scholarly journals Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jugal Mohapatra ◽  
Kyuto Tashiro ◽  
Ryan L Beckner ◽  
Jorge Sierra ◽  
Jessica A Kilgore ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Protein Function ◽  
Post Translational Modification ◽  
Site Specific ◽  
Precise Control ◽  
Protein Ligation ◽  
Adp Ribosylation ◽  
Nuclear Extracts ◽  
Synthesis Strategy ◽  
The Impact

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

scholarly journals Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling

2021 ◽  
Author(s):  
Jugal Mohapatra ◽  
Kyuto Tashiro ◽  
Ryan L Beckner ◽  
Jorge Sierra ◽  
Jessica A Kilgore ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Remodeling ◽  
Protein Function ◽  
Post Translational Modification ◽  
Site Specific ◽  
Precise Control ◽  
Adp Ribosylation ◽  
Nuclear Extracts ◽  
Biochemical Function ◽  
The Impact

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto full-length proteins with precise control over chain length and modification site. A series of semi-synthetic ADP-ribosylated histone proteins are prepared which demonstrate that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. Importantly, we found ALC1 selectively remodels "activated" substrates within heterogeneous nucleosome populations and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a method that is broadly applicable to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

scholarly journals Salicylaldehyde ester-mediated protein semi-synthesis enables studies on the tetra-acetylation of HMGB1

2021 ◽  
Author(s):  
Tongyao Wei ◽  
Jiamei Liu ◽  
Yi Tan ◽  
Ruohan Wei ◽  
Jinzheng Wang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
High Purity ◽  
Protein Function ◽  
Protein A ◽  
High Mobility ◽  
High Mobility Group ◽  
Biological Study ◽  
Post Translational Modifications ◽  
Site Specific ◽  
Synthesis Strategy

To answer how protein post-translational modifications (PTMs) affect protein function, conformation, sta-bility, localization and interaction with binders remains important in the biological study. However, the re-lated study has been dramatically hindered by the difficulty in obtaining homogenous proteins with site-specific PTMs of interest. Herein, we introduce a protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation). This methodology has enabled us to generate Lys (2/6/7/11) tetra-acetylated HMGB1 (high-mobility group box 1) protein, a 25 kDa proin-flammatory protein, in high purity. Further studies revealed that the tetra-acetylation may represent a regu-latory switch to control the HMGB1 signaling pathway by abolishing its interaction with lipopolysaccha-ride (LPS) and accelerating its degradation, consequently preventing cells from pyroptosis and lethality upon infectious injury.

scholarly journals ADP-ribosylation signalling and human disease

Open Biology ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 190041 ◽  
Author(s):  
Luca Palazzo ◽  
Petra Mikolčević ◽  
Andreja Mikoč ◽  
Ivan Ahel
Keyword(s):  
Neurological Disorders ◽  
Fine Tuning ◽  
Molecular Medicine ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Adp Ribosylation ◽  
Damage Repair ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
The Impact

ADP-ribosylation (ADPr) is a reversible post-translational modification of proteins, which controls major cellular and biological processes, including DNA damage repair, cell proliferation and differentiation, metabolism, stress and immune responses. In order to maintain the cellular homeostasis, diverse ADP-ribosyl transferases and hydrolases are involved in the fine-tuning of ADPr systems. The control of ADPr network is vital, and dysregulation of enzymes involved in the regulation of ADPr signalling has been linked to a number of inherited and acquired human diseases, such as several neurological disorders and in cancer. Conversely, the therapeutic manipulation of ADPr has been shown to ameliorate several disorders in both human and animal models. These include cardiovascular, inflammatory, autoimmune and neurological disorders. Herein, we summarize the recent findings in the field of ADPr, which support the impact of this modification in human pathophysiology and highlight the curative potential of targeting ADPr for translational and molecular medicine.

scholarly journals Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0254022
Author(s):  
Samuel Kasson ◽  
Nuwani Dharmapriya ◽  
In-Kwon Kim
Keyword(s):  
Small Molecule ◽  
Stress Responses ◽  
Proof Of Concept ◽  
Post Translational Modification ◽  
Site Specific ◽  
Adp Ribosylation ◽  
Small Molecule Probes ◽  
Cellular Stress Responses ◽  
Specific Activities ◽  
Selective Monitoring

ADP-ribosylation is a key post-translational modification that regulates a wide variety of cellular stress responses. The ADP-ribosylation cycle is maintained by writers and erasers. For example, poly(ADP-ribosyl)ation cycles consist of two predominant enzymes, poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolase (PARG). However, historically, mechanisms of erasers of ADP-ribosylations have been understudied, primarily due to the lack of quantitative tools to selectively monitor specific activities of different ADP-ribosylation reversal enzymes. Here, we developed a new NUDT5-coupled AMP-Glo (NCAG) assay to specifically monitor the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes. We found that NUDT5 selectively cleaves protein-free ADP-ribose, but not protein-bound poly- and mono-ADP-ribosylations, protein-free poly(ADP-ribose) chains, or NAD+. As a proof-of-concept, we successfully measured the kinetic parameters for the exo-glycohydrolase activity of PARG, which releases monomeric ADP-ribose, and monitored activities of site-specific mono-ADP-ribosyl-acceptor hydrolases, such as ARH3 and TARG1. This NCAG assay can be used as a general platform to study the mechanisms of diverse ADP-ribosylation reversal enzymes that release protein-free ADP-ribose as a product. Furthermore, this assay provides a useful tool to identify small-molecule probes targeting ADP-ribosylation metabolism and to quantify ADP-ribose concentrations in cells.

scholarly journals Exploring absent protein function in yeast: assaying post translational modification and human genetic variation

2021 ◽  
Vol 8 (8) ◽  
pp. 164-183
Author(s):  
Christina S. Moesslacher ◽  
Johanna M. Kohlmayr ◽  
Ulrich Stelzl
Keyword(s):  
Protein Function ◽  
Model Organism ◽  
Global Scale ◽  
Test System ◽  
Post Translational Modification ◽  
Protein Activity ◽  
Protein Modifications ◽  
In Vivo Test ◽  
The Impact

Yeast is a valuable eukaryotic model organism that has evolved many processes conserved up to humans, yet many protein functions, including certain DNA and protein modifications, are absent. It is this absence of protein function that is fundamental to approaches using yeast as an in vivo test system to investigate human proteins. Functionality of the heterologous expressed proteins is connected to a quantitative, selectable phenotype, enabling the systematic analyses of mechanisms and specificity of DNA modification, post-translational protein modifications as well as the impact of annotated cancer mutations and coding variation on protein activity and interaction. Through continuous improvements of yeast screening systems, this is increasingly carried out on a global scale using deep mutational scanning approaches. Here we discuss the applicability of yeast systems to investigate absent human protein function with a specific focus on the impact of protein variation on protein-protein interaction modulation.

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

The Impact of Bioconjugation on the Interfacial Activity of a Protein Biosurfactant

2018 ◽  
Author(s):  
Hossam H Tayeb ◽  
Marina Stienecker ◽  
Anton Middelberg ◽  
Frank Sainsbury
Keyword(s):  
Polyethylene Glycol ◽  
Colloidal Stability ◽  
Point Mutations ◽  
Pharmaceutical Formulations ◽  
Industrial Processes ◽  
Parent Molecule ◽  
Site Specific ◽  
Interfacial Activity ◽  
Surface Active ◽  
The Impact

Biosurfactants, are surface active molecules that can be produced by renewable, industrially scalable biologic processes. DAMP4, a designer biosurfactant, enables the modification of interfaces via genetic or chemical fusion to functional moieties. However, bioconjugation of addressable amines introduces heterogeneity that limits the precision of functionalization as well as the resolution of interfacial characterization. Here we designed DAMP4 variants with cysteine point mutations to allow for site-specific bioconjugation. The DAMP4 variants were shown to retain the structural stability and interfacial activity characteristic of the parent molecule, while permitting efficient and specific conjugation of polyethylene glycol (PEG). PEGylation results in a considerable reduction on the interfacial activity of both single and double mutants. Comparison of conjugates with one or two conjugation sites shows that both the number of conjugates as well as the mass of conjugated material impacts the interfacial activity of DAMP4. As a result, the ability of DAMP4 variants with multiple PEG conjugates to impart colloidal stability on peptide-stabilized emulsions is reduced. We suggest that this is due to constraints on the structure of amphiphilic helices at the interface. Specific and efficient bioconjugation permits the exploration and investigation of the interfacial properties of designer protein biosurfactants with molecular precision. Our findings should therefore inform the design and modification of biosurfactants for their increasing use in industrial processes, and nutritional and pharmaceutical formulations.

scholarly journals Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chun-Song Yang ◽  
Kasey Jividen ◽  
Teddy Kamata ◽  
Natalia Dworak ◽  
Luke Oostdyk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Prostate Cancer Cells ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Complex Assembly ◽  
Adp Ribosylation ◽  
Signaling Axis ◽  
Regulate Protein ◽  
Protein Complex Assembly

AbstractAndrogen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

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