Phosphoproteomics analysis of Pseudomonas aeruginosa with PfkA overproduction
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ABSTRACT: To determined the phosphorylation sites of proteins after overproduction of PfkA in MPAO1 via pHERD20T-fpkA, and cells without overproduciton of this protein was used as a control.
Project description:To determined the phosphorylation sites of proteins after overproduction of PfkA, PfkB and PfpC individually in PAO1 via pHERD20T-pfkA, pHERD20T-pfkB and pHERD20T-pfpC.
Project description:The total protein of bone marrw myeloma cells was extracted with cell lysis buffer for IP. YBX1 antibody was used to perform Immunoprecipitation.
Project description:In this study, proteomics was utilized to screen proteins interacting with ENKD1. affinity purification coupled with mass spectrometry (AP-MS) was employed to systematically identify ENKD1-interacting proteins. Following immunoprecipitation and SDS-PAGE separation, the co-purified proteins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This process helps uncover ENKD1's function, its role in cellular pathways.
Project description:Chronic hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis and liver cancer, representing a global health problem for which a functional cure is difficult to achieve. The HBV core protein (HBc) is essential for multiple steps in the viral life cycle; it is the building block of the nucleocapsid in which viral DNA reverse transcription occurs, and it mediates viral–host cell interactions critical to HBV infection persistence. However, systematic studies targeting HBc-interacting proteins remain lacking. An engineered ascorbate peroxidase called APEX2, is genetically targeted to a cellular region of interest and biochemically labeled neighboring proteins within living cells. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry.Here, we combined HBc with the engineered ascorbate peroxidase 2 (APEX2) to systematically identify HBc-related proteins in living cells.
Project description:URI keeps low levels of p53 in a TRIM28-MDM2 dependent manner, maintains SCD1 activity and accumulation of MUFAs, and subsequently promotes resistance to TKIs in cancer cell. URI-p53-SCD1 axis mediates resistance of TKIs and may explain why p53-wild type HCC still showed intrinsic resistance to TKIs. Moreover, the combination therapy identified here may represent a promising strategy for the approximately 41% of patients with advanced HCC who have wild-type p53 and high levels of URI/SCD1.
Project description:Lysine acetylation and ubiquitination are one of many protein modifications and play a crucial role in the biological regulation of many organisms, but little is known about the relationship between acetylation and ubiquitination few. Here, the Isw1 protein is an important member of the chromatin remodeling complex, and we performed single-protein modification mass spectrometry detection of the C. neoformans Isw1 protein and site mutations for both detected modifications. The data showed that the two modifications of Cryptococcus neoformans Isw1 protein have a balance of each other. Acetylation can maintain protein stability and maintain protein function, while ubiquitination can reduce protein level and maintain Isw1 protein expression. The expression level of Isw1 protein leads to resistance to antifungal drugs. These results reveal the resistance mechanism of Isw1 protein of Cryptococcus neoformans to antifungal drugs.
Project description:To identify receptors and pathways active in glioblastoma (GBM) stem like cells (GSCs), we generated and screened thousands of monoclonal antibodies (mAbs) for preferential binding to primary cultures enriched in GSCs. This led to the identification of the integrin alpha 7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression was negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses demonstrated a key biological function of ITGA7 in growth and invasion of GSCs. In addition, we showed that targeting ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling and led to a significant delay of tumor engraftment and strong reduction in size and invasion. Our data underline the potential value of ITGA7 as glioma biomarker and therapeutic target.
Project description:Posttranscriptional and posttranslational modifications play crucial roles in plant immunity. However, how plant fine-tune these two modifications to activate antiviral immunity remains unknown. Here, we report that the m6A methyltransferase TaHAKAI is utilized by wheat yellow mosaic virus (WYMV) to increase viral genomic m6A modification and promotes viral replication. However, TaHAKAI also functions as an E3 ligase that targets the viral RNA silencing suppressor P2 for degradation and inhibits viral infection. A major allele of TaHAKAI in susceptible cultivar reduced the E3 ligase activity but not m6A methyltransferase activity, promoting viral infection. Interestingly, TaHAKAIR attenuates the mRNA stability of TaWPS1, the negative regulator of spike development, to increase panicle length and spikelet number by modulating its m6A modification. Our study reveals a new mechanisms of balancing disease resistance and yield by fine-tuning m6A modification and ubiquitination.
Project description:Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K+ channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutant channel is linked to defective protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The detailed mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K+ channels, however, remains unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent non-glycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, non-glycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.