Quantitative Proteomics and Phosphoproteomics Supports a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis

Summary/AbstractProtein phosphorylation is one of the most prevalent post-translational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-Like Kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation, and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.

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The Intra- and Extra-Telomeric Role of TRF2 in the DNA Damage Response

International Journal of Molecular Sciences ◽

10.3390/ijms22189900 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9900

Author(s):

Siti A. M. Imran ◽

Muhammad Dain Yazid ◽

Wei Cui ◽

Yogeswaran Lokanathan

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Response Factors ◽

Telomere Repeat ◽

Protection Mechanism ◽

Dna Instability ◽

Binding Factor ◽

Damage Response ◽

Dna Break

Telomere repeat binding factor 2 (TRF2) has a well-known function at the telomeres, which acts to protect the telomere end from being recognized as a DNA break or from unwanted recombination. This protection mechanism prevents DNA instability from mutation and subsequent severe diseases caused by the changes in DNA, such as cancer. Since TRF2 actively inhibits the DNA damage response factors from recognizing the telomere end as a DNA break, many more studies have also shown its interactions outside of the telomeres. However, very little has been discovered on the mechanisms involved in these interactions. This review aims to discuss the known function of TRF2 and its interaction with the DNA damage response (DDR) factors at both telomeric and non-telomeric regions. In this review, we will summarize recent progress and findings on the interactions between TRF2 and DDR factors at telomeres and outside of telomeres.

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A Survey of Reported Disease-Related Mutations in the MRE11-RAD50-NBS1 Complex

Cells ◽

10.3390/cells9071678 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1678 ◽

Cited By ~ 2

Author(s):

Samiur Rahman ◽

Marella D. Canny ◽

Tanner A. Buschmann ◽

Michael P. Latham

Keyword(s):

Genomic Stability ◽

Nijmegen Breakage Syndrome ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Response Factors ◽

Strand Breaks ◽

Mrn Complex ◽

Damage Response ◽

Biochemical Level

The MRE11-RAD50-NBS1 (MRN) protein complex is one of the primary vehicles for repairing DNA double strand breaks and maintaining the genomic stability within the cell. The role of the MRN complex to recognize and process DNA double-strand breaks as well as signal other damage response factors is critical for maintaining proper cellular function. Mutations in any one of the components of the MRN complex that effect function or expression of the repair machinery could be detrimental to the cell and may initiate and/or propagate disease. Here, we discuss, in a structural and biochemical context, mutations in each of the three MRN components that have been associated with diseases such as ataxia telangiectasia-like disorder (ATLD), Nijmegen breakage syndrome (NBS), NBS-like disorder (NBSLD) and certain types of cancers. Overall, deepening our understanding of disease-causing mutations of the MRN complex at the structural and biochemical level is foundational to the future aim of treating diseases associated with these aberrations.

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Peptidomics analysis of potato protein hydrolysates: post-translational modifications and peptide hydrophobicity

10.26434/chemrxiv.5664853 ◽

2017 ◽

Author(s):

Shixiang Yao ◽

Chibuike Udenigwe

Keyword(s):

Protein Function ◽

Structural Changes ◽

Protein Hydrolysates ◽

Proteolytic Processing ◽

Methionine Oxidation ◽

Potato Protein ◽

Post Translational Modifications ◽

Peptide Hydrophobicity ◽

Proteins And Peptides

Post-translational modifications (PTMs) often <a></a><a>occur in proteins</a> and play a regulatory role in protein function. However, the role of PTMs in food-derived peptides remains largely unknown. The shotgun peptidomics strategy was employed to identify PTMs in peptides from potato protein hydrolysates. Various hydrophobicity-inducing PTMs were found to be located in different potato peptides, <i>e.g</i>. acetylation of lysine, N-terminal of proteins and peptides, C-terminal amidation, asparagine/glutamine deamidaiton, methylation and trimethylation, methionine oxidation, and N-terminal pyro-glutamate formation. Some of the PTMs are likely formed by chemical reactions that occur during isolation and proteolytic processing of potato proteins. The PTMs enhance peptide hydrophobicity, which can improve bioactivity, decrease solubility and increase the bitterness of peptides. This is the first report that food-derived peptides are widely modified by various PTMs associated with hydrophobicity-inducing structural changes. This finding will enhance understanding of the behaviour of bioactive peptides in biological matrices.

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Phosphoproteomic Analysis of Spiroplasma eriocheiris and Crosstalk with Acetylome Reveals the Role of Post-Translational Modifications in Metabolism

Current Proteomics ◽

10.2174/1570164617666191017140456 ◽

2020 ◽

Vol 17 (5) ◽

pp. 392-403

Author(s):

Peng Liu ◽

Libo Hou ◽

Min Liu ◽

Xuechuan Xu ◽

Qi Gao ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Function ◽

Regulatory Mechanism ◽

Arginine Deiminase ◽

Phosphorylation Sites ◽

Post Translational Modifications ◽

Protein Protein Interaction ◽

Fundamental Biological Process ◽

Cell Skeleton

Background: Post-translational modifications (PTMs) such as phosphorylation are an essential regulatory mechanism of protein function and associated with a range of biological processes beyond genome and transcriptome. Spiroplasma eriocheiris, a wall-less helical bacterium, is one of the smallest known self-replicating bacteria and a novel pathogen of freshwater crustacean. Methods: To study the physiological characteristics and regulatory mechanism of S. eriocheiris, the protein phosphorylation in the bacterium were systematically investigated by iTRAQ analyzed by LC-MS/MS. Data are available via ProteomeXchange with identifier PXD015055. Results: We identified 465 phosphorylation sites in 246 proteins involved in a broad spectrum of fundamental biological process ranging from regulation of metabolic pathways to protein synthesis. Notably, most proteins in glycolysis and all proteins in the arginine deiminase system were phosphorylated. Meanwhile, the cytoskeleton proteins (Fibril, Mrebs and ET-Tu) were all phosphorylated suggest that the phosphorylation also may play a crucial role in cell skeleton formation. We have got a lot of highly conserved proteins and phosphorylation sites by analysis, and those proteins or phosphorylation sites were mainly participated in glucose metabolism and protein synthesis. Crosstalk analysis with protein-protein interaction networks in relation to phosphorylated proteins and acetylated proteins found that the two PTMs are required for playing crucial roles in many physiological processes in S. eriocheiris. By comparing the relative positions of acetylation versus phosphorylation, we found that the two modifications often found close to proximity on the same protein. Conclusions: The results imply that there is previously unreported hidden role of phosphorylation that define the functional state of Spiroplasma.

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Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation

Nucleic Acids Research ◽

10.1093/nar/gku705 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9908-9924 ◽

Cited By ~ 23

Author(s):

Jen-Hao Yang ◽

Yi-Ying Chiou ◽

Shu-Ling Fu ◽

I-Yun Shih ◽

Tsai-Hsuan Weng ◽

...

Keyword(s):

Dna Damage ◽

Rna Processing ◽

Tumor Development ◽

Arginine Methylation ◽

Wild Type ◽

Post Translational Modifications ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Damage Response ◽

Nuclear Ribonucleoprotein

Abstract Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA-binding protein involved in chromatin remodeling, RNA processing and the DNA damage response. In addition, increased hnRNPK expression has been associated with tumor development and progression. A variety of post-translational modifications of hnRNPK have been identified and shown to regulate hnRNPK function, including phosphorylation, ubiquitination, sumoylation and methylation. However, the functional significance of hnRNPK arginine methylation remains unclear. In the present study, we demonstrated that the methylation of two essential arginines, Arg296 and Arg299, on hnRNPK inhibited a nearby Ser302 phosphorylation that was mediated through the pro-apoptotic kinase PKCδ. Notably, the engineered U2OS cells carrying an Arg296/Arg299 methylation-defective hnRNPK mutant exhibited increased apoptosis upon DNA damage. While such elevated apoptosis can be diminished through addition with wild-type hnRNPK, we further demonstrated that this increased apoptosis occurred through both intrinsic and extrinsic pathways and was p53 independent, at least in part. Here, we provide the first evidence that the arginine methylation of hnRNPK negatively regulates cell apoptosis through PKCδ-mediated signaling during DNA damage, which is essential for the anti-apoptotic role of hnRNPK in apoptosis and the evasion of apoptosis in cancer cells.

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PROTEIN DYNAMICS IN PHOSPHORYLATION-MEDIATED ALLOSTERY PROBED BY AMIDE EXCHANGE MASS SPECTROMETRY

COSMOS ◽

10.1142/s0219607713300014 ◽

2013 ◽

Vol 09 (01) ◽

pp. 19-27

Author(s):

MADHUBRATA GHOSH ◽

GANESH S. ANAND

Keyword(s):

Mass Spectrometry ◽

Protein Dynamics ◽

Protein Function ◽

Post Translational Modifications ◽

Exchange Mass Spectrometry ◽

Amide Exchange ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

And Function

A major goal of molecular biology is to correlate molecular structure with function. Since most enzymes and biological catalysts are proteins, the focus for correlating 'form' with 'function' has been entirely on protein macromolecular structure. It is obvious that any understanding of protein function must come through an understanding protein dynamics. Furthermore, all of the regulatory reactions are through changes in dynamics brought about by post-translational modifications, the most important of which is phosphorylation. This review highlights the important role of covalent phosphorylation and noncovalent phosphates in regulating allosteric effects and function through a study of protein dynamics. Mass spectrometry is a relatively new and increasingly important tool for describing protein dynamics. All examples described in this review have been studied by amide hydrogen/deuterium exchange mass spectrometry.

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Unstructured Conformations Are a Substrate Requirement for the Sir2 Family of NAD-dependent Protein Deacetylases

Journal of Biological Chemistry ◽

10.1074/jbc.m508247200 ◽

2005 ◽

Vol 280 (43) ◽

pp. 36073-36078 ◽

Cited By ~ 36

Author(s):

Ahlia N. Khan ◽

Peter N. Lewis

Keyword(s):

Protein Function ◽

Cellular Localization ◽

Substrate Recognition ◽

Histone Deacetylation ◽

Sequence Specificity ◽

Post Translational Modifications ◽

Protein Substrates ◽

Dependent Protein

The regulation of protein function is often achieved through post-translational modifications including phosphorylation, methylation, ubiquitination, and acetylation. The role of acetylation has been most extensively studied in the context of histones, but it is becoming increasingly evident that this modification now includes other proteins. The Sir2 family of NAD-dependent deacetylases was initially recognized as mediating gene silencing through histone deacetylation, but several family members display non-nuclear sub-cellular localization and deacetylate non-histone protein substrates. Although many structural and enzymatic studies of Sir2 proteins have been reported, how substrate recognition is achieved by this family of enzymes is unknown. Here we use in vitro deacetylase assays and a variety of potential substrates to examine the substrate specificity of yeast homologue Hst2. We show that Hst2 is specific for acetyl-lysine within proteins; it does not deacetylate small polycations such as acetyl-spermine or acetylated amino ter-mini of proteins. Furthermore we have found that Hst2 displays conformational rather than sequence specificity, preferentially deacetylating acetyl-lysine within unstructured regions of proteins. Our results suggest that this conformational requirement may be a general feature for substrate recognition in the Sir2 family.

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Role of electrophilic nitrated fatty acids during development and response to abiotic stress processes in plants

Journal of Experimental Botany ◽

10.1093/jxb/eraa517 ◽

2020 ◽

Author(s):

Juan C Begara-Morales ◽

Capilla Mata-Pérez ◽

Maria N Padilla ◽

Mounira Chaki ◽

Raquel Valderrama ◽

...

Keyword(s):

Fatty Acids ◽

Abiotic Stress ◽

Stress Responses ◽

Protein Function ◽

Unsaturated Fatty Acids ◽

Antioxidant Systems ◽

Post Translational Modification ◽

No Donor ◽

Initial Development

Abstract Nitro-fatty acids are generated from the interaction of unsaturated fatty acids and nitric oxide (NO)-derived molecules. The endogenous occurrence and modulation throughout plant development of nitro-linolenic acid (NO2-Ln) and nitro-oleic acid (NO2-OA) suggest a key role for these molecules in initial development stages. In addition, NO2-Ln content increases significantly in stress situations and induces the expression of genes mainly related to abiotic stress, such as genes encoding members of the heat shock response family and antioxidant enzymes. The promoter regions of NO2-Ln-induced genes are also involved mainly in stress responses. These findings confirm that NO2-Ln is involved in plant defense processes against abiotic stress conditions via induction of the chaperone network and antioxidant systems. NO2-Ln signaling capacity lies mainly in its electrophilic nature and allows it to mediate a reversible post-translational modification called nitroalkylation, which is capable of modulating protein function. NO2-Ln is a NO donor that may be involved in NO signaling events and is able to generate S-nitrosoglutathione, the major reservoir of NO in cells and a key player in NO-mediated abiotic stress responses. This review describes the current state of the art regarding the essential role of nitro-fatty acids as signaling mediators in development and abiotic stress processes.

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Autophagy: A Player in response to Oxidative Stress and DNA Damage

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/5692958 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 11

Author(s):

Serena Galati ◽

Christian Boni ◽

Maria Carla Gerra ◽

Mirca Lazzaretti ◽

Annamaria Buschini

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Unfolded Proteins ◽

Cycle Arrest ◽

Dna Damaging Agents ◽

Damage Response ◽

Cellular Stressors ◽

Precise Role ◽

Stress Accumulation

Autophagy is a catabolic pathway activated in response to different cellular stressors, such as damaged organelles, accumulation of misfolded or unfolded proteins, ER stress, accumulation of reactive oxygen species, and DNA damage. Some DNA damage sensors like FOXO3a, ATM, ATR, and p53 are known to be important autophagy regulators, and autophagy seems therefore to have a role in DNA damage response (DDR). Recent studies have partly clarified the pathways that induce autophagy during DDR, but its precise role is still not well known. Previous studies have shown that autophagy alterations induce an increase in DNA damage and in the occurrence of tumor and neurodegenerative diseases, highlighting its fundamental role in the maintenance of genomic stability. During DDR, autophagy could act as a source of energy to maintain cell cycle arrest and to sustain DNA repair activities. In addition, autophagy seems to play a role in the degradation of components involved in the repair machinery. In this paper, molecules which are able to induce oxidative stress and/or DNA damage have been selected and their toxic and genotoxic effects on the U937 cell line have been assessed in the presence of the single compounds and in concurrence with an inhibitor (chloroquine) or an inducer (rapamycin) of autophagy. Our data seem to corroborate the fundamental role of this pathway in response to direct and indirect DNA-damaging agents. The inhibition of autophagy through chloroquine had no effect on the genotoxicity induced by the tested compounds, but it led to a high increase of cytotoxicity. The induction of autophagy, through cotreatment with rapamycin, reduced the genotoxic activity of the compounds. The present study confirms the cytoprotective role of autophagy during DDR; its inhibition can sensitize cancer cells to DNA-damaging agents. The modulation of this pathway could therefore be an innovative approach able to reduce the toxicity of many compounds and to enhance the activity of others, including anticancer drugs.

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