scholarly journals Chromatin proteomic profiling reveals novel proteins associated with histone-marked genomic regions

2015 ◽  
Vol 112 (12) ◽  
pp. 3841-3846 ◽  
Author(s):  
Xiong Ji ◽  
Daniel B. Dadon ◽  
Brian J. Abraham ◽  
Tong Ihn Lee ◽  
Rudolf Jaenisch ◽  
...  
Keyword(s):  
Es Cells ◽  
Cell Types ◽  
The Novel ◽  
Proteomic Profiling ◽  
Novel Proteins ◽  
Disease Mechanisms ◽  
Lysine Residues ◽  
Associated Proteins ◽  
And Function ◽  
Genomic Regions

More than a thousand proteins are thought to contribute to mammalian chromatin and its regulation, but our understanding of the genomic occupancy and function of most of these proteins is limited. Here we describe an approach, which we call “chromatin proteomic profiling,” to identify proteins associated with genomic regions marked by specifically modified histones. We used ChIP-MS to identify proteins associated with genomic regions marked by histones modified at specific lysine residues, including H3K27ac, H3K4me3, H3K79me2, H3K36me3, H3K9me3, and H4K20me3, in ES cells. We identified 332 known and 114 novel proteins associated with these histone-marked genomic segments. Many of the novel candidates have been implicated in various diseases, and their chromatin association may provide clues to disease mechanisms. More than 100 histone modifications have been described, so similar chromatin proteomic profiling studies should prove to be valuable for identifying many additional chromatin-associated proteins in a broad spectrum of cell types.

scholarly journals Controlled Signaling—Insulin-Like Growth Factor Receptor Endocytosis and Presence at Intracellular Compartments

2021 ◽  
Vol 11 ◽  
Author(s):  
Leonie Rieger ◽  
Rosemary O’Connor
Keyword(s):  
Golgi Apparatus ◽  
Cancer Cells ◽  
Growth Factor Receptor ◽  
Cell Types ◽  
Specific Cell ◽  
Receptor Endocytosis ◽  
Intracellular Compartments ◽  
Associated Proteins ◽  
Membrane Compartments ◽  
And Function

Ligand-induced activation of the IGF-1 receptor triggers plasma-membrane-derived signal transduction but also triggers receptor endocytosis, which was previously thought to limit signaling. However, it is becoming ever more clear that IGF-1R endocytosis and trafficking to specific subcellular locations can define specific signaling responses that are important for key biological processes in normal cells and cancer cells. In different cell types, specific cell adhesion receptors and associated proteins can regulate IGF-1R endocytosis and trafficking. Once internalized, the IGF-1R may be recycled, degraded or translocated to the intracellular membrane compartments of the Golgi apparatus or the nucleus. The IGF-1R is present in the Golgi apparatus of migratory cancer cells where its signaling contributes to aggressive cancer behaviors including cell migration. The IGF-1R is also found in the nucleus of certain cancer cells where it can regulate gene expression. Nuclear IGF-1R is associated with poor clinical outcomes. IGF-1R signaling has also been shown to support mitochondrial biogenesis and function, and IGF-1R inhibition causes mitochondrial dysfunction. How IGF-1R intracellular trafficking and compartmentalized signaling is controlled is still unknown. This is an important area for further study, particularly in cancer.

scholarly journals The nucleosome acidic patch and H2A ubiquitination underlie mSWI/SNF recruitment in synovial sarcoma

2020 ◽  
Vol 27 (9) ◽  
pp. 836-845 ◽  
Author(s):  
Matthew J. McBride ◽  
Nazar Mashtalir ◽  
Evan B. Winter ◽  
Hai T. Dao ◽  
Martin Filipovski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synovial Sarcoma ◽  
Direct Interaction ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Selective Targeting ◽  
Associated Proteins ◽  
And Function ◽  
Genomic Regions ◽  
Nucleosome Binding

AbstractInteractions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma–specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.

scholarly journals Peptide affinity analysis of proteins that bind to an unstructured region containing the transactivating domain of the osmoprotective transcription factor NFAT5

2016 ◽  
Vol 48 (11) ◽  
pp. 835-849
Author(s):  
Jenna F. Dumond ◽  
Xue Zhang ◽  
Yuichiro Izumi ◽  
Kevin Ramkissoon ◽  
Guanghui Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
The Novel ◽  
Mrna Transcription ◽  
Cell Extracts ◽  
Expression Of Genes ◽  
Nuclear Extracts ◽  
Potential Binding ◽  
Associated Proteins ◽  
And Function ◽  
Peptide Affinity

NFAT5 is a transcription factor originally identified because it is activated by hypertonicity and that activation increases expression of genes that protect against the adverse effects of the hypertonicity. However, its targets also include genes not obviously related to tonicity. The transactivating domain of NFAT5 is contained in its COOH-terminal region, which is predicted to be unstructured. Unstructured regions are common in transcription factors particularly in transactivating domains where they can bind co-regulatory proteins essential to their function. To identify potential binding partners of NFAT5 from either cytoplasmic or nuclear HEK293 cell extracts, we used peptide affinity chromatography followed by mass spectrometry. Peptide aptamer-baits consisted of overlapping 20 amino acid peptides within the predicted COOH-terminal unstructured region of NFAT5. We identify a total of 351 unique protein preys that associate with at least one COOH-terminal peptide bait from NFAT5 in either cytoplasmic or nuclear extracts from cells incubated at various tonicities (NaCl varied). In addition to finding many proteins already known to associate with NFAT5, we found many new ones whose function suggest novel aspects of NFAT5 regulation, interaction, and function. Relatively few of the proteins pulled down by peptide baits from NFAT5 are generally involved in transcription, and most, therefore, are likely to be specifically related to the regulation of NFAT5 or its function. The novel associated proteins are involved with cancer, effects of hypertonicity on chromatin, development, splicing of mRNA, transcription, and vesicle trafficking.

scholarly journals Chromatin remodeling factor CECR2 forms tissue-specific complexes with CCAR2 and LUZP1

2021 ◽  
Author(s):  
Farshad Niri ◽  
Alaina Terpstra ◽  
Kenji Rowel Quintana Lim ◽  
Heather McDermid
Keyword(s):  
Chromatin Remodeling ◽  
Neural Tube ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Tube Closure ◽  
Loss Of Function ◽  
Cellular Processes ◽  
Novel Proteins ◽  
Chromatin Remodeling Complexes ◽  
And Function

Chromatin remodeling complexes alter chromatin structure to control access to DNA and therefore control cellular processes such as transcription, DNA replication, and DNA repair. CECR2 is a chromatin remodeling factor that plays an important role in neural tube closure and reproduction. Loss-of-function mutations in Cecr2 result primarily in the perinatal lethal neural tube defect exencephaly, with non-penetrant mice that survive to adulthood exhibiting subfertility. CECR2 forms a complex with ISWI proteins SMARCA5 and/or SMARCA1, but further information on the structure and function of the complex is not known. We therefore have identified candidate components of the CECR2-containing remodeling factor (CERF) complex in embryonic stem (ES) cells through mass spectroscopy. Both SMARCA5 and SMARCA1 were confirmed to be present in CERF complexes in ES cells and testis. However, novel proteins CCAR2 and LUZP1 are CERF components in ES cells but not testis. This tissue specificity in mice suggests these complexes may also have functional differences. Furthermore, LUZP1, loss of which is also associated with exencephaly, appears to play a role in stabilizing the CERF complex in ES cells. Keywords: CECR2, LUZP1, CCAR2, Chromatin remodeling factor, Neural tube defects

Identification of a 40 × 10(3) Mr centromere-associated protein in cultured mammalian cells

1990 ◽  
Vol 97 (4) ◽  
pp. 705-713
Author(s):  
R. Balczon ◽  
M.A. Accavitti ◽  
B.R. Brinkley
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Immunoblot Analysis ◽  
Structure And Function ◽  
Interphase Nuclei ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules ◽  
And Function

Monoclonal antibodies were raised against a complex of proteins that was purified following the crosslinking of tubulin to the centromeres of CHO chromosomes using Lomant's reagent. One of the clones, hybridoma 32–9, produced antibodies that reacted with a 40 × 10(3) Mr protein present in the crosslinked complex. Furthermore, immunoblot analysis demonstrated that the 40 × 10(3) Mr antigen was present in various mammalian cell types from several different species. Indirect immunofluorescence using the antibody produced by clone 32–9 demonstrated that the 40 × 10(3) Mr antigen was associated with both spindle and cytoplasmic microtubules. In addition, centromere/kinetochore staining was detected in metaphase-arrested cells, while staining of prekinetochores in interphase nuclei was not observed. Unlike microtubule-associated proteins and microtubule-dependent ATPases, the 40 × 10(3) Mr protein did not copurify with microtubules when tubules were assembled from cellular homogenates using taxol and either GTP or GTP and AMP-PNP. Instead, the 40 × 10(3) Mr protein remained associated with the insoluble cellular material. The 40 × 10(3) Mr antigen could be released from the insoluble pelleted material by extraction with 1 M NaCl. Once solubilized, the 40 × 10(3) Mr protein was able to copurify with microtubules in assembly assays in vitro. This monoclonal antibody should serve as a valuable probe for studies of centromere/kinetochore structure and function.

Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation

1999 ◽  
Vol 277 (5) ◽  
pp. C899-C912 ◽  
Author(s):  
Wanfang Su ◽  
Boris E. Shmukler ◽  
Marina N. Chernova ◽  
Alan K. Stuart-Tilley ◽  
Lucia de Franceschi ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Chromosome 8 ◽  
Human Disorders ◽  
Functional Regulation ◽  
Multiple Species ◽  
Genomic Regions ◽  
Hypotonic Swelling

Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of86Rb into Xenopus oocytes requires extracellular Cl−, is blocked by the diuretic R(+)-[2- n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1 H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.

scholarly journals Vms1p is a release factor for the Ribosome-associated Quality control Complex

2018 ◽  
Author(s):  
Olga Zurita Rendón ◽  
Eric K. Fredrickson ◽  
Conor J. Howard ◽  
Jonathan Van Vranken ◽  
Sarah Fogarty ◽  
...  
Keyword(s):  
Quality Control ◽  
Ubiquitin Proteasome System ◽  
The Novel ◽  
Novel Proteins ◽  
Lysine Residues ◽  
Nascent Chain ◽  
Ubiquitin Proteasome ◽  
Exit Tunnel ◽  
Carboxy Terminal ◽  
Control Complex

Eukaryotic cells employ the Ribosome-associated Quality control Complex (RQC) to maintain homeostasis despite defects that cause ribosomes to stall. The RQC comprises the E3 ubiquitin ligase Ltn1p, the ATPase Cdc48p, and the novel proteins Rqc1p and Rqc2p1–3. Following recognition and subunit splitting of stalled ribosomes, the RQC detects and assembles on 60S subunits that hold incomplete polypeptides linked to a tRNA (60S:peptidyl–tRNA)4–8. Ltn1p cooperates with Rqc1p to facilitate ubiquitination of the incomplete nascent chain, marking it for degradation7,9,10. Rqc2p stabilizes Ltn1p on the 60S3–5,8 and recruits charged tRNAs to the 60S to catalyze elongation of the nascent protein with Carboxy-terminal Alanine and Threonine extensions, or CAT tails, via a mechanism that is distinct from canonical translation4,10. CAT-tailing mobilizes and exposes lysine residues in the nascent chain, especially those stalled within the exit tunnel, thereby supporting efficient ubiquitination10,11. If the ubiquitin-proteasome system is overwhelmed or unavailable, CAT-tailed nascent chains aggregate in the cytosol or within organelles like the mitochondria12–14. Here we identify Vms1p as the tRNA hydrolase that releases nascent polypeptides for extraction and degradation in the RQC pathway.

scholarly journals Characterization and function of medium and large extracellular vesicles from plasma and urine by surface antigens and Annexin V

2019 ◽  
Author(s):  
Ko Igami ◽  
Takeshi Uchiumi ◽  
Saori Ueda ◽  
Kazuyuki Kamioka ◽  
Daiki Setoyama ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Vascular Endothelial Cells ◽  
Annexin V ◽  
Cell Types ◽  
Surface Antigens ◽  
Peptidase Activity ◽  
Proteomic Profiling ◽  
Healthy Human ◽  
Renal Tubular ◽  
And Function

AbstractMedium/large extracellular vesicles (m/lEVs) are released by most cell types and are involved in multiple basic biological processes. Analysis of m/lEV levels in blood or urine may help unravel pathophysiological findings in many diseases. However, it remains unclear how many naturally-occurring m/lEV subtypes exist as well as how their characteristics and functions differ from one another. Here, we identified m/lEVs pelleted from plasma and urine samples by differential centrifugation and showed by flow cytometry that they typically possessed diameters between 200 nm and 800 nm. Using proteomic profiling, we identified several proteins involved in m/lEV biogenesis including adhesion molecules, peptidases and exocytosis regulatory proteins. In healthy human plasma, we could distinguish m/lEVs derived from platelets, erythrocytes, monocytes/macrophages, T and B cells, and vascular endothelial cells using various surface antigens. m/lEVs derived from erythrocytes and monocytes were Annexin V positive. In urine, 50% of m/lEVs were Annexin V negative but contained various membrane peptidases derived from renal tubular villi. Urinary m/lEVs, but not plasma m/lEVs, showed peptidase activity. The method we have developed to characterize cell-derived m/lEVs suggests the possibility of clinical applications.

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