Origins and Evolution of the HET-s Prion-Forming Protein: Searching for Other Amyloid-Forming Solenoids

The HET-s prion-forming domain from the filamentous fungus Podospora anserina is gaining considerable interest since it yielded the first well-defined atomic structure of a functional amyloid fibril. This structure has been identified as a left-handed beta solenoid with a triangular hydrophobic core. To delineate the origins of the HET-s prion-forming protein and to discover other amyloid-forming proteins, we searched for all homologs of the HET-s protein in a database of protein domains and fungal genomes, using a combined application of HMM, psi-blast and pGenThreader techniques, and performed a comparative evolutionary analysis of the N-terminal alpha-helical domain and the C-terminal prion-forming domain of HET-s. By assessing the tandem evolution of both domains, we observed that the prion-forming domain is restricted to Sordariomycetes, with a marginal additional sequence homolog in Arthroderma otae as a likely case of horizontal transfer. This suggests innovation and rapid evolution of the solenoid fold in the Sordariomycetes clade. In contrast, the N-terminal domain evolves at a slower rate (in Sordariomycetes) and spans many diverse clades of fungi. We performed a full three-dimensional protein threading analysis on all identified HET-s homologs against the HET-s solenoid fold, and present detailed structural annotations for identified structural homologs to the prion-forming domain. An analysis of the physicochemical characteristics in our set of structural models indicates that the HET-s solenoid shape can be readily adopted in these homologs, but that they are all less optimized for fibril formation than the P. anserina HET-s sequence itself, due chiefly to the presence of fewer asparagine ladders and salt bridges. Our combined structural and evolutionary analysis suggests that the HET-s shape has “limited scope” for amyloidosis across the wider protein universe, compared to the ‘generic’ left-handed beta helix. We discuss the implications of our findings on future identification of amyloid-forming proteins sharing the solenoid fold

Exploring the impact of intra-tumoural heterogeneity on liquid biopsy cell-free DNA methylation and copy number in head and neck squamous cell carcinoma

Liquid biopsy profiling is gaining increasing promise towards biomarker-led identification and disease stratification of tumours, particularly for tumours displaying significant intra-tumoural heterogeneity (ITH). For head and neck squamous cell carcinoma (HNSCC), which display high levels of genetic ITH, identification of epigenetic modifications and methylation signatures has shown multiple uses in stratification of HNSCC for prognosis, treatment, and HPV status. In this study, we investigated the potential of liquid biopsy methylomics and genomic copy number to profile HNSCC. We conducted multi-region sampling of tumour core, tumour margin and normal adjacent mucosa, as well as plasma cell-free DNA (cfDNA) across 9 HNSCC patients. Collectively, our work highlights the prevalence of methylomic ITH in HNSCC, and demonstrates the potential of cfDNA methylation as a tool for ITH assessment and serial sampling.</p

Correlating novel variable and conserved motifs in the Hemagglutinin protein with significant biological functions

<p>Abstract</p> <p>Background</p> <p>Variations in the influenza Hemagglutinin protein contributes to antigenic drift resulting in decreased efficiency of seasonal influenza vaccines and escape from host immune response. We performed an in silico study to determine characteristics of novel variable and conserved motifs in the Hemagglutinin protein from previously reported H3N2 strains isolated from Hong Kong from 1968–1999 to predict viral motifs involved in significant biological functions.</p> <p>Results</p> <p>14 MEME blocks were generated and comparative analysis of the MEME blocks identified blocks 1, 2, 3 and 7 to correlate with several biological functions. Analysis of the different Hemagglutinin sequences elucidated that the single block 7 has the highest frequency of amino acid substitution and the highest number of co-mutating pairs. MEME 2 showed intermediate variability and MEME 1 was the most conserved. Interestingly, MEME blocks 2 and 7 had the highest incidence of potential post-translational modifications sites including phosphorylation sites, ASN glycosylation motifs and N-myristylation sites. Similarly, these 2 blocks overlap with previously identified antigenic sites and receptor binding sites.</p> <p>Conclusion</p> <p>Our study identifies motifs in the Hemagglutinin protein with different amino acid substitution frequencies over a 31 years period, and derives relevant functional characteristics by correlation of these motifs with potential post-translational modifications sites, antigenic and receptor binding sites.</p

Exploring the impact of intra-tumoural heterogeneity on liquid biopsy cell-free DNA methylation and copy number in head and neck squamous cell carcinoma

Liquid biopsy profiling is gaining increasing promise towards biomarker-led identification and disease stratification of tumours, particularly for tumours displaying significant intra-tumoural heterogeneity (ITH). For head and neck squamous cell carcinoma (HNSCC), which display high levels of genetic ITH, identification of epigenetic modifications and methylation signatures has shown multiple uses in stratification of HNSCC for prognosis, treatment, and HPV status. In this study, we investigated the potential of liquid biopsy methylomics and genomic copy number to profile HNSCC. We conducted multi-region sampling of tumour core, tumour margin and normal adjacent mucosa, as well as plasma cell-free DNA (cfDNA) across 9 HNSCC patients. Collectively, our work highlights the prevalence of methylomic ITH in HNSCC, and demonstrates the potential of cfDNA methylation as a tool for ITH assessment and serial sampling.</p

PrionHome: A Database of Prions and Other Sequences Relevant to Prion Phenomena

Prions are units of propagation of an altered state of a protein or proteins; prions can propagate from organism to organism, through cooption of other protein copies. Prions contain no necessary nucleic acids, and are important both as both pathogenic agents, and as a potential force in epigenetic phenomena. The original prions were derived from a misfolded form of the mammalian Prion Protein PrP. Infection by these prions causes neurodegenerative diseases. Other prions cause non-Mendelian inheritance in budding yeast, and sometimes act as diseases of yeast. We report the bioinformatic construction of the PrionHome, a database of >2000 prion-related sequences. The data was collated from various public and private resources and filtered for redundancy. The data was then processed according to a transparent classification system of prionogenic sequences (i.e., sequences that can make prions), prionoids (i.e., proteins that propagate like prions between individual cells), and other prion-related phenomena. There are eight PrionHome classifications for sequences. The first four classifications are derived from experimental observations: prionogenic sequences, prionoids, other prion-related phenomena, and prion interactors. The second four classifications are derived from sequence analysis: orthologs, paralogs, pseudogenes, and candidate-prionogenic sequences. Database entries list: supporting information for PrionHome classifications, prion-determinant areas (where relevant), and disordered and compositionally-biased regions. Also included are literature references for the PrionHome classifications, transcripts and genomic coordinates, and structural data (including comparative models made for the PrionHome from manually curated alignments). We provide database usage examples for both vertebrate and fungal prion contexts. Using the database data, we have performed a detailed analysis of the compositional biases in known budding-yeast prionogenic sequences, showing that the only abundant bias pattern is for asparagine bias with subsidiary serine bias. We anticipate that this database will be a useful experimental aid and reference resource. It is freely available at: http://libaio.biol.mcgill.ca/prion

A subgroup of microRNAs defines PTEN-deficient, triple-negative breast cancer patients with poorest prognosis and alterations in RB1, MYC, and Wnt signaling

Abstract Background Triple-negative breast cancer (TNBC) represents a heterogeneous group of ER- and HER2-negative tumors with poor clinical outcome. We recently reported that Pten-loss cooperates with low expression of microRNA-145 to induce aggressive TNBC-like lesions in mice. To systematically identify microRNAs that cooperate with PTEN-loss to induce aggressive human BC, we screened for miRNAs whose expression correlated with PTEN mRNA levels and determined the prognostic power of each PTEN-miRNA pair alone and in combination with other miRs. Methods Publically available data sets with mRNA, microRNA, genomics, and clinical outcome were interrogated to identify miRs that correlate with PTEN expression and predict poor clinical outcome. Alterations in genomic landscape and signaling pathways were identified in most aggressive TNBC subgroups. Connectivity mapping was used to predict response to therapy. Results In TNBC, PTEN loss cooperated with reduced expression of hsa-miR-4324, hsa-miR-125b, hsa-miR-381, hsa-miR-145, and has-miR136, all previously implicated in metastasis, to predict poor prognosis. A subgroup of TNBC patients with PTEN-low and reduced expression of four or five of these miRs exhibited the worst clinical outcome relative to other TNBCs (hazard ratio (HR) = 3.91; P < 0.0001), and this was validated on an independent cohort (HR = 4.42; P = 0.0003). The PTEN-low/miR-low subgroup showed distinct oncogenic alterations as well as TP53 mutation, high RB1-loss signature and high MYC, PI3K, and β-catenin signaling. This lethal subgroup almost completely overlapped with TNBC patients selected on the basis of Pten-low and RB1 signature loss or β-catenin signaling-high. Connectivity mapping predicted response to inhibitors of the PI3K pathway. Conclusions This analysis identified microRNAs that define a subclass of highly lethal TNBCs that should be prioritized for aggressive therapy

Circulating tumour DNA detects somatic variants contributing to spatial and temporal intratumural heterogeneity in head and neck squamous cell carcinoma

Background: As circulating tumour DNA (ctDNA) liquid biopsy analysis is increasingly incorporated into modern oncological practice, establishing the impact of genomic intra-tumoural heterogeneity (ITH) upon data output is paramount. Despite advances in other cancer types the evidence base in head and neck squamous cell carcinoma (HNSCC) remains poor. We sought to investigate the utility of ctDNA to detect ITH in HNSCC.Methods: In a pilot cohort of 9 treatment-naïve HNSCC patients, DNA from two intra-tumoural sites (core and margin) was whole-exome sequenced. A 9-gene panel was designed to perform targeted sequencing on pre-treatment plasma cell-free DNA and selected post-treatment samples.Results: Rates of genomic ITH among the 9 patients was high. COSMIC variants from 19 TCGA HNSCC genes demonstrated an 86.9% heterogeneity rate (present in one tumour sub-site only). Across all patients, cell-free DNA (ctDNA) identified 12.9% (range 7.5-19.8%) of tumour-specific variants, of which 55.6% were specific to a single tumour sub-site only. CtDNA identified 79.0% (range: 55.6-90.9%) of high-frequency variants (tumour VAF&gt;5%). Analysis of ctDNA in serial post-treatment blood samples in patients who suffered recurrence demonstrated dynamic changes in both tumour-specific and acquired variants that predicted recurrence ahead of clinical detection.Conclusion: We demonstrate that a ctDNA liquid biopsy identified spatial genomic ITH in HNSCC and reliably detected high-frequency driver mutations. Serial sampling allowed post-treatment surveillance and early identification of treatment failure

Consensus on Molecular Subtypes of High-grade Serous Ovarian Carcinoma

Purpose: The majority of ovarian carcinomas are of high-grade serous histology, which is associated with poor prognosis. Surgery and chemotherapy are the mainstay of treatment, and molecular characterization is necessary to lead the way to targeted therapeutic options. To this end, various computational methods for gene expression-based subtyping of high-grade serous ovarian carcinoma (HGSOC) have been proposed, but their overlap and robustness remain unknown. Experimental Design: We assess three major subtype classifiers by meta-analysis of publicly available expression data, and assess statistical criteria of subtype robustness and classifier concordance. We develop a consensus classifier that represents the subtype classifications of tumors based on the consensus of multiple methods, and outputs a confidence score. Using our compendium of expression data, we examine the possibility that a subset of tumors are unclassifiable based on currently proposed subtypes. Results: HGSOC subtyping classifiers exhibit moderate pairwise concordance across our data compendium (58.9%-70.9%, p \u3c 10-5) and are associated with overall survival in a metaanalysis across datasets (p \u3c 10-5). Current subtypes do not meet statistical criteria for robustness to re-clustering across multiple datasets (Prediction Strength \u3c 0.6). A new subtype classifier is trained on concordantly classified samples to yield a consensus classification of patient tumors that correlates with patient age, survival, tumor purity, and lymphocyte infiltration. Conclusion: A new consensus ovarian subtype classifier represents the consensus of methods, and demonstrates the importance of classification approaches for cancer that do not require all tumors to be assigned to a distinct subtype

The anti-tumour activity of DNA methylation inhibitor 5-aza-2′-deoxycytidine is enhanced by the common analgesic paracetamol through induction of oxidative stress

The DNA demethylating agent 5-aza-2′-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects and its use in solid tumours is debated. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E2 pathway, inadvertently providing cancer cells with survival potential, while the addition of paracetamol offsets this effect. Secondly, in the presence of paracetamol, DAC treatment leads to glutathione depletion and finally to accumulation of ROS and/or mitochondrial superoxide, both of which have the potential to restrict tumour growth. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. The sensitizing effect of paracetamol supplementation is specific to DAC but not its analogue 5-azacitidine. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy