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1.
Genome Res ; 30(4): 635-646, 2020 04.
Article in English | MEDLINE | ID: mdl-32188699

ABSTRACT

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell-specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein-protein relationships and protein functions at the chromatin template.


Subject(s)
Chromatin Assembly and Disassembly , Chromatin/genetics , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Genetic Loci , Proteome , Proteomics , Transcription, Genetic , Chromatin Immunoprecipitation Sequencing , Genomic Library , Protein Binding , Proteomics/methods , RNA Polymerase II/metabolism , Transcription Factors/metabolism , Yeasts/genetics , Yeasts/metabolism
2.
Angew Chem Int Ed Engl ; 57(43): 14164-14168, 2018 10 22.
Article in English | MEDLINE | ID: mdl-30188611

ABSTRACT

Ubiquitin-fold modifier 1 (UFM1) is a reversible post-translational modifier that is covalently attached to target proteins through an enzymatic cascade and removed by designated proteases. Abnormalities in this process, referred to as Ufmylation, have been associated with a variety of human diseases. Given this, the UFM1-specific enzymes represent potential therapeutic targets; however, understanding of their biological function has been hampered by the lack of chemical tools for activity profiling. To address this unmet need, a diversifiable platform for UFM1 activity-based probes (ABPs) utilizing a native chemical ligation (NCL) strategy was developed, enabling the generation of a variety of tools to profile both UFM1 conjugating and deconjugating enzymes. The use of the probes is demonstrated in vitro and in vivo for monitoring UFM1 enzyme reactivity, opening new research avenues.


Subject(s)
Molecular Probes , Peptide Hydrolases/metabolism , Protein Processing, Post-Translational , Proteins/metabolism , Chromatography, High Pressure Liquid , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Electroporation , HeLa Cells , Humans , Proteins/chemistry
3.
Angew Chem Int Ed Engl ; 57(29): 8958-8962, 2018 07 16.
Article in English | MEDLINE | ID: mdl-29771001

ABSTRACT

SUMO is a post-translational modifier critical for cell cycle progression and genome stability that plays a role in tumorigenesis, thus rendering SUMO-specific enzymes potential pharmacological targets. However, the systematic generation of tools for the activity profiling of SUMO-specific enzymes has proven challenging. We developed a diversifiable synthetic platform for SUMO-based probes by using a direct linear synthesis method, which permits N- and C-terminal labelling to incorporate dyes and reactive warheads, respectively. In this manner, activity-based probes (ABPs) for SUMO-1, SUMO-2, and SUMO-3-specific proteases were generated and validated in cells using gel-based assays and confocal microscopy. We further expanded our toolbox with the synthesis of a K11-linked diSUMO-2 probe to study the proteolytic cleavage of SUMO chains. Together, these ABPs demonstrate the versatility and specificity of our synthetic SUMO platform for in vitro and in vivo characterization of the SUMO protease family.


Subject(s)
Peptide Hydrolases/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , HeLa Cells , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Models, Molecular , Peptide Hydrolases/analysis , Peptides/chemistry , Peptides/metabolism , Proteolysis , Small Ubiquitin-Related Modifier Proteins/chemistry , Solid-Phase Synthesis Techniques , Substrate Specificity
4.
Nat Commun ; 9(1): 1119, 2018 03 16.
Article in English | MEDLINE | ID: mdl-29549242

ABSTRACT

Signal transduction by small ubiquitin-like modifier (SUMO) regulates a myriad of nuclear processes. Here we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery results in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identify the anaphase-promoting complex/cyclosome (APC/C), a master regulator of metaphase to anaphase transition. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation is crucial for accurate progression of cells through mitosis and increases APC/C ubiquitylation activity toward a subset of its targets, including the newly identified target KIF18B. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal how SUMO and ubiquitin cooperate to drive mitosis.


Subject(s)
Apc4 Subunit, Anaphase-Promoting Complex-Cyclosome/metabolism , Mitosis/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation/genetics , Ubiquitins/metabolism , Apc4 Subunit, Anaphase-Promoting Complex-Cyclosome/genetics , Cell Line, Tumor , HCT116 Cells , HeLa Cells , Humans , Kinesins/metabolism , RNA Interference , RNA, Small Interfering/genetics , Signal Transduction/physiology , Small Ubiquitin-Related Modifier Proteins/genetics , Ubiquitin-Activating Enzymes/genetics , Ubiquitination/genetics , Ubiquitins/genetics
5.
Proc Natl Acad Sci U S A ; 114(35): E7255-E7261, 2017 08 29.
Article in English | MEDLINE | ID: mdl-28808029

ABSTRACT

Proteins can be modified by multiple posttranslational modifications (PTMs), creating a PTM code that controls the function of proteins in space and time. Unraveling this complex PTM code is one of the great challenges in molecular biology. Here, using mass spectrometry-based assays, we focus on the most common PTMs-phosphorylation and O-GlcNAcylation-and investigate how they affect each other. We demonstrate two generic crosstalk mechanisms. First, we define a frequently occurring, very specific and stringent phosphorylation/O-GlcNAcylation interplay motif, (pSp/T)P(V/A/T)(gS/gT), whereby phosphorylation strongly inhibits O-GlcNAcylation. Strikingly, this stringent motif is substantially enriched in the human (phospho)proteome, allowing us to predict hundreds of putative O-GlcNAc transferase (OGT) substrates. A set of these we investigate further and show them to be decent substrates of OGT, exhibiting a negative feedback loop when phosphorylated at the P-3 site. Second, we demonstrate that reciprocal crosstalk does not occur at PX(S/T)P sites, i.e., at sites phosphorylated by proline-directed kinases, which represent 40% of all sites in the vertebrate phosphoproteomes.


Subject(s)
Phosphorylation/physiology , Protein Processing, Post-Translational/physiology , Acetylglucosamine/metabolism , Amino Acid Motifs/physiology , Amino Acid Sequence , Glycosylation , Humans , Mass Spectrometry/methods , N-Acetylglucosaminyltransferases/metabolism , N-Acetylglucosaminyltransferases/physiology , Proline , Proteins/metabolism , Proteolysis , Serine , Signal Transduction , Threonine
7.
J Biol Chem ; 288(44): 31728-37, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-24030825

ABSTRACT

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.


Subject(s)
I-kappa B Kinase/chemistry , Multienzyme Complexes/chemistry , Ubiquitin-Protein Ligases/chemistry , Ubiquitin/chemistry , Ubiquitination/physiology , Catalysis , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
9.
Chembiochem ; 13(15): 2251-8, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-23011887

ABSTRACT

Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.


Subject(s)
Endopeptidases/metabolism , Fluorescent Dyes/chemistry , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitination , Biotin/chemistry , Biotinylation , Catalytic Domain , Cell Line, Tumor , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , Humans , Solid-Phase Synthesis Techniques
10.
Chembiochem ; 13(14): 2056-60, 2012 Sep 24.
Article in English | MEDLINE | ID: mdl-22927162

ABSTRACT

A litter of pups: The synthesis and in vitro evaluation of new Pup-based fluorogenic substrates for Dop, the mycobacterial depupylase, are described. A full-length Pup-amidomethylcoumarin conjugate as well as an amino-terminus-truncated analogue exhibited high sensitivity and specificity towards hydrolysis by Dop. The substrates developed here might find application as high-throughput screening assay reagents for the identification of Dop inhibitors.


Subject(s)
Amidohydrolases/metabolism , Bacterial Proteins/metabolism , Fluorescent Dyes/chemistry , Mycobacterium tuberculosis/drug effects , Ubiquitins/metabolism , Virulence Factors/metabolism , Amidohydrolases/antagonists & inhibitors , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Biocatalysis , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , High-Throughput Screening Assays , Hydrolysis , Kinetics , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Peptides/metabolism , Substrate Specificity , Ubiquitins/antagonists & inhibitors , Virulence Factors/antagonists & inhibitors
11.
J Med Chem ; 49(8): 2549-57, 2006 Apr 20.
Article in English | MEDLINE | ID: mdl-16610798

ABSTRACT

In an effort to establish the structural requirements for agonism, neutral antagonism, and inverse agonism at the human histamine H(3) receptor (H(3)R) we have prepared a series of higher homologues of histamine in which the terminal nitrogen of the side chain has been either mono- or disubstituted with several aliphatic, alicyclic, and aromatic moieties or incorporated in cyclic systems. The novel ligands have been pharmacologically investigated in vitro for their affinities on the human H(3)R and H(4)R subtypes by radioligand displacement experiments and for their intrinsic H(3)R activities via a CRE-mediated beta-galactosidase reporter gene assay. Subtle changes of the substitution pattern at the side chain nitrogen alter enormously the pharmacological activity of the ligands, resulting in a series of compounds with a wide spectrum of pharmacological activities. Among the several neutral H(3)R antagonists identified within this series, compounds 2b and 2h display an H(3)R affinity in the low nanomolar concentration range (pK(i) values of 8.1 and 8.4, respectively). A very potent and selective H(3)R agonist (1l, pEC(50) = 8.9, alpha = 0.94) and a very potent, though not highly selective, H(3)R inverse agonist (2k, pIC(50) = 8.9, alpha = -0.97) have been identified as well.


Subject(s)
Amines/pharmacology , Histamine/pharmacology , Receptors, Histamine H3/drug effects , Amines/chemistry , Histamine/chemical synthesis , Histamine/chemistry , Humans , Ligands , Molecular Structure , Receptors, G-Protein-Coupled/drug effects , Receptors, Histamine/drug effects , Receptors, Histamine H4 , Structure-Activity Relationship
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