A Computational and Informatics Framework for the Analysis of Affinity Purification Mass Spectrometry Data and Reconstruction of Protein Interaction Networks.

Affinity purification coupled with mass spectrometry (AP-MS) is a high-throughput approach to detect protein interactions, where a protein complex is affinity-purified using an antibody targeted against a clonally modified member of the complex that expresses an epitope tag (bait) and analyzed using protein mass spectrometry. In most cases, co-purifying proteins (prey) include a large number of non-specific interactors, hence methods to discern bona fide interactions from non-specific background are critical for the successful application of AP-MS technology. In this thesis, I present a computational and informatics framework for streamlined analysis of AP-MS data, which includes (i) a pipeline for scoring and detecting potentially bona fide protein interactions (SPrInt), (ii) an integrated network reconstruction tool (PInt), (iii) a database of standardized negative control experiments that assist in scoring interactions (the CRAPome) and (iv) a protein interaction database that aggregates uniformly scored interactions (the RePrInt). SPrInt implements two novel scoring functions that are complementary to previously published models and several visualizations that assist in filtering data. PInt facilitates systematic network reconstruction and analysis by integrating prior knowledge from protein interaction databases and providing tools to dissect large networks into constituent sub-networks. In summary, SPrInt and PInt are versatile tools for analyzing a wide variety of AP-MS data sets. Small-scale AP-MS studies may not capture the complete set of non-specific interactions due to limited availability of negative controls. Fortunately, negative controls are largely bait-independent and can be aggregated to increase the coverage and characterization of the background. Accordingly, we created the first, large-scale, publicly accessible repository of negative controls (the CRAPome, www.crapome.org) and demonstrated its utility using a benchmark dataset. Current protein interaction databases are created using curated lists of protein interactions. While manual curation is limited in its scope, computational curation often leads to high false positives. We present an alternative (data driven) approach for creating protein interaction databases by aggregating raw data and making available uniformly scored interactions (the RePrInt). We also present a novel pipeline for comprehensive network reconstruction that includes a model to merge evidence from multiple datasets.PhDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111349/1/dattam_1.pd

Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WT versus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.This work was supported by the National Institutes of Health (NIH) through the National Heart, Lung, and Blood Institute (NHLBI) grant R01HL122352 awarded to J.J., as well as the National Institute of General Medical Sciences (NIGMS) grant R01GM094231 and the National Cancer Institute (NCI) grant U24CA210967 awarded to A.I.N. R.K. is supported by the NCI support grant P30CA046592 awarded to the University of Michigan Rogel Cancer Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.S

Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models.

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases

The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans

<i>SKS1</i> transcript levels are regulated in response to nitrogen and glucose levels.

<p>A) Analysis of <i>SKS1</i> mRNA levels in a wild-type strain of the filamentous Σ1278b background in normal media relative to media limited in nitrogen and glucose. B) Analysis of <i>SKS1</i> mRNA levels in the indicated homozygous diploid deletion mutants under normal and low nitrogen/glucose conditions. Fold-change in mRNA relative to wild type is indicated with standard error; actual values are listed below the graph.</p

Network connectivity of Sks1p kinase signaling.

<p>A) Network connectivity map constructed by expanding connections involving Sks1p-dependent phosphoproteins through genetic and physical interactions from KEGG, BioGrid, and GeneMania. Clusters of proteins enriched for the indicated KEGG signaling pathways are shown. Numbered proteins identify Sks1p and a subset of proteins exhibiting Sks1p-dependent phosphorylation by mass spectrometry (shaded red) within the context of the larger connected network. As indicated, the KEGG glycolysis/gluconeogenesis pathway was enriched in the subset of Sks1p-dependent phosphoproteins identified by mass spectrometry. Proteins within the network clustered around B) the KEGG glycolysis/gluconeogenesis pathway and C) the KEGG cell cycle and meiosis pathways are highlighted.</p

Quantitative phosphoproteomic analysis of the Sks1p signaling network in the filamentous Σ1278b genetic background.

<p>A) Schematic overview of major steps in the SILAC-based mass spectrometric analysis of Sks1p signaling; strain auxotrophies are indicated in the control and kinase-dead strains. B) A listing of proteins exhibiting decreased or increased phosphopeptide abundance after enrichment from a strain carrying the <i>sks1-K39R</i> kinase-dead allele relative to a strain carrying wild-type <i>SKS1</i>.</p

Phenotypic analysis of homozygous diploid mutants corresponding to a subset of Sks1p-dependent phosphoproteins identified by mass spectrometry.

<p>A) The indicated homozygous diploid deletion mutants exhibit surface-spread pseudohyphal filamentation phenotypes on SLAD and SLALD medium relative to a wild-type strain. The degree of observed pseudohyphal growth is indicated, with the “−” corresponding to an absence of filamentation and “+++” indicating the strongest observed pseudohyphal growth. Scale bars representing 2 mm are indicated. The <i>pda1</i>Δ/Δ strain formed slightly smaller colonies than wild type, and <i>npr3</i>Δ/Δ mutants formed markedly small colonies. B) Amino acid sequences identifying Sks1p-dependent phosphorylation sites selected for further phenotypic analysis. The ratio indicates abundance upon phosphopeptide enrichment from the <i>sks1-K39R</i> mutant relative to wild-type. All homozygous diploid integrated point mutants demonstrated a significant fitness defect compared to wild-type when grown in liquid media under nitrogen-limiting (SLAD) or nitrogen- and glucose-limiting (SLALD) conditions as indicated by C) the long-term cell titer and D) growth curves for each condition. Full values for the growth curves shown here are provided in Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004183#pgen.1004183.s005" target="_blank">Tables S3</a> through <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004183#pgen.1004183.s008" target="_blank">S6</a>.</p

Previously unreported phosphosites from peptides differentially abundant in <i>sks1-K39R</i>.

a<p>Phosphosites indicating a localization probability of <i>p</i>>0.75.</p>b<p>Phosphopeptide abundance as <i>sks1-K39R</i>/WT ratio.</p

HSC70 is a chaperone for wild-type and mutant cardiac myosin binding protein C

Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover