A soluble truncated tau species related to cognitive dysfunction is elevated in the brain of cognitively impaired human individuals.

Neurofibrillary tangles are a pathological hallmark of Alzheimer's disease, and their levels correlate with the severity of cognitive dysfunction in humans. However, experimental evidence suggests that soluble tau species cause cognitive deficits and memory impairment. Our recent study suggests that caspase-2 (Casp2)-catalyzed tau cleavage at aspartate 314 mediates synaptic dysfunction and memory impairment in mouse and cellular models of neurodegenerative disorders. Δtau314, the C-terminally-truncated cleavage products, are soluble and present in human brain. In addition, levels of Δtau314 proteins are elevated in the brain of the cognitively impaired individuals compared to the cognitively normal individuals, indicating a possible role for Δtau314 proteins in cognitive deterioration. Here we show that (1) Δtau314 proteins are present in the inferior temporal gyrus of human brains; (2) Δtau314 proteins are generated from all six tau splicing isoforms, (3) levels of both Casp2 and Δtau314 proteins are elevated in cognitively impaired individuals compared to cognitively normal individuals, and (4) levels of Δtau314 proteins show a modest predictive value for dementia. These findings advance our understanding of the characteristics of Δtau314 proteins and their relevance to cognitive dysfunction and shed light on the contribution of Casp2-mediated Δtau314 production to cognitive deterioration.

Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells.

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

Bridging the Chromosome-centric and Biology/Disease-driven Human Proteome Projects: Accessible and Automated Tools for Interpreting the Biological and Pathological Impact of Protein Sequence Variants Detected via Proteogenomics.

The Chromosome-centric Human Proteome Project (C-HPP) seeks to comprehensively characterize all protein products coded by the genome, including those expressed sequence variants confirmed via proteogenomics methods. The closely related Biology/Disease-driven Human Proteome Project (B/D-HPP) seeks to understand the biological and pathological associations of expressed protein products, especially those carrying sequence variants that may be drivers of disease. To achieve these objectives, informatics tools are required that interpret potential functional or disease implications of variant protein sequence detected via proteogenomics. Toward this end, we have developed an automated workflow within the Galaxy for Proteomics (Galaxy-P) platform, which leverages the Cancer-Related Analysis of Variants Toolkit (CRAVAT) and makes it interoperable with proteogenomic results. Protein sequence variants confirmed by proteogenomics are assessed for potential structure-function effects as well as associations with cancer using CRAVAT's rich suite of functionalities, including visualization of results directly within the Galaxy user interface. We demonstrate the effectiveness of this workflow on proteogenomic results generated from an MCF7 breast cancer cell line. Our free and open software should enable improved interpretation of the functional and pathological effects of protein sequence variants detected via proteogenomics, acting as a bridge between the C-HPP and B/D-HPP.

N6-methyladenine is an epigenetic marker of mammalian early life stress.

Recent evidence described 6-methyladenine (6 mA) as a novel epigenetic regulator in a variety of multicellular species, including rodents; however, its capacity to influence gene expression in the mammalian brain remains unknown. We examined if 6 mA is present and regulated by early life stress associated with predator odor exposure (POE) within the developing rat amygdala. Our results provide evidence that 6 mA is present in the mammalian brain, is altered within the Htr2a gene promoter by early life stress and biological sex, and increased 6 mA is associated with gene repression. These data suggest that methylation of adenosine within mammalian DNA may be used as an additional epigenetic biomarker for investigating the development of stress-induced neuropathology.

An Accessible Proteogenomics Informatics Resource for Cancer Researchers.

Proteogenomics has emerged as a valuable approach in cancer research, which integrates genomic and transcriptomic data with mass spectrometry-based proteomics data to directly identify expressed, variant protein sequences that may have functional roles in cancer. This approach is computationally intensive, requiring integration of disparate software tools into sophisticated workflows, challenging its adoption by nonexpert, bench scientists. To address this need, we have developed an extensible, Galaxy-based resource aimed at providing more researchers access to, and training in, proteogenomic informatics. Our resource brings together software from several leading research groups to address two foundational aspects of proteogenomics: (i) generation of customized, annotated protein sequence databases from RNA-Seq data; and (ii) accurate matching of tandem mass spectrometry data to putative variants, followed by filtering to confirm their novelty. Directions for accessing software tools and workflows, along with instructional documentation, can be found at z.umn.edu/canresgithub. Cancer Res; 77(21); e43-46. ©2017 AACR.

Tet-mediated formation of 5-hydroxymethylcytosine in RNA.

Oxidation of 5-methylcytosine in DNA by ten-eleven translocation (Tet) family of enzymes has been demonstrated to play a significant role in epigenetic regulation in mammals. We found that Tet enzymes also possess the activity of catalyzing the formation of 5-hydroxymethylcytidine (5-hmrC) in RNA in vitro. In addition, the catalytic domains of all three Tet enzymes as well as full-length Tet3 could induce the formation of 5-hmrC in human cells. Moreover, 5-hmrC was present at appreciable levels (∼1 per 5000 5-methylcytidine) in RNA of mammalian cells and tissues. Our results suggest the involvement of this oxidation in RNA biology.

An ultraviolet-radiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background.

People with pale skin, red hair, freckles and an inability to tan--the 'red hair/fair skin' phenotype--are at highest risk of developing melanoma, compared to all other pigmentation types. Genetically, this phenotype is frequently the product of inactivating polymorphisms in the melanocortin 1 receptor (MC1R) gene. MC1R encodes a cyclic AMP-stimulating G-protein-coupled receptor that controls pigment production. Minimal receptor activity, as in red hair/fair skin polymorphisms, produces the red/yellow pheomelanin pigment, whereas increasing MC1R activity stimulates the production of black/brown eumelanin. Pheomelanin has weak shielding capacity against ultraviolet radiation relative to eumelanin, and has been shown to amplify ultraviolet-A-induced reactive oxygen species. Several observations, however, complicate the assumption that melanoma risk is completely ultraviolet-radiation-dependent. For example, unlike non-melanoma skin cancers, melanoma is not restricted to sun-exposed skin and ultraviolet radiation signature mutations are infrequently oncogenic drivers. Although linkage of melanoma risk to ultraviolet radiation exposure is beyond doubt, ultraviolet-radiation-independent events are likely to have a significant role. Here we introduce a conditional, melanocyte-targeted allele of the most common melanoma oncoprotein, BRAF(V600E), into mice carrying an inactivating mutation in the Mc1r gene (these mice have a phenotype analogous to red hair/fair skin humans). We observed a high incidence of invasive melanomas without providing additional gene aberrations or ultraviolet radiation exposure. To investigate the mechanism of ultraviolet-radiation-independent carcinogenesis, we introduced an albino allele, which ablates all pigment production on the Mc1r(e/e) background. Selective absence of pheomelanin synthesis was protective against melanoma development. In addition, normal Mc1r(e/e) mouse skin was found to have significantly greater oxidative DNA and lipid damage than albino-Mc1r(e/e) mouse skin. These data suggest that the pheomelanin pigment pathway produces ultraviolet-radiation-independent carcinogenic contributions to melanomagenesis by a mechanism of oxidative damage. Although protection from ultraviolet radiation remains important, additional strategies may be required for optimal melanoma prevention.