which can be used for high throughput biodosimetry screening. In this study, we compare the results of different biodosimetry methods on the same irradiated cells to assess the suitability of current biomarkers and push forward the idea of employing a multiparametric approach to achieve an accurate dose and risk estimation.

Chromosomal instability--mechanisms and consequences.

Chromosomal instability is defined as a state of numerical and/or structural chromosomal anomalies in cells. Numerous studies have documented the incidence of chromosomal instability, which acutely or chronically may lead to accelerated ageing (tissue-wide or even organismal), cancer or other genetic disorders. Potential mechanisms leading to the generation of chromosome-genome instability include erroneous/inefficient DNA repair, chromosome segregation defects, spindle assembly defects, DNA replication stress, telomere shortening/dysfunction - to name a few. Understanding the cellular and molecular mechanisms for chromosomal instability in various human cells and tissues will be useful in elucidating the cause for many age associated diseases including cancer. This approach holds a great promise for the cytogenetic assays not only for prognosis but also for diagnostic purposes in clinical settings. In this review, a multi-dimensional approach has been attempted to portray the complexity behind the incidence of chromosome-genome instability including evolutionary implications at the species level for some of the mechanisms of chromosomal instability.

Plumbagin alters telomere dynamics, induces DNA damage and cell death in human brain tumour cells.

Natural plant products may possess much potential in palliative therapy and supportive strategies of current cancer treatments with lesser cytotoxicity to normal cells compared to conventional chemotherapy. In the current study, anti-cancer properties of plumbagin, a plant-derived naphthoquinone, on brain cancer cells were determined. Plumbagin treatment resulted in the induction of DNA damage, cell cycle arrest and apoptosis, followed by suppression of the colony forming ability of the brain tumour cells. These effects were substantiated by upregulation of PTEN, TNFRSF1A and downregulation of E2F1 genes, along with a drop in MDM2, cyclin B1, survivin and BCL2 protein expression. Plumbagin induced elevated levels of caspase-3/7 activity as well. For the first time, we show here that plumbagin inhibits telomerase in brain tumour cells and results in telomere shortening following chronic long-term treatment. This observation implies considerable cytotoxicity of plumbagin towards cancer cells with higher telomerase activity. Collectively, our findings suggest plumbagin as a potential chemotherapeutic phytochemical in brain tumour treatment modalities.

Clinico-Pathological Correlation of ß-Catenin and Telomere Dysfunction in Head and Neck Squamous Cell Carcinoma Patients.

BACKGROUND: Tumorigenesis is a complex process of accumulated alteration in function of multiple genes and pathways. Wnt signalling pathway is involved in various differentiation events during embryonic development and is conserved in various species. OBJECTIVE: A multicentre collaborative initiative is undertaken to study the occurrence, prognosis and molecular mechanism of HNSCC (Head and Neck Squamous Cell Carcinoma) which is highly prevalent in eastern parts of India. From a large cohort of HNSCC tissue repository, 67 cases were selected for multi-parametric investigation. RESULTS: 67 cases showed stable ß-catenin expression. We have seen correlation, if any, of the transcription factor - ß-catenin, telomere maintenance and shelterin complex proteins - TRF2, Rap1 and hTert with respect to tumor differentiation and telomere dysfunction. Immunohistochemistry of ß-catenin protein showed stable and high expression in tumor when compared to stroma. MDSCC (Moderately Differentiated Squamous cell carcinoma) cases expressed nuclear expression of ß-catenin in invasive fronts and showed increased genomic instability. Higher frequency of Anaphase bridges was observed ranging from <3% in normal cut margin to 13% in WDSCC (Well differentiated squamous cell carcinoma) and 18% in MDSCC (Moderately differentiated Squamous cell carcinoma). There was significant decrease in telomere length in MDSCC (<4) when compared to the normal cut margin samples (<7). Quantitative Real Time-PCR confirmed a significant correlationship between stable ß-catenin expression and poor clinical and pathological outcome. CONCLUSION: The Stabilisation and accumulation of ß-catenin was significant and correlated well with de-differentiation process as well as prognosis and therapy outcome of the patients in the cohort. Expression status of molecular markers such as ß-catenin, hTert, TRF2 and RAP1 correlate significantly with the process of tumorigenesis and prognosis and may play a role in therapeutic management of Head and neck patients.

Physicochemical and toxicological characteristics of urban aerosols during a recent Indonesian biomass burning episode.

Air particulate matter (PM) samples were collected in Singapore from 21 to 29 October 2010. During this time period, a severe regional smoke haze episode lasted for a few days (21-23 October). Physicochemical and toxicological characteristics of both haze and non-haze aerosols were evaluated. The average mass concentration of PM2.5 (PM with aerodynamic diameter of ≤2.5 µm) increased by a factor of 4 during the smoke haze period (107.2 µg/m(3)) as compared to that during the non-smoke haze period (27.0 µg/m(3)). The PM2.5 samples were analyzed for 16 priority polycyclic aromatic hydrocarbons (PAHs) listed by the United States Environmental Protection Agency and 10 transition metals. Out of the seven PAHs known as potential or suspected carcinogens, five were found in significantly higher levels in smoke haze aerosols as compared to those in the background air. Metal concentrations were also found to be higher in haze aerosols. Additionally, the toxicological profile of the PM2.5 samples was evaluated using a human epithelial lung cell line (A549). Cell viability and death counts were measured after a direct exposure of PM2.5 samples to A459 cells for a period of 48 h. The percentage of metabolically active cells decreased significantly following a direct exposure to PM samples collected during the haze period. To provide further insights into the toxicological characteristics of the aerosol particles, glutathione levels, as an indirect measure of oxidative stress and caspase-3/7 levels as a measure of apoptotic death, were also evaluated.