scholarly journals Global Analysis of Protein Sumoylation inSaccharomyces cerevisiae

2004 ◽  
Vol 279 (44) ◽  
pp. 45662-45668 ◽  
Author(s):  
James A. Wohlschlegel ◽  
Erica S. Johnson ◽  
Steven I. Reed ◽  
John R. Yates
Keyword(s):  
Mass Spectrometry ◽  
Chromatin Remodeling ◽  
Global Analysis ◽  
Eukaryotic Cells ◽  
Transcriptional Elongation ◽  
Biological Processes ◽  
Pol Ii ◽  
Macromolecular Complexes ◽  
Cellular Factors ◽  
Biological Patterns

Although the modification of cellular factors by SUMO is an essential process inSaccharomyces cerevisiae, the identities of the substrates remain largely unknown. Using a mass spectrometry-based approach, we have identified 271 new SUMO targets. These substrates play roles in a diverse set of biological processes and greatly expand the scope of SUMO regulation in eukaryotic cells. Transcription appears to be the most prevalent process associated with sumoylation with novel SUMO substrates found in basal transcription machinery for RNA polymerases I, II, and III, pol II transcriptional elongation complexes, and a variety of chromatin remodeling, chromatin modifying, and chromatin silencing complexes. Additionally, our global analysis has revealed a number of interesting biological patterns in the list of SUMO targets including a clustering of sumoylation targets within macromolecular complexes.

Negotiating the nucleosome: factors that allow RNA polymerase II to elongate through chromatinThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

2007 ◽  
Vol 85 (4) ◽  
pp. 426-434 ◽  
Author(s):  
Jennifer A. Armstrong
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Peer Review Process ◽  
Histone Variants ◽  
Transcriptional Elongation ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Pol Ii ◽  
Assembly Factors

Initiation by RNA polymerase II (Pol II) involves a host of enzymes, and the process of elongation appears similarly complex. Transcriptional elongation through chromatin requires the coordinated efforts of Pol II and its associated transcription factors: C-terminal domain kinases, elongation complexes, chromatin-modifying enzymes, chromatin remodeling factors, histone chaperones (nucleosome assembly factors), and histone variants. This review examines the following: (i) the consequences of the encounter between elongating Pol II and a nucleosome, and (ii) chromatin remodeling factors and nucleosome assembly factors that have recently been identified as important for the elongation stage of transcription.

scholarly journals Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

2021 ◽  
Author(s):  
Lingjun Li ◽  
Yatao Shi ◽  
Zihui Li ◽  
Bin Wang ◽  
Xudong Shi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Global Analysis ◽  
Protein Structures ◽  
Isotopic Labeling ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Mouse Tissues ◽  
Global Mapping

Abstract Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 1,198 citrullination sites and 108 homocitrullination sites from 619 and 79 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation to further decipher their physiological and pathological roles.

Global analysis of protein expression in muscle tissues of dermatomyositis/polymyosisits patients demonstrated an association between dysferlin and human leucocyte antigen A

Rheumatology ◽  
2019 ◽  
Vol 58 (8) ◽  
pp. 1474-1484 ◽  
Author(s):  
Yizhi Xiao ◽  
Honglin Zhu ◽  
Liya Li ◽  
Siming Gao ◽  
Di Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Muscle Tissue ◽  
Inflammatory Cell ◽  
Global Analysis ◽  
Inflammatory Cell Infiltration ◽  
Cell Infiltration ◽  
Biological Processes ◽  
Monocyte Chemoattractant Protein 1 ◽  
Transmembrane Glycoprotein ◽  
Muscle Tissues

Abstract Objectives DM and PM are characterized by myofibre damage with inflammatory cell infiltration due to the strong expressions of MHC class I HLA-A and monocyte chemoattractant protein-1 (MCP-1). Dysferlin (DYSF) is a transmembrane glycoprotein that anchors in the sarcolemma of myofibres. DYSF mutation is closely associated with inherited myopathies. This study aimed to determine the role of DYSF in the development of DM/PM. Methods Mass spectrometry was performed in muscle tissues from DM/PM patients and controls. The DYSF levels in muscle tissue, peripheral blood cells and serum were detected by Western blotting, IF, flow cytometry or ELISA. Double IF and co-immunoprecipitation were used to investigate the relationship between DYSF and HLA-A. Results Mass spectrometry and bioinformatics analysis findings suggested the dysregulated proteins in DM/PM patients participated in common biological processes and pathways, such as the generation of precursor metabolites and energy. DYSF was upregulated in the muscle tissue and serum of DM/PM patients. DYSF was mainly expressed in myofibres and co-localized with HLA-A and MCP-1. DYSF and HLA-A expressions were elevated in myocytes and endothelial cells after being stimulated by patient serum and IFN-β. However, no direct interactions were found between DYSF and HLA-A by co-immunoprecipitation. Conclusion Our study revealed the dysregulated proteins involved in common and specific biological processes in DM/PM patient samples. DYSF is upregulated and exhibits a potential role along with that of HLA-A and MCP-1 in inflammatory cell infiltration and muscle damage during the development of DM/PM.

scholarly journals The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae

Genetics ◽  
2021 ◽  
Author(s):  
Emily Biernat ◽  
Jeena Kinney ◽  
Kyle Dunlap ◽  
Christian Rizza ◽  
Chhabi K Govind
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Biological Processes ◽  
Strongly Correlated ◽  
Coding Sequences ◽  
Pol Ii ◽  
Chromatin Remodeling Complex ◽  
Nucleosomal Dna

Abstract RSC (Remodels the Structure of Chromatin) is a conserved ATP-dependent chromatin remodeling complex that regulates many biological processes, including transcription by RNA polymerase II (Pol II). We report that RSC contributes in generating accessible nucleosomes in transcribed coding sequences (CDSs). RSC MNase ChIP-seq data revealed that RSC-bound nucleosome fragments were very heterogenous (∼80 bp to 180 bp) compared to a sharper profile displayed by the MNase inputs (140 bp to 160 bp), supporting the idea that RSC promotes accessibility of nucleosomal DNA. Notably, RSC binding to + 1 nucleosomes and CDSs, but not with -1 nucleosomes, strongly correlated with Pol II occupancies, suggesting that RSC enrichment s CDSs is linked to transcription. We also observed that Pol II associates with nucleosomes throughout transcribed CDSs, and similar to RSC, Pol II-protected fragments were highly heterogenous, consistent with the idea that Pol II interacts with remodeled nucleosomes in CDSs. This idea is supported by the observation that the genes harboring high-levels of Pol II in their CDSs were the most strongly affected by ablating RSC function. Additionally, rapid nuclear depletion of Sth1 decreases nucleosome accessibility and results in accumulation of Pol II in highly transcribed CDSs. This is consistent with a slower clearance of elongating Pol II in cells with reduced RSC function, and is distinct from the effect of RSC depletion on PIC assembly. Altogether, our data provide evidence in support of the role of RSC in promoting Pol II elongation, in addition to its role in regulating transcription initiation.

scholarly journals Enabling global analysis of protein citrullination and homocitrullination via biotin thiol tag-assisted mass spectrometry

2022 ◽  
Author(s):  
Lingjun Li ◽  
Yatao Shi ◽  
Zihui Li ◽  
Bin Wang ◽  
Xudong Shi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Global Analysis ◽  
Protein Structures ◽  
Isotopic Labeling ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Disease Pathogenesis ◽  
Post Translational Modifications ◽  
Mouse Tissues ◽  
Global Mapping

Abstract Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 691 citrullination sites and 81 homocitrullination sites from 432 and 63 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation for further deciphering their physiological and pathological roles.

scholarly journals Mass spectrometry-based analysis of glycoproteins and its clinical applications in cancer biomarker discovery

2017 ◽  
Vol 4 (7) ◽  
pp. 203-215
Author(s):  
Farid Abu Shammala
Keyword(s):  
Mass Spectrometry ◽  
Posttranslational Modifications ◽  
Biomarker Discovery ◽  
Global Analysis ◽  
Structural Identification ◽  
Clinical Applications ◽  
Biological Processes ◽  
Complex Sample ◽  
Systematic Identification ◽  
Glycoprotein Glycosylation

Most proteins are glycosylated, glycosylation is one of the most important posttranslational modifications of proteins and plays essential roles in various biological processes. Aberration in the glycan moieties of glycoproteins is associated with many diseases. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. With recent advances in proteomics, analytical and computational technologies, glycoproteomics, the global analysis of glycoproteins, is rapidly emerging as a subfield of proteomics with high biological and clinical relevance. Glycoproteomics integrates glycoprotein enrichment and proteomics technologies to support the systematic identification and quantification of glycoproteins in a complex sample. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. Mass spectrometry (MS) has become a powerful tool for mapping glycoprotein glycosylation and detailed glycan structural determination. Especially, tandem mass spectrometry can provide highly informative fragments for structural identification of glycoproteins. This review provides an overview of the development of MS technologies and their applications in identification of abnormal glycoproteins and glycans in human serum to screen cancer biomarkers in recent years.

scholarly journals The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Emily Biernat ◽  
Jeena Kinney ◽  
Kyle Dunlap ◽  
Christian Rizza ◽  
Chhabi K Govind
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Biological Processes ◽  
Strongly Correlated ◽  
Rna Pol Ii ◽  
Coding Sequences ◽  
Pol Ii ◽  
Chromatin Remodeling Complex ◽  
Nucleosomal Dna

RSC (Remodels the Structure of Chromatin) is a conserved ATP-dependent chromatin remodeling complex that regulates many biological processes, including transcription by RNA polymerase II (Pol II). We report that not only RSC binds to nucleosomes in coding sequences (CDSs) but also remodels them to promote transcription. RSC MNase ChIP-seq data revealed that RSC-protected fragments were very heterogenous (~80 bp to 180 bp) compared to the sharper profile displayed by the MNase inputs (140 bp to 160 bp), supporting the idea that RSC activity promotes accessibility of nucleosomal DNA. Importantly, RSC binding to +1 nucleosomes and CDSs, but not with -1 nucleosomes, strongly correlated with Pol II occupancies suggesting that the RSC enrichment in CDSs is important for efficient transcription. This is further supported by a similar heterogenous distribution of Pol II-protected fragments. As such, the genes harboring high-levels of RSC in their CDSs were the most strongly affected by ablating RSC function. Altogether, this study provides a mechanism by which RSC-mediated remodeling aids in RNA Pol II traversal though coding sequence nucleosomes in vivo.

scholarly journals DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii287-iii287
Author(s):  
Hiroaki Katagi ◽  
Nozomu Takata ◽  
Yuki Aoi ◽  
Yongzhan Zhang ◽  
Emily J Rendleman ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Xenograft Model ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Transcriptional Elongation ◽  
Pontine Glioma ◽  
Elongation Complex ◽  
Pol Ii ◽  
Repair Genes ◽  
Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

scholarly journals Tat-SF1 Protein Associates with RAP30 and Human SPT5 Proteins

1999 ◽  
Vol 19 (9) ◽  
pp. 5960-5968 ◽  
Author(s):  
Jae B. Kim ◽  
Yuki Yamaguchi ◽  
Tadashi Wada ◽  
Hiroshi Handa ◽  
Phillip A. Sharp
Keyword(s):  
Recombinant Proteins ◽  
Transcriptional Activity ◽  
Transcription Activation ◽  
Cellular Proteins ◽  
Pol Ii ◽  
Nuclear Extracts ◽  
Cellular Factors ◽  
Immunodeficiency Virus

ABSTRACT The potent transactivator Tat recognizes the transactivation response RNA element (TAR) of human immunodeficiency virus type 1 and stimulates the processivity of elongation of RNA polymerase (Pol) II complexes. The cellular proteins Tat-SF1 and human SPT5 (hSPT5) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins. In nuclear extracts, small fractions of both hSPT5 and Pol II are associated with Tat-SF1 protein. Surprisingly, the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat-SF1, while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat-SF1. Overexpression of Tat-SF1 and hSPT5 specifically stimulates the transcriptional activity of Tat in vivo. These results suggest that Tat-SF1 and hSPT5 are indispensable cellular factors supporting Tat-specific transcription activation and that they may interact with RAP30 in controlling elongation.

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