Aggregation of huntingtin protein arising from expanded polyglutamine (polyQ) sequences in the exon-1 region of mutant huntingtin plays a central role in the pathogenesis of Huntington's disease. The huntingtin aggregation pathways are of therapeutic and diagnostic interest, but obtaining critical information from the physiologically relevant htt exon-1 (Httex1) protein has been challenging. Using biophysical techniques and an expression and purification protocol that generates clean, monomeric Httex1, we identified and mapped three distinct aggregation pathways: 1) unseeded in solution; 2) seeded in solution; and 3) membrane-mediated. In solution, aggregation proceeded in a highly stepwise manner, in which the individual domains (N terminus containing 17 amino acids (N17), polyQ, and proline-rich domain (PRD)) become ordered at very different rates. The aggregation was initiated by an early oligomer requiring a pathogenic, expanded Gln length and N17 α-helix formation. In the second phase, β-sheet forms in the polyQ. The slowest step is the final structural maturation of the PRD. This stepwise mechanism could be bypassed by seeding, which potently accelerated aggregation and was a prerequisite for prion-like spreading Remarkably, membranes could catalyze aggregation even more potently than seeds, in a process that caused significant membrane damage. The N17 governed membrane-mediated aggregation by anchoring Httex1 to the membrane, enhancing local concentration and promoting collision via two-dimensional diffusion. Considering its central roles in solution and in membrane-mediated aggregation, the N17 represents an attractive target for inhibiting multiple pathways. Our approach should help evaluate such inhibitors and identify diagnostic markers for the misfolded forms identified here.

BackgroundHypoxia is a critical aspect of the glioma microenvironment and has been associated with poor prognosis and resistance to various therapies. However, the mechanisms responsible for hypoxic survival of glioma cells remain unclear. Recent studies strongly suggest that microRNAs act as critical mediators of the hypoxic response. We thus hypothesized their prominent role in hypoxia resistance in glioblastoma (GBM) and aimed to identify those.ResultsWith this study, we present the first detailed analysis of small RNA transcriptome of cell line U87MG, a grade IV glioma cell line, and its alteration under hypoxic condition. Based on deep sequencing and microarray data, we identify a set of hypoxia regulated microRNAs, with the miR-210-3p and its isomiRs showing highest induction in GBM cell lines U87MG and U251MG. We show miR-210-3p, miR-1275, miR-376c-3p, miR-23b-3p, miR-193a-3p and miR-145-5p to be up-regulated, while miR-92b-3p, miR-20a-5p, miR-10b-5p, miR-181a-2-3p and miR-185-5p are down-regulated by hypoxia. Interestingly, certain hypoxia-induced miRNAs are also known to be over-expressed in GBM tumors, suggesting that hypoxia may be one of the factors involved in establishing the miRNA signature of GBM. Transcription factor binding sites for Hypoxia inducible factor 1 A (HIF1A) were identified in the promoter region (5 kb upstream) of 30 hypoxia-induced miRNAs. HIF-1A over-expression and silencing studies show regulation of specific miRNAs, including miR-210-3p, to be HIF1A dependent. On the other hand, miR-210-3p leads to an increase in transcriptional activity of HIF and its target genes vascular endothelial growth factor (VEGF) and carbonic anhydrase 9 (CA9). MiR-210-3p levels were found to be high in GBM patient samples and showed good correlation with the known hypoxia markers CA9 and VEGF. We show that miR-210-3p promotes hypoxic survival and chemoresistance in GBM cells and targets a negative regulator of hypoxic response, HIF3A. Additionally, a total of 139 novel miRNAs were discovered by the analysis of deep sequencing data and three of these were found to be differentially expressed under hypoxia.ConclusionsOverall, our study reveals a novel miRNA signature of hypoxia in GBM and suggests miR-210-3p to be an oncogenic player and a novel potential intrinsic marker of hypoxia in glioblastoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-686) contains supplementary material, which is available to authorized users.

Down syndrome is associated with genome-wide perturbation of gene expression, which may be mediated by epigenetic changes. We perform an epigenome-wide association study on neonatal bloodspots comparing 196 newborns with Down syndrome and 439 newborns without Down syndrome, adjusting for cell-type heterogeneity, which identifies 652 epigenome-wide significant CpGs (P < 7.67 × 10−8) and 1,052 differentially methylated regions. Differential methylation at promoter/enhancer regions correlates with gene expression changes in Down syndrome versus non-Down syndrome fetal liver hematopoietic stem/progenitor cells (P < 0.0001). The top two differentially methylated regions overlap RUNX1 and FLI1, both important regulators of megakaryopoiesis and hematopoietic development, with significant hypermethylation at promoter regions of these two genes. Excluding Down syndrome newborns harboring preleukemic GATA1 mutations (N = 30), identified by targeted sequencing, has minimal impact on the epigenome-wide association study results. Down syndrome has profound, genome-wide effects on DNA methylation in hematopoietic cells in early life, which may contribute to the high frequency of hematological problems, including leukemia, in children with Down syndrome.

Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

MicroRNAs control cellular processes by regulating expression of their target genes. Here we report that neuro-epithelial transforming gene 1 (NET1) is a target of tumor suppressor microRNA 22 (miR-22). miR-22 is downregulated in peripheral blood mononuclear cells derived from chronic myeloid leukemia (CML) patients and in CML cell line K562. NET1 was identified as one of the targets of miR-22 using both in vitro and in vivo experiments. Either mutations or naturally occurring single-nucleotide polymorphisms in NET1 3 0 -UTR that map at the miR-22 binding site were found to affect binding of miR-22 to NET1 mRNA. Over expression of NET1 in K562 cells resulted in increased proliferation. However decreased proliferation and alteration in cell cycle were observed on either overexpression of miR-22 or knockdown of NET1 expression respectively. We also found that overexpression of miR-22 or NET1 knockdown inhibits actin fiber formation, probably by downregulation of NET1 as NET1 knockdown also resulted in depletion of actin fiber formation. We suggest that the oncogenic properties of CML cells are probably due to deregulated expression of NET1 as a result of altered expression of miR-22.