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Identification of new interaction partners of D. discoideum BirA-UBXD9

ABSTRACT: The largest family of p97 cofactors is the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. We analysed one member of this family, the largely uncharacterised UBXD9 protein. To shed a light on the UBXD9 interactome, we used independent approaches to identify new interacting proteins. As a result, we confirmed p97 (also known as valosin-containing protein, VCP) as an interaction partner of UBXD9 and identified new interaction partners.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Dictyostelium Discoideum (slime Mold)

TISSUE(S): Cell Culture

SUBMITTER: Jana Riehl

LAB HEAD: Ludwig Eichinger

PROVIDER: PXD027162 | Pride | 2021-08-26

REPOSITORIES: Pride

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			Action	DRS
	20180825_Q2_UC_CollID_653_ProjID_1367_Eichinger_Sample_11.raw	Raw
	20180825_Q2_UC_CollID_653_ProjID_1367_Eichinger_Sample_12.raw	Raw
	20200701_Q2_UC_ColID_954_projID_2145_Eichinger_sample_01.raw	Raw
	20200701_Q2_UC_ColID_954_projID_2145_Eichinger_sample_02.raw	Raw
	20200701_Q2_UC_ColID_954_projID_2145_Eichinger_sample_03.raw	Raw

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Project description:Right ventricular heart failure (RVF) associated with pulmonary hypertension (PH) is characterized by a distinct gene expression pattern when compared with functional compensatory hypertrophy. Carvedilol treatment after RVF has been established reduces right ventricle (RV) hypertrophy and improves the RV function. In addition, carvedilol treatment has been shown to alter the gene expression of select genes. We sought to identify, on a genome-wide basis, the effect of carvedilol on gene expression. RVF was induced in male Sprague-Dawley rats by the combination of VEGF-receptor blockade and chronic hypoxia; thereafter, one group was treated with carvedilol. RNA was isolated from the RV and subjected to microarray analysis. A prediction analysis of the carvedilol-treated RVs showed that carvedilol treated RVs most resembled in their expression pattern the RVF pattern. However, an analysis beyond the boundaries of the prediction set revealed a small set of genes associated with carvedilol reversal of RVF. Pathway analysis of this set of genes revealed expression changes of genes involved in cardiac hypertrophy, mitochondrial dysfunction, protein ubiquitination, and sphingolipid metabolism. Genes encoding proteins in the cardiac hypertrophy and protein ubiquitination pathways were downregulated in the RV by carvedilol, while genes encoding proteins in the mitochondrial dysfunction and sphingolipid metabolism pathways were upregulated by carvedilol. Male Sprague-Dawley rats (Harlan Laboratories Inc., Indianapolis, IN) weighing 200g were injected subcutaneously with SU5416 suspended in 0.5% (w/v) carboxymethylcellulose sodium, 0.9% (w/v) sodium chloride, 0.4% (w/v) polysorbate 80, and 0.9% (v/v) benzyl alcohol in deionized water. Rats were given a single injection of SU5416 (20 mg/kg) at the beginning of the 6-week experiment. The animals were then exposed to chronic hypoxia (simulated altitude of 5,000 m in a nitrogen dilution chamber) for four weeks; thereafter the animals were kept at the altitude of Richmond, VA (sea level) for another two weeks. Hypoxia-only rats were exposed to chronic hypoxia for four weeks. Carvedilol (15 mg/kg; Sigma-Aldrich, St. Louis, MO) was dissolved in 20% dimethyl sulfoxide and water and administered once daily per oral gavage for 4 weeks, beginning after return to room air breathing. Six rats per treatment group were randomly assigned. Rats were euthanized with Euthanasia-III Solution (390 mg/ml pentobarbital sodium, 50 mg/ml phenytoin sodium; Med-Pharmex, Inc., Pomona, CA) and hearts were removed. The RV was dissected from the LV and both ventricles were snap-frozen in liquid nitrogen. Total RNA was isolated from approximately 30 mg of snap-frozen rat heart tissue using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA). Hearts were homogenized with Buffer RLT and beta-mercaptoethanol in an MP FastPrepM-BM-.-24 Lysing Matrix D tube (MP Biomedicals LLC, Solon, OH), and then RNA was isolated and purified following the manufacturerM-bM-^@M-^Ys protocol. RNA concentration was determined using a NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, DE). All samples had an A260/A280 ratio between 1.9 and 2.1. The amplification and hybridization process is as follows. 500 ng of total RNA was amplified and labeled with Cyanine-5 and 500 ng of universal rat reference RNA (Stratagene, Santa Clara, CA) was amplified and labeled with Cyanine-3 using the Agilent QuickAmp Labeling kit (Agilent Technologies Inc., Santa Clara, CA) to produce labeled cRNA following the manufacturerM-bM-^@M-^Ys protocol. After amplification and labeling, the dye incorporation was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). All ratios were greater than 8.0 pmole dye per mg cRNA per the manufacturerM-bM-^@M-^Ys recommendation. 825 ng of sample and 825 ng of reference RNA were combined and incubated with an Agilent Whole Rat Genome 4x44k microarray slide (Agilent Technologies Inc., Wilmington, DE) for 17 hours at 65M-BM-0C. Following hybridization, slides were washed following the manufacturerM-bM-^@M-^Ys protocol and scanned using an Axon GenePix 4200A scanner (Axon Instruments, Union City, CA) at a resolution of 5 mM. The raw data were generated using GenePix Pro 5.0 software (Axon Instruments, Union City, CA). Raw expression data files were uploaded into R and normalized using the Bioconductor marray package by the Lowess normalization algorithm, then exported to BRB Array Tools for analysis.

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Identification of new interaction partners of D. discoideum BirA-UBXD9

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