Huntington’s disease (HD) is a debilitating neurodegenerative disorder affecting an individual’s cognitive and motor abilities. HD is caused by a mutation in the huntingtin gene producing a toxic polyglutamine-expanded protein (mHTT) and leading to degeneration in the striatum and cortex. Yet, the molecular signatures that underlie tissue-specific vulnerabilities remain unclear. Here, we investigate this aspect by leveraging multi-epitope protein interaction assays, subcellular fractionation, thermal proteome profiling, and genetic modifier assays. The use of human cell, mouse, and fly models afforded capture of distinct subcellular pools of epitope-enriched and tissue-dependent interactions linked to dysregulated cellular pathways and disease relevance. We established an HTT association with nearly all subunits of the transcriptional regulatory Mediator complex (20/26), with preferential enrichment of MED15 in the tail domain. Using HD and KO models, we find HTT modulates the subcellular localization and assembly of the Mediator. We demonstrated striatal enriched and functional interactions with regulators of calcium homeostasis and chromatin remodeling, whose disease relevance was supported by HD fly genetic modifiers assays. Altogether, we offer insights into tissue- and localization-dependent (m)HTT functions and pathobiology.

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress—DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-β expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.

BACKGROUND:Males and females exhibit distinct anatomic and functional characteristics of the heart, predisposing them to specific disease states.METHODS:We identified microRNAs (miRNAs/miR) with sex-differential expression in mouse hearts.RESULTS:Four conserved miRNAs are present in a single locus on the X-chromosome and are expressed at higher levels in females than males. We show miRNA, miR-871, is responsible for decreased expression of the protein SRL (sarcalumenin) in females. SRL is involved in calcium signaling, and we show it contributes to differences in electrophysiology between males and females. miR-871 overexpression mimics the effects of the cardiac physiology of conditional cardiomyocyte-specific Srl-null mice. Inhibiting miR-871 with an antagomir in females shortened ventricular repolarization. The human orthologue of miR-871, miR-888, coevolved with the SRL 3 ' untranslated region and regulates human SRL.CONCLUSIONS:These data highlight the importance of sex-differential miRNA mechanisms in mediating sex-specific functions and their potential relevance to human cardiac diseases.

Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

Sex disparities in cardiac homeostasis and heart disease are well documented, with differences attributed to actions of sex hormones. However, studies have indicated sex chromosomes act outside of the gonads to function without mediation by gonadal hormones. Here, we performed transcriptional and proteomics profiling to define differences between male and female mouse hearts. We demonstrate, contrary to current dogma, cardiac sex disparities are controlled not only by sex hormones but also through a sex-chromosome mechanism. Using Turner syndrome (XO) and Klinefelter (XXY) models, we find the sex-chromosome pathway is established by X-linked gene dosage. We demonstrate cardiac sex disparities occur at the earliest stages of heart formation, a period before gonad formation. Using these datasets, we identify and define a role for alpha-1B-glycoprotein (A1BG), showing loss of A1BG leads to cardiac defects in females, but not males. These studies provide resources for studying sex-biased cardiac disease states.

ABSTRACTMale and female disease states differ in their prevalence, treatment responses, and survival rates. In cardiac disease, women almost uniformly fare far worse than men. Though sex plays a critical role in cardiac disease, the mechanisms underlying sex differences in cardiac homeostasis and disease remain unexplained. Here, in adult and embryonic hearts we reveal sex-specific transcriptomes and proteomes and show that cardiac sex differences are predominately accounted for by post-transcriptional mechanisms. We found differential expression of male-female proteins in the cardiomyocytes. Using a quantitative proteomics-based approach, we characterized differential sex-specific enriched cardiac proteins, protein complexes, and biological sex processes in the context of global genetic diversity of the Collaborative Cross, an established surrogate for human diversity. We also found that sex differences in cardiac protein expression are established by both hormonal and sex chromosomal mechanisms. We have demonstrated the onset of sex-biased protein expression and discovered that sex disparities in heart tissue occur at the earliest stages of heart development at a period that preceeds mammalian gonadal development. Collectively, these findings may explain why congenital heart disease, a leading cause of death whose origin is often developmental, is sex biased. Our results reveal molecular foundations for differences in cardiac tissue that underlie sex disparities in health, disease, and treatment outcomes.

Dynamically shifting protein-protein interactions (PPIs) regulate cellular responses to viruses and the resulting immune signaling. Here, we use thermal proximity coaggregation (TPCA) mass spectrometry to characterize the on-off behavior of PPIs during infection with herpes simplex virus 1 (HSV-1), a virus with an ancient history of coevolution with hosts. Advancing the TPCA analysis to infer associations de novo, we build a time-resolved portrait of thousands of host-host, virus-host, and virus-virus PPIs. We demonstrate that, early in infection, the DNA sensor IFI16 recruits the active DNA damage response kinase, DNA-dependent protein kinase (DNA-PK), to incoming viral DNA at the nuclear periphery. We establish IFI16 T149 as a substrate of DNA-PK upon viral infection or DNA damage. This phosphorylation promotes IFI16-driven cytokine responses. Together, we characterize the global dynamics of PPIs during HSV-1 infection, uncovering the co-regulation of IFI16 and DNA-PK functions as a missing link in immunity to herpesvirus infection.

The integrity and regulation of the nuclear lamina is essential for nuclear organization and chromatin stability, with its dysregulation being linked to laminopathy diseases and cancer. Although numerous posttranslational modifications have been identified on lamins, few have been ascribed a regulatory function. Here, we establish that lamin B1 (LMNB1) acetylation at K134 is a molecular toggle that controls nuclear periphery stability, cell cycle progression, and DNA repair. LMNB1 acetylation prevents lamina disruption during herpesvirus type 1 (HSV-1) infection, thereby inhibiting virus production. We also demonstrate the broad impact of this site on laminar processes in uninfected cells. LMNB1 acetylation negatively regulates canonical nonhomologous end joining by impairing the recruitment of 53BP1 to damaged DNA. This defect causes a delay in DNA damage resolution and a persistent activation of the G1/S checkpoint. Altogether, we reveal LMNB1 acetylation as a mechanism for controlling DNA repair pathway choice and stabilizing the nuclear periphery.

Mutations of the KRAS gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic KRAS colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching KRAS genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.

KRAS is a frequently mutated oncogene and activating missense mutations have been shown to have a causative role in tumor formation. More than a third of human colon cancers are driven by KRAS mutations. Here, we integrated genomic, transcriptomic and proteomic information to study isogenic cell lines and primary tumor samples with defined KRAS genotypes. We combined RNA‐seq data with top‐down and bottom‐up proteomic analyses to measure differential expression within the proteome, and KRAS proteoforms specifically. Integration of these three complementary omics datasets provided us with a list of molecular entities showing differential expression in response to the mutational status of KRAS. Overall, the results show a strong enrichment for kinases, consistent with the established effect of KRAS mutations in constitutive activation of the MAPK signaling pathway. In addition, proteins associated with cytoskeleton reorganization seemed to be upregulated in the presence of the KRAS G13D mutant allele. This could be involved in increasing cell motility and lead to a more invasive phenotype. Furthermore, we developed a targeted top‐down assay for the detection and quantitation of endogenous KRAS proteoforms to measure allele‐specific modification patterns. Such quantitative measurements (both targeted and untargeted) provide new insights into colorectal cancer phenotypes through the measurement of cancer‐specific protein variants.Support or Funding InformationThis work was supported by Federal Funds from the National Cancer Institute (Office of Cancer Clinical Proteomics Research), National Institutes of Health, under Contract No. HHSN261200800001E, and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under Grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health.

Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25-twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu.

Abstract Mutational activation of KRAS is associated with increased cellular proliferation and tumorigenicity, and metabolic reprogramming. We have used a mass spectrometry analysis by multiple reaction monitoring (MRM) to quantify the expression of 62 proteins encompassing glycolysis (21 proteins), TCA cycle (14 proteins), pentose phosphate pathway (10 proteins), serine biosynthesis (4 proteins), and 13 other proteins involved in metabolism. We applied this MRM panel to the colon tumor cell line DLD-1 (one mutant (G13D) KRAS allele and one WT allele) and the isogenic derivative cell lines DKO-1 (G13D, -) and DKs-8 (+,-). DLD-1 and DKO-1 cells produce lactate at a significantly higher rate than the Dks-8 cells, indicating metabolic reprogramming reflecting a “Warburg” phenotype. MRM panel comparisons of these cell lines revealed that KRAS mutation was associated with the increased expression of glucose and glutamine transporters, glycolytic enzymes, enzymes involved in serine biosynthesis, and a few TCA cycle enzymes. Comparison of the isogenic KRAS mutant/WT pair to each other and to the DLD-1 parental cell line revealed distinct expression changes for the three different KRAS mutant/WT combinations, which suggests that mutant and WT KRAS play distinct roles in metabolic reprogramming. The MRM panel was also used to analyze 16 human formalin-fixed, paraffin-embedded (FFPE) Stage II colorectal tumors. Although the expression of the metabolic proteins varied from patient to patient, a consensus group of 7 metabolic proteins, including enzymes of glycolysis, were significantly increased in KRAS mutant tumors. These results demonstrate that mutational activation of KRAS induces metabolic reprogramming via signaling through both BRAF and PI3K signaling pathways in colorectal cancer cells by the altered expression of metabolic proteins. (Supported by NIH Grant U24CA159988) Citation Format: Josiah E. Hutton, III, Lisa J. Zimmerman, Robbert J. Slebos, Ming Li, Daniel C. Liebler. Multiplexed mass spectrometry analysis of KRAS-associated metabolic reprogramming in colorectal tumors and cell lines. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B28. doi: 10.1158/1557-3125.RASONC14-B28