Tyrosine-Based Signals Regulate the Assembly of Daple⋅PARD3 Complex at Cell-Cell Junctions.

Polarized distribution of organelles and molecules inside a cell is vital for a range of cellular processes and its loss is frequently encountered in disease. Polarization during planar cell migration is a special condition in which cellular orientation is triggered by cell-cell contact. We demonstrate that the protein Daple (CCDC88C) is a component of cell junctions in epithelial cells which serves like a cellular "compass" for establishing and maintaining contact-triggered planar polarity. Furthermore, these processes may be mediated through interaction with the polarity regulator PARD3. This interaction, mediated by Daple's PDZ-binding motif (PBM) and the third PDZ domain of PARD3, is fine-tuned by tyrosine phosphorylation on Daple's PBM by receptor and non-receptor tyrosine kinases, such as Src. Hypophosphorylation strengthens the interaction, whereas hyperphosphorylation disrupts it, thereby revealing an unexpected role of Daple as a platform for signal integration and gradient sensing for tyrosine-based signals within the planar cell polarity pathway

Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism.

Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems

Proteomic analyses of Urine Exosomes reveal New Biomarkers of Diabetes in Pregnancy.

ObjectiveTo evaluate 24 hour urine exosome protein content changes among pregnant US subjects with diabetes and obesity during early pregnancy.MethodsThe exosome proteome content from 24 hour urine samples of pregnant subjects with gestational diabetes mellitus (GDM, N=8) and pre-gestational Type 2 diabetes (PGD, N = 10) were compared with control samples (CTRL, N = 10) obtained at week 20 of pregnancy. Differences in exosome protein load between groups was identified by liquid chromatography/mass spectrometry, analyzed by linear regression in negative binomial distribution, visualized in MetaboAnalyst (version 3.0), and validated by western immunoblotting.ResultsAt the 20th week of pregnancy, we identified 646, 734 and 856 proteins in exosomes from 24 hour urine samples of patients from the CTRL, GDM and PGD groups, respectively. S100 calcium binding protein A9, damage associated molecular pattern (DAMP) signal, was found to be significantly increased in both GDM and PGD subjects. In GDM subjects the peptide counts for S100A9 protein independently correlated with maternal obesity and macrosomia of the newborn infants. Early to late pregnancy developmental changes in the GDM group were shown to utilize pathways and protein expression levels differently from those in PGD or CTRL groups.ConclusionsUrinary exosome proteomic analysis non-invasively provides insights into maternal changes during diabetic pregnancy. Exosome biomarkers early in pregnancy can be potentially used to better understand pathophysiologic mechanisms of diabetes at a cellular level, and to distinguish between gestational and pre-gestational diabetes at the pathway level. This information can aid intervention efforts to improve pregnancy outcomes in women with diabetes

An endogenous peptide marker differentiates SOD1 stability and facilitates pharmacodynamic monitoring in SOD1 amyotrophic lateral sclerosis

The discovery of novel biomarkers has emerged as a critical need for therapeutic development in amyotrophic lateral sclerosis (ALS). For some subsets of ALS, such as the genetic superoxide dismutase 1 (SOD1) form, exciting new treatment strategies, such as antisense oligonucleotide-mediated (ASO-mediated) SOD1 silencing, are being tested in clinical trials, so the identification of pharmacodynamic biomarkers for therapeutic monitoring is essential. We identify increased levels of a 7-amino acid endogenous peptide of SOD1 in cerebrospinal fluid (CSF) of human SOD1 mutation carriers but not in other neurological cases or nondiseased controls. Levels of peptide elevation vary based on the specific SOD1 mutation (ranging from 1.1-fold greater than control in D90A to nearly 30-fold greater in V148G) and correlate with previously published measurements of SOD1 stability. Using a mass spectrometry-based method (liquid chromatography-mass spectrometry), we quantified peptides in both extracellular samples (CSF) and intracellular samples (spinal cord from rat) to demonstrate that the peptide distinguishes mutation-specific differences in intracellular SOD1 degradation. Furthermore, 80% and 63% reductions of the peptide were measured in SOD1G93A and SOD1H46R rat CSF samples, respectively, following treatment with ASO, with an improved correlation to mRNA levels in spinal cords compared with the ELISA measuring intact SOD1 protein. These data demonstrate the potential of this peptide as a pharmacodynamic biomarker

Amplification of Inflammation by Lubricin Deficiency Implicated in Incident, Erosive Gout Independent of Hyperuricemia

Objective In gout, hyperuricemia promotes urate crystal deposition that stimulates the NLRP3 inflammasome and IL-1β-mediated arthritis. Incident gout without background hyperuricemia is rarely reported. To identify hyperuricemia-independent mechanisms driving gout incidence and progression, we characterized erosive urate crystalline inflammatory arthritis meeting ACR/EULAR gout classification criteria in a normouricemic young adult female. Methods Whole genome sequencing, quantitative proteomics, whole blood RNA-seq, and IL-1β-induced murine knee synovitis characterized proband candidate genes, biomarkers, and pathogenic mechanisms. Results Lubricin was attenuated in proband serum, associated with elevated acute phase reactants and inflammatory whole blood transcripts and transcriptional pathways. The proband had predicted damaging gene variants of NLRP3 and of Inter-Alpha-Trypsin Inhibitor Heavy Chain 3, an inhibitor of lubricin-degrading Cathepsin G. Proband serum protein interactome network changes supported enhanced lubricin degradation, with Cathepsin G activity increased relative to its inhibitors SERPINB6 and Thrombospondin1. TLR2 activation suppressed cultured human synovial fibroblast lubricin mRNA and release (p\u3c0.01). Lubricin blunted urate crystal precipitation, and IL-1β induction of xanthine oxidase and urate in cultured macrophages (p\u3c0.001). In lubricin-deficient mice, IL-1β knee injection increased xanthine oxidase positive synovial resident M1 macrophages (p\u3c0.05). Conclusion We linked normouricemic erosive gout to attenuated lubricin, with impaired control of Cathepsin G activity, compounded by deleterious NLRP3 variants. Lubricin suppressed monosodium urate crystallization, and blunted IL-1β-induced increases in macrophage xanthine oxidase and urate. Collective activities of articular lubricin that could limit incident and erosive gouty arthritis independently of hyperuricemia are subject to disruption by inflammation, activated Cathepsin G, and synovial fibroblast TLR2 signaling

Genetic and molecular interactors of ABA insensitive1 gene (abi1) of Arabidopsis thaliana

grantor: University of TorontoThe plant hormone abscisic acid (ABA) is important in numerous functions ranging from the establishment of seed dormancy and germination to protecting adult plants from a variety of environmental stresses. Previously in ' Arabidopsis', mutants with reduced sensitivity to applied ABA (' abi') have been described. One ABA insensitive mutation, designated ' abi1', identifies a gene that encodes a type 2C protein phosphatase suggesting the phosphorylation status of the plant is important for correct ABA action. To further understand the role of ABI1 in ABA signal transduction and to identify new genes involved in ABA action, an 'abi1' mutant background was used to screen for mutations that further reduce ABA responsiveness in 'Arabidopsis'. This 'ab i1' 'e_'nhancer (' abe') screen resulted in the isolation of mutations in 6 genetic loci. Three complementation groups define the previously identified ABA response loci 'ABI3', 'ABI4' and 'ABI5'. Mutations at two loci identify new genes designated 'ABE1' and 'ABE2' and the last complementation group was found to be allelic to the 'CTR1' gene, a known negative regulator of ethylene signaling in 'Arabidopsis'. This last result suggests that ABA and ethylene may have overlapping interactions that define the sensitivity of the plant seeds to these growth factors. Interestingly, in a parallel study it was discovered that a number of known mutants that reduce ethylene sensitivity also show altered ABA responsiveness. Since mutations that confer ethylene insensitivity cause seed ABA hypersensitivity and mutations that confer constitutive ethylene response cause increased seed ABA insensitivity, I proposed that ethylene signaling functions in the negative regulation of seed ABA responses. Furthermore, studies involving the interaction between ABA and ethylene in the root suggest ABA may signal directly through ethylene signal transduction pathway in an ethylene independent manner.Ph.D

Data from proteome analysis of Lasiodiplodia theobromae (Botryosphaeriaceae)

Trunk disease fungi are a global problem affecting many economically important fruiting trees. The Botryosphaeriaceae are a family of trunk disease fungi that require detailed biochemical characterization in order to gain insight into their pathogenicity. The application of a modified Folch extraction to protein extraction from the Botryosphaeriaceae Lasiodiplodia theobromae generated an unprecedented data set of protein identifications from fragmentation analysis and de novo peptide sequencing of its proteome. This article contains data from protein identifications obtained from a database-dependent fragmentation analysis using three different proteomics algorithms (MSGF, Comet and X! Tandem via the SearchGUI proteomics pipeline program) and de novo peptide sequencing. Included are data sets of gene ontology annotations using an all-Uniprot ontology database, as well as a Saccharomyces cerevisiae-only and a Candida albicans-only ontology database, in order to discern between those proteins involved in common functions with S. cerevisiae and those in common with the pathogenic yeast C. albicans. Our results reveal the proteome of L. theobromae contains more ontological categories in common to C. albicans, yet possesses a much wider metabolic repertoire than any of the yeasts studied in this work. Many novel proteins of interest were identified for further biochemical characterization and annotation efforts, as further discussed in the article referencing this article (1). Interactive Cytoscape networks of molecular functions of identified peptides using an all-Uniprot ontological database are included. Data, including raw data, are available via ProteomeXchange with identifier PXD005283. Keywords: Trunk-disease fungi, LC-nanoESI-MS, Proteomics, de novo peptide sequencing, Gene ontolog

Cortactin as a target for FAK in the regulation of focal adhesion dynamics.

Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement