Tandem mass tag proteomic and untargeted metabolomic profiling reveals altered serum and CSF biochemical datasets in iron deficient monkeys.

The effects of early-life iron deficiency anemia (IDA) extend past the blood and include both short- and long-term adverse effects on many tissues including the brain. Prior to IDA, iron deficiency (ID) can cause similar tissue effects, but a sensitive biomarker of iron-dependent brain health is lacking. To determine serum and CSF biomarkers of ID-induced metabolic dysfunction we performed proteomic and metabolomic analysis of serum and CSF at 4- and 6- months from a nonhuman primate model of infantile IDA. LC/MS/MS analyses identified a total of 227 metabolites and 205 proteins in serum. In CSF, we measured 210 metabolites and 1,560 proteins. Data were either processed from a Q-Exactive (Thermo Scientific, Waltham, MA) through Progenesis QI with accurate mass and retention time comparisons to a proprietary small molecule database and Metlin or with raw files imported directly from a Fusion Orbitrap (Thermo Scientific, Waltham, MA) through Sequest in Proteome Discoverer 2.4.0.305 (Thermo Scientific, Waltham, MA) with peptide matches through the latest Rhesus Macaque HMDB database. Metabolite and protein identifiers, p-values, and q-values were utilized for molecular pathway analysis with Ingenuity Pathways Analysis (IPA). We applied multiway distance weighted discrimination (DWD) to identify a weighted sum of the features (proteins or metabolites) that distinguish ID from IS at 4-months (pre-anemic period) and 6-months of age (anemic).

SFPQ Promotes Lung Cancer Malignancy via Regulation of CD44 v6 Expression.

Mesenchymal stem cells (MSCs) contribute to tumor pathogenesis and elicit antitumor immune responses in tumor microenvironments. Nuclear proteins might be the main players in these processes. In the current study, combining spatial proteomics with ingenuity pathway analysis (IPA) in lung non-small cell (NSC) cancer MSCs, we identify a key nuclear protein regulator, SFPQ (Splicing Factor Proline and Glutamine Rich), which is overexpressed in lung cancer MSCs and functions to promote MSCs proliferation, chemical resistance, and invasion. Mechanistically, the knockdown of SFPQ reduces CD44v6 expression to inhibit lung cancer MSCs stemness, proliferation in vitro, and metastasis in vivo. The data indicates that SFPQ may be a potential therapeutic target for limiting growth, chemotherapy resistance, and metastasis of lung cancer.

Proteomic analysis of the IPF mesenchymal progenitor cell nuclear proteome identifies abnormalities in key nodal proteins that underlie their fibrogenic phenotype.

IPF is a progressive fibrotic lung disease whose pathogenesis remains incompletely understood. We have previously discovered pathologic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients. IPF MPCs display a distinct transcriptome and create sustained interstitial fibrosis in immune deficient mice. However, the precise pathologic alterations responsible for this fibrotic phenotype remain to be uncovered. Quantitative mass spectrometry and interactomics is a powerful tool that can define protein alterations in specific subcellular compartments that can be implemented to understand disease pathogenesis. We employed quantitative mass spectrometry and interactomics to define protein alterations in the nuclear compartment of IPF MPCs compared to control MPCs. We identified increased nuclear levels of PARP1, CDK1, and BACH1. Interactomics implicated PARP1, CDK1, and BACH1 as key hub proteins in the DNA damage/repair, differentiation, and apoptosis signaling pathways respectively. Loss of function and inhibitor studies demonstrated important roles for PARP1 in DNA damage/repair, CDK1 in regulating IPF MPC stemness and self-renewal, and BACH1 in regulating IPF MPC viability. Our quantitative mass spectrometry studies combined with interactomic analysis uncovered key roles for nuclear PARP1, CDK1, and BACH1 in regulating IPF MPC fibrogenicity.

Multiomic profiling of iron-deficient infant monkeys reveals alterations in neurologically important biochemicals in serum and cerebrospinal fluid before the onset of anemia.

The effects of iron deficiency (ID) during infancy extend beyond the hematologic compartment and include short- and long-term adverse effects on many tissues including the brain. However, sensitive biomarkers of iron-dependent brain health are lacking in humans. To determine whether serum and cerebrospinal fluid (CSF) biomarkers of ID-induced metabolic dysfunction are concordant in the pre/early anemic stage of ID before anemia in a nonhuman primate model of infantile iron deficiency anemia (IDA). ID (n = 7), rhesus infants at 4 mo (pre-anemic period) and 6 mo of age (anemic) were examined. Hematological, metabolomic, and proteomic profiles were generated via HPLC/MS at both time points to discriminate serum biomarkers of ID-induced brain metabolic dysfunction. We identified 227 metabolites and 205 proteins in serum. Abnormalities indicating altered liver function, lipid dysregulation, and increased acute phase reactants were present in ID. In CSF, we measured 210 metabolites and 1,560 proteins with changes in ID infants indicative of metabolomic and proteomic differences indexing disrupted synaptogenesis. Systemic and CSF proteomic and metabolomic changes were present and concurrent in the pre-anemic and anemic periods. Multiomic serum and CSF profiling uncovered pathways disrupted by ID in both the pre-anemic and anemic stages of infantile IDA, including evidence for hepatic dysfunction and activation of acute phase response. Parallel changes observed in serum and CSF potentially provide measurable serum biomarkers of ID that reflect at-risk brain processes prior to progression to clinical anemia.

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.

Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA.

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2'-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2'-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2-3 and 3-4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35-400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.

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.

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis.

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1ß (IL-1ß). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1ß production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1ß secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.