Changes In Post-translational Modifications Research Articles

Metabolic pathways, genomic alterations, and post-translational modifications in pulmonary hypertension and cancer as therapeutic targets and biomarkers.

Pulmonary hypertension (PH) can lead to right ventricular hypertrophy, significantly increasing mortality rates. This study aims to clarify PH-specific metabolites and their impact on genomic and post-translational modifications (PTMs) in cancer, evaluating DHA and EPA's therapeutic potential to mitigate oxidative stress and inflammation. Data from 289,365 individuals were analyzed using Mendelian randomization to examine 1,400 metabolites' causal roles in PH. Anti-inflammatory and antioxidative effects of DHA and EPA were tested in RAW 264.7 macrophages and cancer cell lines (A549, HCT116, HepG2, LNCaP). Genomic features like CNVs, DNA methylation, tumor mutation burden (TMB), and PTMs were analyzed. DHA and EPA's effects on ROS production and cancer cell proliferation were assessed. We identified 57 metabolites associated with PH risk and examined key tumor-related pathways through promoter methylation analysis. DHA and EPA significantly reduced ROS levels and inflammatory markers in macrophages, inhibited the proliferation of various cancer cell lines, and decreased nuclear translocation of SUMOylated proteins during oxidative stress and inflammatory responses. These findings suggest a potential anticancer role through the modulation of stress-related nuclear signaling, as well as a regulatory function on cellular PTMs. This study elucidates metabolic and PTM changes in PH and cancer, indicating DHA and EPA's role in reducing oxidative stress and inflammation. These findings support targeting these pathways for early biomarkers and therapies, potentially improving disease management and patient outcomes.

Characterizing age-related changes in intact mitochondrial proteoforms in murine hearts using quantitative top-down proteomics

BackgroundCardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence of CVDs increases markedly with age. Due to the high energetic demand, the heart is highly sensitive to mitochondrial dysfunction. The complexity of the cardiac mitochondrial proteome hinders the development of effective strategies that target mitochondrial dysfunction in CVDs. Mammalian mitochondria are composed of over 1000 proteins, most of which can undergo post-translational modifications (PTMs). Top-down proteomics is a powerful technique for characterizing and quantifying proteoform sequence variations and PTMs. However, there are still knowledge gaps in the study of age-related mitochondrial proteoform changes using this technique. In this study, we used top-down proteomics to identify intact mitochondrial proteoforms in young and old hearts and determined changes in protein abundance and PTMs in cardiac aging.MethodsIntact mitochondria were isolated from the hearts of young (4-month-old) and old (24-25-month-old) mice. The mitochondria were lysed, and mitochondrial lysates were subjected to denaturation, reduction, and alkylation. For quantitative top-down analysis, there were 12 runs in total arising from 3 biological replicates in two conditions, with technical duplicates for each sample. The collected top-down datasets were deconvoluted and quantified, and then the proteoforms were identified.ResultsFrom a total of 12 LC-MS/MS runs, we identified 134 unique mitochondrial proteins in the different sub-mitochondrial compartments (OMM, IMS, IMM, matrix). 823 unique proteoforms in different mass ranges were identified. Compared to cardiac mitochondria of young mice, 7 proteoforms exhibited increased abundance and 13 proteoforms exhibited decreased abundance in cardiac mitochondria of old mice. Our analysis also detected PTMs of mitochondrial proteoforms, including N-terminal acetylation, lysine succinylation, lysine acetylation, oxidation, and phosphorylation. Data are available via ProteomeXchange with the identifier PXD051505.ConclusionBy combining mitochondrial protein enrichment using mitochondrial fractionation with quantitative top-down analysis using ultrahigh-pressure liquid chromatography (UPLC)-MS and label-free quantitation, we successfully identified and quantified intact proteoforms in the complex mitochondrial proteome. Using this approach, we detected age-related changes in abundance and PTMs of mitochondrial proteoforms in the heart.

Effects of P301L-TAU on post-translational modifications of microtubules in human iPSC-derived cortical neurons and TAU transgenic mice.

JOURNAL/nrgr/04.03/01300535-202508000-00025/figure1/v/2024-09-30T120553Z/r/image-tiff TAU is a microtubule-associated protein that promotes microtubule assembly and stability in the axon. TAU is missorted and aggregated in an array of diseases known as tauopathies. Microtubules are essential for neuronal function and regulated via a complex set of post-translational modifications, changes of which affect microtubule stability and dynamics, microtubule interaction with other proteins and cellular structures, and mediate recruitment of microtubule-severing enzymes. As impairment of microtubule dynamics causes neuronal dysfunction, we hypothesize cognitive impairment in human disease to be impacted by impairment of microtubule dynamics. We therefore aimed to study the effects of a disease-causing mutation of TAU (P301L) on the levels and localization of microtubule post-translational modifications indicative of microtubule stability and dynamics, to assess whether P301L-TAU causes stability-changing modifications to microtubules. To investigate TAU localization, phosphorylation, and effects on tubulin post-translational modifications, we expressed wild-type or P301L-TAU in human MAPT -KO induced pluripotent stem cell-derived neurons (iNeurons) and studied TAU in neurons in the hippocampus of mice transgenic for human P301L-TAU (pR5 mice). Human neurons expressing the longest TAU isoform (2N4R) with the P301L mutation showed increased TAU phosphorylation at the AT8, but not the p-Ser-262 epitope, and increased polyglutamylation and acetylation of microtubules compared with endogenous TAU-expressing neurons. P301L-TAU showed pronounced somatodendritic presence, but also successful axonal enrichment and a similar axodendritic distribution comparable to exogenously expressed 2N4R-wildtype-TAU. P301L-TAU-expressing hippocampal neurons in transgenic mice showed prominent missorting and tauopathy-typical AT8-phosphorylation of TAU and increased polyglutamylation, but reduced acetylation, of microtubules compared with non-transgenic littermates. In sum, P301L-TAU results in changes in microtubule PTMs, suggestive of impairment of microtubule stability. This is accompanied by missorting and aggregation of TAU in mice but not in iNeurons. Microtubule PTMs/impairment may be of key importance in tauopathies.

The Impacts of Osmotic Stress During the Critical Window of Development on Protein and Histone PTM Alterations in California grunion

The California Grunion, Leuresthes tenuis, can experience a broad range of salinities during their early life stages, with their nursery grounds measuring salinities of 21 to 42ppt under normal conditions. Due to the unique subterrestrial incubation of their eggs within the sand of California beaches, it is not unlikely for this marine fish to hatch in non-seawater conditions, whereafter the larvae may transition to relatively diluted estuaries or concentrated harbors to develop as juveniles for several months. Consequently, they are an ideal system to study the impacts of ecologically relevant salinity stress on the alteration of proteins and histone post-translational modifications (PTMs) during early development. The study of the relationship between the critical window of development (CWD), in which sexual bipotential is lost, and the longevity of protein landscape and histone PTM changes has not been widely explored. The CWD is of interest as this is when the gametes of this species become set. Therefore, we hypothesize that histone PTM changes that occur during this time may thus be a form of heritable epigenetics. Thus, hypo-and-hyperosmotic exposures of L. tenuis hatchlings during this window, and past it, attempt to identify if the CWD is relevant to the presence and persistence of protein and histone PTM changes under salinity stress. We hypothesize that exposure to osmotic stress during the CWD will result in induced compensatory mechanisms in the protein landscape of the California grunion larvae, as well as persistent alterations in the histone PTMs in the larvae. To elucidate the relationship between these factors, California grunion larvae were exposed in replicate to one of three chronic salinity treatments (16ppt, 32ppt, or 40ppt) for the duration of their CWD (68dph), with 16ppt and 40ppt inducing hypoosmotic and hyperosmotic stress respectively. Chronic post-CWD exposures and recovery times until 98dph were used to determine whether the CWD or prolonged salinity exposures were key to alterations in the histone PTM and protein landscape of the larvae. After each timepoint, L. tenuis were culled and processed for histone PTM enrichment and proteomic analysis. At the time of culling, there were no significant differences in survivorship between replicates and experimental groups, with statistically significant different deaths between groups occurring only shortly after hatching. Skyline and MSstats were used in the analysis of the statistical significance of differential regulation of proteins and histone PTMs under stress conditions versus the controls. Of special interest is the osmoregulatory mechanisms involved in the myo-inositol biosynthesis pathway, with analysis focusing on the abundance of histone acetylation, amidation, ubiquitylation, and 4-hydroxynonelation in histone PTMs, as the accumulations of these have been found to be correlated with osmotic stress in some fish tissues. Funding provided by NSF IOS-2209383 and NSF GRFP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Monitoring phosphorylation and acetylation of CRISPR-mediated HiBiT-tagged endogenous proteins

Intracellular pathways transduce signals through changes in post-translational modifications (PTMs) of effector proteins. Among the approaches used to monitor PTM changes are immunoassays and overexpression of recombinant reporter genes. Genome editing by CRISPR/Cas9 provides a new means to monitor PTM changes by inserting reporters onto target endogenous genes while preserving native biology. Ideally, the reporter should be small in order not to interfere with the processes mediated by the target while sensitive enough to detect tightly expressed proteins. HiBiT is a 1.3 kDa reporter peptide capable of generating bioluminescence through complementation with LgBiT, an 18 kDa subunit derived from NanoLuc. Using HiBiT CRISPR/Cas9-modified cell lines in combination with fluorescent antibodies, we developed a HiBiT-BRET immunoassay (a.k.a. Immuno-BRET). This is a homogeneous immunoassay capable of monitoring post-translational modifications on diverse protein targets. Its usefulness was demonstrated for the detection of phosphorylation of multiple signaling pathway targets (EGFR, STAT3, MAPK8 and c-MET), as well as chromatin containing histone H3 acetylation on lysine 9 and 27. These results demonstrate the ability to efficiently monitor endogenous biological processes modulated by post-translational modifications using a small bioluminescent peptide tag and fluorescent antibodies, providing sensitive quantitation of the response dynamics to multiple stimuli.

Palmitic Acid Induces Posttranslational Modifications of Tau Protein in Alzheimer’s Disease–Related Epitopes and Increases Intraneuronal Tau Levels

Metabolic diseases derived from an unhealthy lifestyle have been linked with an increased risk for developing cognitive impairment and even Alzheimer’s disease (AD). Although high consumption of saturated fatty acids such as palmitic acid (PA) has been associated with the development of obesity and type II diabetes, the mechanisms connecting elevated neuronal PA levels and increased AD marker expression remain unclear. Among other effects, PA induces insulin resistance, increases intracellular calcium and reactive oxygen species (ROS) production, and reduces the NAD+/NADH ratio, resulting in decreased activity of the deacetylase Sirtuin1 (SIRT1) in neurons. These mechanisms may affect signaling pathways that impact the posttranslational modifications (PTMs) of the tau protein. To analyze the role played by PA in inducing the phosphorylation and acetylation of tau, we examined PTM changes in human tau in differentiated neurons from human neuroblastoma cells. We found changes in the phosphorylation state of several AD-related sites, namely, S199/202 and S214, that were mediated by a mechanism associated with the dysregulated activity of the kinases GSK3β and mTOR. PA also increased the acetylation of residue K280 and elevated total tau level after long exposure time. These findings provide information about the mechanisms by which saturated fatty acids cause tau PTMs that are similar to those observed in association with AD biochemical changes.Graphical

The interplay of post-translational protein modifications in Arabidopsis leaves during photosynthesis induction.

Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM) and have been studied at depth at the level of individual proteins. In contrast, a global picture of the light-dependent PTMome dynamics is lacking, leaving the response of a large proportion of cellular function undefined. Here, we investigated the light-dependent metabolome and proteome changes in Arabidopsis rosettes in a time resolved manner to dissect their kinetic interplay, focusing on phosphorylation, lysine acetylation, and cysteine-based redox switches. Of over 24 000 PTM sites that were detected, more than 1700 were changed during the transition from dark to light. While the first changes, as measured 5 min after onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveal connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insight into the extent by which photosynthesis reprograms global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.

Epigenetic dysregulation from chromosomal transit in micronuclei

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1–4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Acidotic and hypoxic tumor microenvironment induces changes to histone acetylation and methylation in oral squamous cell carcinoma.

Hypoxia and acidosis are ubiquitous hallmarks of the tumor microenvironment (TME), and in most solid cancers they have been linked to rewired cancer cell metabolism. These TME stresses are linked to changes in histone post-translational modifications (PTMs) such as methylation and acetylation, which lead to tumorigenesis and drug resistance. Hypoxic and acidotic TME cause changes in histone PTMs by impacting the activities of histone-modifying enzymes. These alterations are yet to be extensively explored in oral squamous cell carcinoma (OSCC), one of the most prevalent cancers in developing countries. Hypoxic, acidotic, and hypoxia with acidotic TME affecting histone acetylation and methylation in the CAL27 OSCC cell line was studied using LC-MS-based proteomics. The study identified several well-known histone marks, in the context of their functionality in gene regulation, such as H2AK9Ac, H3K36me3, and H4K16Ac. The results provide insights into the histone acetylation and methylation associated with hypoxic and acidotic TME, causing changes in their level in a position-dependent manner in the OSCC cell line. Hypoxia and acidosis, separately and in combination, cause differential impacts on histone methylation and acetylation in OSCC. The work will helpuncovertumor cell adaptation to these stress stimuli in connection with histone crosstalk events.

Protein post-translational modification in SARS-CoV-2 and host interaction.

SARS-CoV-2 can cause lung diseases, such as pneumonia and acute respiratory distress syndrome, and multi-system dysfunction. Post-translational modifications (PTMs) related to SARS-CoV-2 are conservative and pathogenic, and the common PTMs are glycosylation, phosphorylation, and acylation. The glycosylation of SARS-CoV-2 mainly occurs on spike (S) protein, which mediates the entry of the virus into cells through interaction with angiotensin-converting enzyme 2. SARS-CoV-2 utilizes glycans to cover its epitopes and evade the immune response through glycosylation of S protein. Phosphorylation of SARS-CoV-2 nucleocapsid (N) protein improves its selective binding to viral RNA and promotes viral replication and transcription, thereby increasing the load of the virus in the host. Succinylated N and membrane(M) proteins of SARS-CoV-2 synergistically affect virus particle assembly. N protein regulates its affinity for other proteins and the viral genome through acetylation. The acetylated envelope (E) protein of SARS-CoV-2 interacts with bromodomain-containing protein 2/4 to influence the host immune response. Both palmitoylation and myristoylation sites on S protein can affect the virus infectivity. Papain-like protease is a domain of NSP3 that dysregulates host inflammation by deubiquitination and impinges host IFN-I antiviral immune responses by deISGylation. Ubiquitination of ORF7a inhibits host IFN-α signaling by blocking STAT2 phosphorylation. The methylation of N protein can inhibit the formation of host stress granules and promote the binding of N protein to viral RNA, thereby promoting the production of virus particles. NSP3 macrodomain can reverse the ADP-ribosylation of host proteins, and inhibit the cascade immune response with IFN as the core, thereby promoting the intracellular replication of SARS-CoV-2. On the whole, PTMs have fundamental roles in virus entry, replication, particle assembly, and host immune response. Mutations in various SARS-CoV-2 variants, which lead to changes in PTMs at corresponding sites, cause different biological effects. In this paper, we mainly reviewed the effects of PTMs on SARS-CoV-2 and host cells, whose application is to inform the strategies for inhibiting viral infection and facilitating antiviral treatment and vaccine development for COVID-19.

TiCPG - a strategy for the simultaneous enrichment of reversibly modified cysteine peptides, phosphopeptides, and sialylated N-Glycopeptides to study cytokines stimulated beta-cells

Diverse post-translational modifications (PTMs) regulate protein function and interaction to fine-tune biological processes. Reversible phosphorylation, cysteines (Cys) modifications, and N-linked glycosylation are all essentially involved in cellular signaling pathways, such as those initiated by the action of pro-inflammatory cytokines, which can induce pancreatic β-cell death and diabetes. Here we have developed a novel strategy for the simultaneous and comprehensive characterization of the proteome and three PTMs including reversibly modified Cysteines (rmCys), phosphorylation, and sialylated N-linked glycosylation from low amount of sample material. This strategy, termed TiCPG, is based on a combination of chemical labeling and titanium dioxide (TiO2) chromatography. We applied the TiCPG strategy to study the proteome and the three PTMs changes in β-cells subject to pro-inflammatory cytokines stimulation. It enabled quantitative analysis of 8346 rmCys sites, 10,321 phosphosites and 962 sialylated N-glycosites from 5496 proteins. Significant regulation was found on 100 proteins at the expression level, while 3020 PTM peptide isoforms from 1468 proteins were significantly regulated. The three PTMs were involved in cytokine mediated β-cell apoptosis, such as the NFκB and the inducible NO synthase signaling pathways. Overall, the TiCPG strategy is a cheap, straightforward, and powerful tool for studies targeting the three PTMs described above. SIGNIFICANCE: The present study presents a fast and easy method for quantitative assessment of the proteome and three PTMs from minimal amount of sample material. This simple method provides comprehensive and significant knowledge on biological systems and cellular signaling with relatively low analysis time, suitable for younger researchers and researchers that do not have direct access to LC-MSMS in their laboratories. From sub-milligram amount of material, we were able to map known cellular signaling events of proinflammatory cytokine effect on beta-cells and to discover novel PTMs involved in several known signaling pathways.

Differential Histone Posttranslational Modifications Induced by DNA Hypomethylating Agents.

IntroductionThe prototype DNA hypomethylating agents 5-azacytidine (5AC) and decitabine (DAC) are currently FDA-approved for treatment of blood and bone marrow disorders like myelodysplastic syndrome. 5AC and DAC are considered similar drugs and were shown to induce histone modifications that modulate gene expression. The aim of this study is to compare the effect of both drugs on histone acetylation and methylation at multiple histone amino acids residues.MethodsMass spectrometry was used to compare the effect of both drugs on 95 different histone posttranslational modifications (PTMs) in leukemia cells. ChIP-Seq analysis was used to compare the impact of both drugs on the genome-wide acetylation of the H3K9 mark using primary leukemia cells from six de-identified AML patients.ResultsBoth DAC and 5AC induced histone PTMs in different histone isoforms like H1.4, H2A, H3, H3.1, and H4. Changes in both histone methylation and acetylation were observed with both drugs; however, there were distinct differences in the histone modifications induced by the two drugs. Since both drugs were shown to increase the activity of the HDAC SIRT6 previously, we tested the effect of 5AC on the acetylation of H3K9, the physiological substrate SIRT6, using ChIP-Seq analysis and compared it to the previously published DAC-induced changes. Significant H3K9 acetylation changes (P< .05) were detected at 925 genes after 5AC treatment vs only 182 genes after DAC treatment. Nevertheless, the gene set modified by 5AC was different from that modified by DAC with only ten similar genes modulated by both drugs.ConclusionDespite similarity in chemical structure and DNA hypomethylating activity, 5AC and DAC induced widely different histone PTMs and considering them interchangeable should be carefully evaluated. The mechanism of these histone PTM changes is not clear and may involve modulation of the activity or the expression of the enzymes inducing histone PTMs.

Post-translational Modification in Muscular Dystrophies.

Muscular dystrophies are a complex group of inherited neuromuscular disorders that progressively lead to a loss of muscle fibers and mobility and muscle weakness; over time, they evolve to an increasing level of disability. Muscular dystrophies are mostly caused by genetic mutations in proteins responsible for maintaining sarcolemma structures, such as an absence or reductions of dystrophin expression, conditions which are strictly related to muscular disorders that affect most people with this disease. Along the years, with the recent advances in the understanding of muscular dystrophies, it has been shown that many changes in Post-Translational Modifications (PTMs) of muscle proteins are associated with muscular dystrophies, wherein pathogenic alterations in the modulation of these muscle proteins are directly related to the incidence of this disease. An increase in the identification of the genetic bases and molecular mechanisms involved in the most common form of muscular dystrophies, including PTMs changes, holds potential to develop new therapeutic approaches. In this chapter we will describe the most common muscular dystrophies and changes in PTM processes such as phosphorylation and glycosylation that are very important in the evolution of the disease, highlighting the lack of mass spectrometry-based (MS-based) studies of these PTMs, suggesting that the application of this technique could reveal important informations about the molecular mechanisms of muscular dystrophies.

Quantitative Proteome and PTMome Analysis of Arabidopsis thaliana Root Responses to Persistent Osmotic and Salinity Stress.

Abiotic stresses such as drought result in large annual economic losses around the world. As sessile organisms, plants cannot escape the environmental stresses they encounter but instead must adapt to survive. Studies investigating plant responses to osmotic and/or salt stress have largely focused on short-term systemic responses, leaving our understanding of intermediate to longer-term adaptation (24 h to d) lacking. In addition to protein abundance and phosphorylation changes, evidence suggests reversible lysine acetylation may also be important for abiotic stress responses. Therefore, to characterize the protein-level effects of osmotic and salt stress, we undertook a label-free proteomic analysis of Arabidopsis thaliana roots exposed to 300 mM mannitol and 150 mM NaCl for 24 h. We assessed protein phosphorylation, lysine acetylation and changes in protein abundance, detecting significant changes in 245, 35 and 107 total proteins, respectively. Comparison with available transcriptome data indicates that transcriptome- and proteome-level changes occur in parallel, while post-translational modifications (PTMs) do not. Further, we find significant changes in PTMs, and protein abundance involve different proteins from the same networks, indicating a multifaceted regulatory approach to prolonged osmotic and salt stress. In particular, we find extensive protein-level changes involving sulfur metabolism under both osmotic and salt conditions as well as changes in protein kinases and transcription factors that may represent new targets for drought stress signaling. Collectively, we find that protein-level changes continue to occur in plant roots 24 h from the onset of osmotic and salt stress and that these changes differ across multiple proteome levels.

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages.

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.

Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

Role of Post-translational Modifications in Influenza A Virus Life Cycle and Host Innate Immune Response.

Throughout various stages of its life cycle, influenza A virus relies heavily on host cellular machinery, including the post-translational modifications (PTMs) system. During infection, influenza virus interacts extensively with the cellular PTMs system to aid in its successful infection and dissemination. The complex interplay between viruses and the PTMs system induces global changes in PTMs of the host proteome as well as modifications of specific host or viral proteins. The most common PTMs include phosphorylation, ubiquitination, SUMOylation, acetylation, methylation, NEDDylation, and glycosylation. Many PTMs directly support influenza virus infection, whereas others contribute to modulating antiviral responses. In this review, we describe current knowledge regarding the role of PTMs in different stages of the influenza virus replication cycle. We also discuss the concerted role of PTMs in antagonizing host antiviral responses, with an emphasis on their impact on viral pathogenicity and host range.

Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle

Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite. SIGNIFICANCE: Trypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.

Comparative Proteomic Analysis of Histone Modifications upon Acridone Derivative 8a-Induced CCRF-CEM Cells by Data Independent Acquisition.

The lead compound acridone derivative 8a showed potent antiproliferative activity by inducing DNA damage through direct stacking with DNA bases and triggering ROS in CCRF-CEM cells. To define the chromatin alterations during DNA damage sensing and repair, a detailed quantitative map of single and coexisting histone post-translational modifications (PTMs) in CCRF-CEM cells affected by 8a was performed by the Data Independent Acquisition (DIA) method on QE-plus. A total of 79 distinct and 164 coexisting histone PTMs were quantified, of which 16 distinct histone PTMs were significantly altered when comparing 8a-treated cells with vehicle control cells. The changes in histone PTMs were confirmed by Western blotting analysis for three H3 and one H4 histone markers. The up-regulated dimethylation on H3K9, H3K36, and H4K20 implied that CCRF-CEM cells might accelerate DNA damage repair to counteract the DNA lesion induced by 8a, which was verified by an increment in the 53BP1 foci localization at the damaged DNA. Most of the significantly altered PTMs were involved in transcriptional regulation, including down-regulated acetylation on H3K18, H3K27, and H3K122, and up-regulated di- and trimethylation on H3K9 and H3K27. This transcription-silencing phenomenon was associated with G2/M cell cycle arrest after 8a treatment by flow cytometry. This study shows that the DIA proteomics strategy provides a sensitive and accurate way to characterize the coexisting histone PTMs changes and their cross-talk in CCRF-CEM cells after 8a treatment. Specifically, histone PTMs rearrange transcription-silencing, and cell cycle arrest DNA damage repair may contribute to the mechanism of epigenetic response affected by 8a.

A post-translational modification signature defines changes in soluble tau correlating with oligomerization in early stage Alzheimer\u2019s disease brain

Tau is a microtubule-binding protein that can receive various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Hyperphosphorylation of tau is linked to its aggregation and the formation of neurofibrillary tangles (NFTs), which are a hallmark of Alzheimer’s disease (AD). While more than 70 phosphorylation sites have been detected previously on NFT tau, studies of oligomeric and detergent-soluble tau in human brains during the early stages of AD are lacking. Here we apply a comprehensive electrochemiluminescence ELISA assay to analyze twenty-five different PTM sites as well as tau oligomerization in control and sporadic AD brain. The samples were classified as Braak stages 0–I, II or III–IV, corresponding to the progression of microscopically detectable tau pathology throughout different brain regions. We found that soluble tau multimers are strongly increased at Braak stages III–IV in all brain regions under investigation, including the temporal cortex, which does not contain NFTs or misfolded oligomers at this stage of pathology. We additionally identified five phosphorylation sites that are specifically and consistently increased across the entorhinal cortex, hippocampus and temporal cortex in the same donors. Three of these sites correlate with tau multimerization in all three brain regions, but do not overlap with the epitopes of phospho-sensitive antibodies commonly used for the immunohistochemical detection of NFTs. Our results thus suggest that soluble multimers are characterized by a small set of specific phosphorylation events that differ from those dominating in mature NFTs. These findings shed light on early PTM changes of tau during AD pathogenesis in human brains.