Project description:Many of the best-studied actin regulatory proteins use non-covalent means to modulate the properties of actin. Yet, actin is also susceptible to covalent modifications of its amino acids. Recent work is increasingly revealing that actin processing and its covalent modifications regulate important cellular events. In addition, numerous pathogens express enzymes that specifically use actin as a substrate to regulate their hosts' cells. Actin post-translational alterations have been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight specific co-translational and post-translational modifications of actin and discuss the current understanding of the role that these modifications play in regulating actin.
Project description:DNA is highly vulnerable to spontaneous and environmental timely damage in living cells. DNA damage may cause genetic instability and increase cancer risk if the damages are not repaired timely and efficiently. Human cells possess several DNA damage response (DDR) mechanisms to protect the integrity of their genome. Clarification of the mechanisms underlying the DNA damage response following lethal damage will facilitate the identification of therapeutic targets for cancers. Histone post-translational modifications (PTMs) have been indicated to play different roles in the repair of DNA damage. In this context, histone PTMs regulate recruitment of downstream effectors, and facilitate appropriate repair response. This review outlines the current understanding of different histone PTMs in response to DNA damage repair, besides, enumerates the role of new type PTMs such as histone succinylation and crotonylation in regulating DNA damage repair processes.
Project description:Monocyclic aromatic amines are widespread environmental contaminants with multiple sources such as combustion products, pharmaceuticals, and pesticides. Their phenolic metabolites are converted intracellularly to electrophilic quinone imines upon autoxidation and can embed in the cellular matrix through a transimination reaction that leaves a redox-active residue as a substituent of lysine side-chain amino groups. To demonstrate the occurrence of this process within the cellular nucleus, Chinese hamster ovary AA8 cells were treated with the para-phenol of 3,5-dimethylamine, after which the histone proteins were isolated, derivatized, and subjected to tryptic digestion. The resulting peptides were analyzed by tandem mass spectrometry to determine which lysines were modified. Nine residues in histones H2A, H2B, and H4 were identified; these were located in histone tails, close to where DNA makes contact with the nuclear core particle, elsewhere on the protein surface, and deep within the core. Kinetics of disappearance of the modified lysines in cultured cells was determined using isotope-dilution mass spectrometry. AA8 cells were also transfected with the genetically encoded hydrogen peroxide biosensor HyPer in constructs that lead to expression of HyPer in different cellular compartments. Challenging the resulting cells with the dimethylaminophenol resulted in sustained fluorescence emission in each of the compartments, demonstrating ongoing production of H2O2. The kinetics of modified lysine loss determined by mass spectrometry was consistent with persistence of HyPer fluorescence emission. We conclude that the para-phenol of 3,5-dimethylamine can become stably integrated into the histone proteins, which are minimally repaired, if at all, and function as a persistent source of intracellular H2O2.
Project description:Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants.
Project description:BackgroundEpigenetic (including DNA and histone) modifications occur in a variety of neurological disorders. If epigenetic features of brain autopsy material are to be studied, it is critical to understand the post-mortem stability of the modifications.MethodsPig and mouse brain tissue were formalin-fixed and paraffin-embedded, or frozen after post-mortem delays of 0, 24, 48, and 72?h. Epigenetic modifications frequently reported in the literature were studied by DNA agarose gel electrophoresis, DNA methylation enzyme-linked immunosorbent assays, Western blotting, and immunohistochemistry. We constructed a tissue microarray of human neocortex samples with devitalization or death to fixation times ranging from <?60?min to 5?days.ResultsIn pig and mouse brain tissue, we found that DNA cytosine modifications (5mC, 5hmC, 5fC, and 5caC) were stable for ??72?h post-mortem. Histone methylation was generally stable for ??48?h (H3K9me2/K9me3, H3K27me2, H3K36me3) or ??72?h post-mortem (H3K4me3, H3K27me3). Histone acetylation was generally less stable. The levels of H3K9ac, H3K27ac, H4K5ac, H4K12ac, and H4K16ac declined as early as ??24?h post-mortem, while the levels of H3K14ac did not change at ??48?h. Immunohistochemistry showed that histone acetylation loss occurred primarily in the nuclei of large neurons, while immunoreactivity in glial cell nuclei was relatively unchanged. In the human brain tissue array, immunoreactivity for DNA cytosine modifications and histone methylation was stable, while subtle changes were apparent in histone acetylation at 4 to 5?days post-mortem.ConclusionWe conclude that global epigenetic studies on human post-mortem brain tissue are feasible, but great caution is needed for selection of post-mortem delay matched controls if histone acetylation is of interest.
Project description:Analyses of histone H3 from 10 rat tissues using a Middle Down proteomics platform revealed tissue-specific differences in their expression and global PTM abundance. ESI/FTMS with electron capture dissociation showed that, in general, these proteins were hypomodified in heart, liver and testes. H3.3 was hypermodified compared to H3.2 in some, but not all tissues. In addition, a novel rat testes-specific H3 protein was identified with this approach.
Project description:Histone acetylation adds an acetyl group on the lysine residue commonly found within the N-terminal tail protruding from the histone core of the nucleosome, and is important for chromosome structure and function in gene transcription and chromatin remodeling. Acetylation may also occur on other residues additional to lysine, but have not been thoroughly investigated at the proteomics level. Here we report a wide tolerance acetylation study mimicking the addition of 42 ± 0.5 Da delta mass modification on undefined amino acid residues of histones by shotgun proteomics using liquid chromatography-tandem mass spectrometry. A multi-blind spectral alignment algorithm with a wide peptide tolerance revealed frequent occurrence of 42 ± 0.5 Da modifications at lysine (K), serine (S) and threonine (T) residues in human histones from kidney tissues. Precision delta mass analysis identified acetylation (42.011 ± 0.004 Da) and trimethylation (42.047 ± 0.002 Da) modifications within the delta mass range. A specific antibody was produced to validate the acetylated T22 of human histone H3 (H3T22ac) by immune assays. Thus, we demonstrated that the wide tolerance acetylation approach identified histone acetylation as well as modification variants commonly associated with acetylation at undefined residues additional to lysine.
Project description:Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.
Project description:BackgroundThe concept that individual traits can be acquired and transmitted by the germline through epigenetic mechanisms has gained recognition in the past years. However, epigenetic marks in sperm have not been are not well identified.ResultsUsing a novel proteomic approach that combines peptide-based bottom-up and intact protein top-down tandem mass spectrometry, we report the identification of epigenetic marks on histones and protamines in adult mouse sperm. We identified a total of 26 post-translational modifications (PTMs) on specific residues of the core histones H2B, H3 and H4, and the linker histone H1, four of which had not been described previously in any tissue or cell line. We also detected 11 novel PTMs on the protamines PRM1 and PRM2 and observed that they are present in specific combinations on individual protamines.ConclusionsBoth histones and protamines carry multiple PTMs in the adult mouse sperm. On protamines, specific PTM combinations might form a 'protamine code' similar to the 'histone code'. These findings suggest a potential role for PTMs on sperm histones and protamines in epigenetic signatures underlying transgenerational inheritance.
Project description:Amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) are devastating neurodegenerative diseases involving the progressive degeneration of neurons. No cure is available for patients diagnosed with these diseases. A prominent feature of both ALS and PD is the accumulation of protein inclusions in the cytoplasm of degenerating neurons; however, the particular proteins constituting these inclusions vary: the RNA-binding proteins TDP-43 and FUS are most notable in ALS, while α-synuclein aggregates into Lewy bodies in PD. In both diseases, genetic causes fail to explain the occurrence of a large proportion of cases, and thus, both are considered mostly sporadic. Despite mounting evidence for a possible role of epigenetics in the occurrence and progression of ALS and PD, epigenetic mechanisms in the context of these diseases remain mostly unexplored. Here we comprehensively delineate histone post-translational modification (PTM) profiles in ALS and PD yeast proteinopathy models. Remarkably, we find distinct changes in histone modification profiles for each. We detect the most striking changes in the context of FUS aggregation: changes in several histone marks support a global decrease in gene transcription. We also detect more modest changes in histone modifications in cells overexpressing TDP-43 or α-synuclein. Our results highlight a great need for the inclusion of epigenetic mechanisms in the study of neurodegeneration. We hope our work will pave the way for the discovery of more effective therapies to treat patients suffering from ALS, PD, and other neurodegenerative diseases.