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 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 Biallelic ADPRHL2 mutations in complex neuropathy affect ADP ribosylation and DNA damage response

2021 ◽  
Vol 4 (11) ◽  
pp. e202101057
Author(s):  
Danique Beijer ◽  
Thomas Agnew ◽  
Johannes Gregor Matthias Rack ◽  
Evgeniia Prokhorova ◽  
Tine Deconinck ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Function ◽  
Damage Control ◽  
Axonal Neuropathy ◽  
Vital Role ◽  
Adp Ribosylation ◽  
Function Characterization ◽  
Ataxia Syndrome ◽  
Protein Instability

ADP ribosylation is a reversible posttranslational modification mediated by poly(ADP-ribose)transferases (e.g., PARP1) and (ADP-ribosyl)hydrolases (e.g., ARH3 and PARG), ensuring synthesis and removal of mono-ADP-ribose or poly-ADP-ribose chains on protein substrates. Dysregulation of ADP ribosylation signaling has been associated with several neurodegenerative diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease. Recessive ADPRHL2/ARH3 mutations are described to cause a stress-induced epileptic ataxia syndrome with developmental delay and axonal neuropathy (CONDSIAS). Here, we present two families with a neuropathy predominant disorder and homozygous mutations in ADPRHL2. We characterized a novel C26F mutation, demonstrating protein instability and reduced protein function. Characterization of the recurrent V335G mutant demonstrated mild loss of expression with retained enzymatic activity. Although the V335G mutation retains its mitochondrial localization, it has altered cytosolic/nuclear localization. This minimally affects basal ADP ribosylation but results in elevated nuclear ADP ribosylation during stress, demonstrating the vital role of ADP ribosylation reversal by ARH3 in DNA damage control.

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 HPF1-dependent histone ADP-ribosylation triggers chromatin relaxation to promote the recruitment of repair factors at sites of DNA damage

2021 ◽  
Author(s):  
Rebecca Smith ◽  
Siham Zentout ◽  
Catherine Chapuis ◽  
Gyula Timinszky ◽  
Sebastien Huet
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Dna Lesions ◽  
End Joining ◽  
Adp Ribosylation ◽  
Damage Response ◽  
Non Homologous End Joining ◽  
Chromatin Relaxation ◽  
The Impact

PARP1 activity is regulated by its cofactor HPF1. The binding of HPF1 on PARP1 controls the grafting of ADP-ribose moieties on serine residues of proteins nearby the DNA lesions, mainly PARP1 and histones. However, the impact of HPF1 on DNA repair regulated by PARP1 remains unclear. Here, we show that HPF1 controls both the number and the length of the ADP-ribose chains generated by PARP1 at DNA lesions. We demonstrate that HPF1-dependent histone ADP-ribosylation, rather than auto-modification of PARP1, triggers the rapid unfolding of the chromatin structure at the DNA damage sites and promotes the recruitment of the repair factors CHD4 and CHD7. Together with the observation that HPF1 contributes to efficient repair both by homologous recombination and non-homologous end joining, our findings highlight the key roles played by this PARP1 cofactor at early stages of the DNA damage response.

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 Quantitatively profiling acetylome of DNA repair proteins in early DNA damage

2020 ◽  
Author(s):  
Shiqin Li ◽  
Lin Zhou ◽  
Xinli Liu ◽  
Bingbing Shi ◽  
Tingting Huang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Function ◽  
Critical Role ◽  
Repair Process ◽  
Post Translational Modification ◽  
Affinity Enrichment ◽  
Dna Repair Proteins ◽  
Liquid Chromatography Tandem Mass ◽  
And Function

Abstract Background：Lysine acetylation is a reversible regulated post-translational modification that can regulate the stability, localization, and function of proteins in multiple cellular processes. However, the regulative mechanism of acetylation on the repair proteins in the early DNA damage is not fully understood. Methods：We performed a global proteome and acetylome of DNA repair proteins in DNA damage in 1 h after treated with epirubicin by using high affinity enrichment and high-resolution liquid chromatography–tandem mass spectrometry approaches. Results: 190 Kac sites in 50 repair proteins were identified in cells treated with epirubicin as compared to the control. 42 acetylated lysine sites and 24 deacetylated lysine sites were observed in 21 and 16 repair proteins, respectively. 7 repair proteins simultaneously contained both acetylated and deacetylated lysine sites. 11 acetylation sites were located in the function domains of 7 repair proteins that might reveal mechanisms by which acetylations alter DDR protein function. In 17 repair proteins, the induced acetylation changes were for the first time identified in the present study. Conclusion: The proteome and acetylome results indicated that fast acetylation or deacetylation on these repair proteins might play a critical role in the early DNA damage repair process.

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.

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