ABSTRACT: This project was aimed to identify lipid droplet (LD) membrane proteins that are ubiquitylated. We isolated LDs from the livers of control and alcohol-fed rats and extracted LD membrane proteins.
Project description:We describe a method that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation to better understand the role of this crosstalk. The procedure requires 3 days when starting from cell pellets and can yield more than 8000 SUMO sites and 3500 ubiquitin sites from 16 mg of cell extract.
Project description:Lipid droplet (LD) function is regulated by a complement of integral and peripheral proteins that associate with the LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to U2OS and Huh7 cells identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins.
Project description:Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the CD79B subunit of the B cell receptor (BCR). When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL. To identify molecular determinants of sensitivity to Pola-V, we used CRISPR-Cas9 screening for genes that modulated the toxicity of Pola-V for lymphomas or the surface expression of its target, CD79B. Our results reveal a striking impact of CD79B glycosylation on Pola-V epitope availability on the lymphoma cell surface and on Pola-V toxicity. Genetic, pharmacological, and enzymatic approaches that remove terminal sialic acid residues from N-linked glycans enhanced lymphoma killing by Pola-V. Pola-V toxicity was also modulated by KLHL6, a ubiquitin ligase that targets CD79B for degradation in normal and malignant germinal center B cells, explaining its recurrent inactivation in germinal center-derived lymphomas. Our findings suggest precision medicine strategies to optimize Pola-V as a lymphoma therapeutic.
Project description:The number of known proteins associated with plant lipid droplets (LDs) is small compared to other organelles. Many questions of LD biosynthesis and degradation remain open, also due to lack of candidate LD proteins whose characterization could help to elucidate their function in those processes. We performed a proteomic screen on LDs isolated from Nicotiana tabacum pollen tubes. Proteins that were highly enriched in the LD fraction compared to the total or cytosolic fraction where verified for LD localization via transient expression in tobacco pollen tubes. We also compared the isoforms of typical LD proteins found in the pollen tubes on a qualitative level to the isoforms found in tobacco seeds.
Project description:The number of known proteins associated with plant lipid droplets (LDs) is small compared to other organelles. Many questions of LD biosynthesis and degradation remain open, also due to lack of candidate LD proteins whose characterization could help to elucidate their function in those processes. We performed a proteomic screen on LDs isolated from Nicotiana tabacum pollen tubes. Proteins that were highly enriched in the LD fraction compared to the total or cytosolic fraction where verified for LD localization via transient expression in tobacco pollen tubes.
Project description:Multilocular adipocytes are a hallmark of thermogenic adipose tissue, but the factors that enforce this cellular phenotype are unknown. Here, we show that an adipocyte-specific product of the Clstn3 locus (CLSTN3b) present only in placental mammals facilitates the rapid utilization of stored triglyceride by limiting lipid droplet (LD) size. CLSTN3b is an integral ER protein that localizes to ER-LD contact sites via a conserved hairpin domain. Mice lacking CLSTN3b have altered LD morphology and increased lipid accumulation in BAT, as well as heightened sensitivity to cold challenge, despite having no defect in adrenergic signaling. Conversely, forced expression of CLSTN3b promotes a multilocular LD phenotype in cultured cells and BAT and facilitates triglyceride utilization. Mechanistically, CLSTN3b associates with CIDE proteins and impairs their ability to transfer lipid between droplets, thereby limiting LD expansion. These findings define a molecular mechanism that maximizes LD surface area to facilitate lipid utilization in thermogenic adipocytes.
Project description:We developed a strategy that allows the identification of NEDP1 dependent NEDDylation sites under endogenous expression of wild type NEDD8. We combined the use of anti-diGly antibodies that recognise both ubiquitin and NEDD8 modified peptides upon trypsin digestion with short treatment of cells with the ubiquitin E1 inhibitor MLN7243 (UAEi), that dramatically reduces ubiquitin but not NEDD8 modification. By eliminating the majority of ubiquitin-derived diGly peptides upon MLN7243 treatment, we would be able to quantify NEDP1 dependent diGly peptides. Extracts from parental and NEDP1 knockout HCT116 cells both treated with UAEi were used for the isolation of diGly modified peptides and mass spectrometry analysis.
Project description:Post-translational modification of proteins by ubiquitin (UQ) and UQ-like modifiers is emerging as a central pathway in DNA replication. We previously showed that chromatin in the vicinity of replisomes is rich in SUMO and depleted in UQ, whereas an opposite pattern is observed in mature chromatin. How this SUMO-rich/UQ-low environment is maintained at replisomes is not known. Here we identify USP7 as a SUMO deubiquitinase that localizes to replication forks and is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 prevents their ubiquitination. Chemical inhibition or genetic deletion of USP7 leads to the accumulation of UQ on SUMOylated proteins, which are displaced from the replisomes. Our findings provide a model to explain the differential accumulation of SUMO and UQ in replication forks versus mature chromatin, and identify an essential role of USP7 that should be taken into account for the use of USP7 inhibitors as anticancer agents.
Project description:Identification of nuclear CSF-1R, H3K4me1 and H3K4me3 localization on chromatin in human primary monocytes and modification of this localization during monocyte differentiation into macrophage induced by 100ng/mL CSF-1 during 6 hours (1 donor) or 72h (3 donors). Identification of EGR1 chromatin localization in human primary monocytes (3 donors) Comparison of nuclear CSF-1R chromatin localization in monocytes from 1 healthy donor and 2 chronic myelomonocytic leukemia patients.
Project description:MS/MS characterization of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state that together with structural and biochemical studies reveal a novel ubiquitin-binding patch on RPN10 that directs K84 ubiquitylation.