In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in D. melanogaster and S. cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sub-lethal, stress response pathway that is tailored to an organism's environmental norms. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse, and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species, highlights the physiological significance of this protein conformation and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events.
Fusobacterium nucleatum is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal cancer and other cancer settings. F. nucleatum can adhere to and invade host cells in a manner that varies across F. nucleatum strains and host cell phenotypes. Here, we performed pairwise cocultures between three F. nucleatum strains and two immortalized primary host cell types (human colonic epithelial [HCE] cells and human carotid artery endothelial [HCAE] cells) followed by transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate transcriptional and epigenetic host cell responses. We observed that F. nucleatum-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNF, CXCL8, CXCL1, and CCL20. Furthermore, we identified genes strongly upregulated in a cell line-specific manner. In HCE cells, overexpressed genes included UBD and DUOX2/DUOXA2, associated with p53 degradation-mediated proliferation and intestinal reactive oxygen species (ROS) production, respectively. In HCAE cells, overexpressed genes included EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon F. nucleatum exposure. We used the ChIP-seq data to annotate chromatin states genome wide and found significant chromatin remodeling following F. nucleatum exposure in HCAE cells, with increased frequencies of active enhancer and low-signal/quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to F. nucleatum exposure and extensive host cell epigenomic changes specific to host cell type. IMPORTANCE Fusobacterium nucleatum is a bacterium normally found in the healthy oral cavity but also has an emerging role in colorectal cancer and other cancer settings. The host-microbe interactions of F. nucleatum and its involvement in tumor initiation, progression, and treatment resistance are not fully understood. We explored host cell changes that occur in response to F. nucleatum. We identified key genes differentially expressed in response to various conditions of F. nucleatum exposure and determined that the conserved host cell response to F. nucleatum was dominated by increased inflammation and chemokine gene expression. Additionally, we found extensive host cell epigenomic changes as a novel aspect of host modulation associated with F. nucleatum exposure. These results extend our understanding of F. nucleatum as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of F. nucleatum.
The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.
The Human Leukocyte Antigen (HLA) gene locus plays a fundamental role in human immunity, and it is established that certain HLA alleles are disease determinants. Previously, we have identified prevalent HLA class I and class II alleles, including DPA1*02:02, in two small patient cohorts at the COVID-19 pandemic onset.
We have since analyzed a larger public patient cohort data (n = 126 patients) with controls, associated demographic and clinical data. By combining the predictive power of multiple in silico HLA predictors, we report on HLA-I and HLA-II alleles, along with their associated risk significance.
We observe HLA-II DPA1*02:02 at a higher frequency in the COVID-19 positive cohort (29%) when compared to the COVID-negative control group (Fisher’s exact test [FET] p = 0.0174). Having this allele, however, does not appear to put this cohort’s patients at an increased risk of hospitalization. Inspection of COVID-19 disease severity outcomes, including admission to intensive care, reveal nominally significant risk associations with A*11:01 (FET p = 0.0078) and C*04:01 (FET p = 0.0087). The association with severe disease outcome is especially evident for patients with C*04:01, where disease prognosis measured by mechanical ventilation-free days was statistically significant after multiple hypothesis correction (Bonferroni p = 0.0323). While prevalence of some of these alleles falls below statistical significance after Bonferroni correction, COVID-19 patients with HLA-I C*04:01 tend to fare worse overall. This HLA allele may hold potential clinical value.
Pancreatic neuroendocrine neoplasms (PNENs) are biologically and clinically heterogeneous. Here, we use a multi-omics approach to uncover the molecular factors underlying this heterogeneity. Transcriptomic analysis of 84 PNEN specimens, drawn from two cohorts, is substantiated with proteomic profiling and identifies four subgroups: Proliferative, PDX1-high, Alpha cell-like and Stromal/Mesenchymal. The Proliferative subgroup, consisting of both well- and poorly differentiated specimens, is associated with inferior overall survival probability. The PDX1-high and Alpha cell-like subgroups partially resemble previously described subtypes, and we further uncover distinctive metabolism-related features in the Alpha cell-like subgroup. The Stromal/Mesenchymal subgroup exhibits molecular characteristics of YAP1/WWTR1(TAZ) activation suggestive of Hippo signaling pathway involvement in PNENs. Whole-exome sequencing reveals subgroup-enriched mutational differences, supported by activity inference analysis, and identifies hypermorphic proto-oncogene variants in 14.3% of sequenced PNENs. Our study reveals differences in cellular signaling axes that provide potential directions for PNEN patient stratification and treatment strategies.
The expression of BCL6 in B cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase involved in the degradation of BCL6 and is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of Burkitt lymphoma (BL) patients. FBXO11 mutations impaired BCL6 degradation and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of either one or two copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B cell lymphoma onset, providing experimental evidence that FBXO11 is a haplo-insufficient oncosuppressor in B cell lymphoma. In WT and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degrader or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches such as BCL6 degraders and direct MYC inhibition could be exploited as a targeted therapy for BL.
TET2 loss-of-function mutations are recurrent events in a wide range of hematological malignancies and a physiologic occurrence in blood cells of healthy older adults. It is currently unknown what determines if a person harboring a somatic TET2 mutation will progress to myelodysplastic syndrome or acute myeloid leukemia. Here we develop a zebrafish tet2 mutant through which we show that tet2 loss leads to restricted hematopoietic differentiation combined with a modest upregulation of p53, which is also characteristic of many inherited bone marrow failure syndromes. Uniquely in the context of emergency hematopoiesis by external stimuli, such as infection or cytokine stimulation, lack of tet2 leads hematopoietic stem cells to undergo excessive proliferation, resulting in an accumulation of immature cells, which are poised to become leukemogenic following additional genetic/epigenetic perturbations. This same phenomenon observed in zebrafish extends to human hematopoietic stem cells, identifying TET2 as a critical relay switch in the context of stress hematopoiesis.
Androgen receptor (AR) belongs to the steroid hormone receptor group of ligand-activated transcription factors in the nuclear receptor superfamily. AR mediates the action of physiological and exogenous androgens to regulate the expression of a network of genes in target tissues that are essential for the development and maintenance of the male phenotype and reproductive function as well as the function of numerous other tissues in both males and females. AR is ubiquitously expressed throughout the body. AR is a modular protein that comprises an N-terminal domain (NTD) that contains all of its transcriptional activity, a DNA-binding domain, a flexible hinge region, and a C-terminal ligand-binding domain (LBD). All clinically approved hormonal therapies target the AR LBD, either directly with antiandrogens and selective AR modulators or indirectly by reducing levels of androgens. Pathological conditions related to AR dysfunction involve altered levels of androgens and structural alterations in the AR. These include mutations, polymorphisms in the polyglutamine tract of the NTD, and alternative splicing of AR to yield constitutively active receptors. From the extensive list of AR-related diseases, herein we describe prostate cancer, androgen-insensitivity syndrome, polycystic ovary syndrome, breast cancer, and a few more pathological conditions in more detail.
Steroid hormone receptors are ligand-activated transcription factors, acting as master regulators of gene expression. Steroid receptors mediate formation of large protein complexes by recruiting coregulatory proteins and transcriptional machinery to specific genomic regions. Unlike the structured ligand-binding or DNA-binding domains, the N-terminal domain (NTD), where many of these protein-protein interactions occur, contains extended regions of intrinsic disorder. Interactions in the NTD and allosteric binding elsewhere induce temporary and reversible changes in the NTD structure, substantially influencing the repertoire of potential binding partners. This structural plasticity is key for the steroid receptors to coordinate intra- and intercellular signals into a tissue specific response. Designing small molecule inhibitors against intrinsically disordered proteins (IDP) in general has proven difficult as structural information is limited. While some progress has been made in this area, only recently has any molecule targeting IDPs progressed beyond the preclinical stage. Here we summarize the discovery and development of sintokamides, niphatenones, and EPI compounds which target the intrinsically disordered NTD of the androgen receptor. These are the first drugs to target the NTD of any steroid receptor, and EPI-506 and EPI-7386 remain the only compounds that bind to an IDP to have been tested in clinical trials.