Global Post-translational Modifications Research Articles

Reinforcement of repressive marks in the chicken primordial germ cell epigenetic signature: divergence from basal state resetting in mammals

BackgroundIn mammals, primordial germ cells (PGCs), the embryonic precursors of the germline, arise from embryonic or extra-embryonic cells upon induction by the surrounding tissues during gastrulation, according to mechanisms which are elucidated in mice but remain controversial in primates. They undergo genome-wide epigenetic reprogramming, consisting of extensive DNA demethylation and histone post-translational modification (PTM) changes, toward a basal, euchromatinized state. In contrast, chicken PGCs are specified by preformation before gastrulation based on maternally-inherited factors. They can be isolated from the bloodstream during their migration to the genital ridges. Our prior research highlighted differences in the global epigenetic profile of cultured chicken PGCs compared with chicken somatic cells and mammalian PGCs. This study investigates the acquisition and evolution of this profile during development.ResultsQuantitative analysis of global DNA methylation and histone PTMs, including their distribution, during key stages of chicken early development revealed divergent PGC epigenetic changes compared with mammals. Unlike mammalian PGCs, chicken PGCs do not undergo genome-wide DNA demethylation or exhibit a decrease in histone H3 lysine 9 dimethylation. However, chicken PGCs show 5‑hydroxymethylcytosine loss, macroH2A redistribution, and chromatin decompaction, mirroring mammalian processes. Chicken PGCs initiate their epigenetic signature during migration, progressively accumulating high global levels of H3K9me3, with preferential enrichment in inactive genome regions. Despite apparent global chromatin decompaction, abundant heterochromatin marks, including repressive histone PTMs, HP1 variants, and DNA methylation, persists in chicken PGCs, contrasting with mammalian PGCs.ConclusionsChicken PGCs’ epigenetic signature does not align with the basal chromatin state observed in mammals, suggesting a departure from extensive epigenetic reprogramming. Despite disparities in early PGC development, the persistence of several epigenetic features shared with mammals implies their involvement in chromatin-regulated germ cell properties, with the distinctive elevation of chicken-specific H3K9me3 potentially participating in these processes.

Post-translational modifications of proteins in cardiovascular diseases examined by proteomic approaches.

Over 400 different types of post-translational modifications (PTMs) have been reported and over 200 various types of PTMs have been discovered using mass spectrometry (MS)-based proteomics. MS-based proteomics has proven to be a powerful method capable of global PTM mapping with the identification of modified proteins/peptides, the localization of PTM sites and PTM quantitation. PTMs play regulatory roles in protein functions, activities and interactions in various heart related diseases, such as ischemia/reperfusion injury, cardiomyopathy and heart failure. The recognition of PTMs that are specific to cardiovascular pathology and the clarification of the mechanisms underlying these PTMs at molecular levels are crucial for discovery of novel biomarkers and application in a clinical setting. With sensitive MS instrumentation and novel biostatistical methods for precise processing of the data, low-abundance PTMs can be successfully detected and the beneficial or unfavorable effects of specific PTMs on cardiac function can be determined. Moreover, computational proteomic strategies that can predict PTM sites based on MS data have gained an increasing interest and can contribute to characterization of PTM profiles in cardiovascular disorders. More recently, machine learning- and deep learning-based methods have been employed to predict the locations of PTMs and explore PTM crosstalk. In this review article, the types of PTMs are briefly overviewed, approaches for PTM identification/quantitation in MS-based proteomics are discussed and recently published proteomic studies on PTMs associated with cardiovascular diseases are included.

Global profiling of ribosomal protein acetylation reveals essentiality of acetylation homeostasis in maintaining ribosome assembly and function.

Acetylation is a global post-translational modification that regulates various cellular processes. Bacterial acetylomic studies have revealed extensive acetylation of ribosomal proteins. However, the role of acetylation in regulating ribosome function remains poorly understood. In this study, we systematically profiled ribosomal protein acetylation and identified a total of 289 acetylated lysine residues in 52 ribosomal proteins (r-proteins) from Salmonella Typhimurium. The majority of acetylated lysine residues of r-proteins were found to be regulated by both acetyltransferase Pat and metabolic intermediate acetyl phosphate. Our results show that acetylation plays a critical role in the assembly of the mature 70S ribosome complex by modulating r-proteins binding to rRNA. Moreover, appropriate acetylation is important for the interactions between elongation factors and polysomes, as well as regulating ribosome translation efficiency and fidelity. Dysregulation of acetylation could alter bacterial sensitivity to ribosome-targeting antibiotics. Collectively, our data suggest that the acetylation homeostasis of ribosomes is crucial for their assembly and function. Furthermore, this mechanism may represent a universal response to environmental signals across different cell types.

A Strategy to Characterize the Global Landscape of Histone Post-Translational Modifications Within Tissues of Nonmodel Organisms.

Histone post-translational modifications (PTMs) are epigenetic marks that play a critical role in the expression and maintenance of DNA, but they remain largely uninvestigated in nonmodel organisms due to technical challenges. To begin alleviating this issue, we developed a workflow for histone PTM analysis in Mozambique tilapia (Oreochromis mossambicus), being a widespread and environmentally hardy fish, using mass spectrometry methods. By incorporating multiple protein digestion methods into the preparation of each sample, we reliably quantified 214 biologically relevant histone PTMs. All of these histone PTMs, collectively referred to as the global histone PTM landscape, were characterized in the gills, kidney, and testes of this fish. By comparing the global histone PTM landscape between the three tissues, we found that 91.59% of histone PTMs were tissue-dependent. The workflow and tools for histone PTM analysis described in this study are now publicly available and enable comprehensive investigation into the influence of environmental stress on histone PTMs in nonmodel organisms. Given the functionality and flexibility of histone PTMs, we anticipate that the study of histone PTMs in ecologically relevant contexts will provide ground-breaking insights into comparative physiology and evolution.

Data on multiple post-translational modifications in Alzheimer's disease

This article describes the data obtained for global post-translational modifications (PTMs) profiled for Alzheimer's Disease (AD) from two distinct human brain regions and one cerebrospinal fluid (CSF) sample. The PTM profiling was performed to identify phosphorylation, O-GluNAcetylation, methylation, acetylation and citrullination using three publicly available LC-MS/MS raw data sets (PRIDE ID: PXD004010, PXD002516, PXD004863). A total of 1,857 PTM harbouring proteins with 4,961 unique post-translationally modified peptides were identified. Among the modified peptides, 75 corresponded uniquely to proteins identified from CSF samples. The data is related to the research article "Dissecting Alzheimer's disease molecular substrates by proteomics and discovery of novel post-translational modifications (PTMs)".

Bifidobacterium longum subsp. infantis Shifts Global Transcriptional and Post-translational Modification Signals in Preterm Infant Gut Epithelial Cells

Objectives Bifidobacterium longum subsp. infantis (B. infantis) is an important human infant gut symbiont that confers benefits to the infant by shaping the gut microbiome in vivo. Here, we investigated the transcriptomic and post-translational modification (PTM) impacts of B. infantis on host gut epithelial cells representative of preterm infants, in vitro.MethodsRNA isolated from preterm infant intestinal epithelial cells (HIEC-6) exposed to sterile, cell-free B. infantis supernatants or controls (no supernatant) for 20 hours was subjected to RNA-sequencing to assess global transcriptional changes (n = 6/group). Using the same approach, HIEC-6 cells were incubated for 3 or 20 hours, with or without supernatant (10% v/v; n = 3/group), before harvest for cellular proteins and subsequent western blotting. Gels were probed for α-tubulin, acetylated α-tubulin, Extracellular signal-Regulated Kinase (ERK), and phosphorylated ERK as markers of PTM activity.ResultsGlobal transcriptomic profiles were significantly different between cells exposed to supernatant and those that were not (P = 0.006) and gene pathways involved in anti-inflammatory responses and chemotaxis were significantly upregulated (P < 0.01). Western blotting revealed significant increases in acetyl-α-tubulin and phospho-ERK in cells exposed to supernatant relative to the control group (P < 0.05), while total α-tubulin and ERK concentrations were not significantly different (P > 0.05).ConclusionsThese findings demonstrate the ability of B. infantis to alter global transcriptional and PTM responses affecting inflammation, intracellular trafficking of α-tubulin, and transcription factor recruitment by ERK phosphorylation. These pathways regulate key cellular processes such as cell proliferation, differentiation, adhesion, migration and survival, providing molecular insights into the ability of B. infantis to regulate transcriptional and PTM processes in vitro.Funding SourcesThis work was funded by the Department of Nutrition, the College of Agriculture, Biotechnology, & Natural Resources, the Nevada Agricultural Experiment Station, and the Vice President for Research and Innovation, as well as the NICHD, R03HD105881, and grants from the NIGMS (GM103440 and GM104944) from the NIH.

Targeting Histone Deacetylase‐7 as a Novel Therapeutic Strategy for Glioblastoma Multiforme

BackgroundGlioblastoma multiforme (GBM) is the most aggressive primary brain tumor. GBM has an ominous prognosis with a survival rate of 14‐15 months after diagnosis and despite worldwide initiatives to optimize therapeutic approaches, GBM is still among the most challenging diseases to treat and the fastest to relapse in clinical oncology. The inevitable treatment resistance and relapse are mainly attributed to the presence of glioma stem cells (GSCs), which have the ability to self‐renew, indefinitely proliferate and to differentiate into different cell types. We hypothesize that HDAC7, as an epigenetic regulator, is playing a crucial mechanistic role in GBM and constitutes a novel druggable target for this universally fatal disease. By uncovering the HDAC7 interactome in the GSC nucleus, we provide mechanistic insights into the role of HDAC7 in regulating cancer stemness and possible unprecedented targeting strategies.MethodologyWe quantified the HDAC7 expression in tissue samples from 80 GBM patients admitted at Rhode Island Hospital using Custom Nanostring Panels and correlated the HDAC7 expression to overall patient survival. Also, we compared HDAC7 mRNA expression in GBM to the non‐tumor samples from TCGA data using GlioVis webtool. Next, we performed siRNA knock‐down (KD) of HDAC7 in GSCs followed by RNA‐seq and transcriptomic, gene enrichment and stemness enrichment analysis. We used mass spectrometry (Mod Spec) in si‐HDAC7 KD versus Control to quantify the potential enzymatic de‐acetylation properties of HDAC7 on the global post‐translational modifications of histones. Finally, to unravel the protein partners for HDAC7 in the GSC nucleus, we performed Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME).ResultsSurvival analysis of GBM patients showed significant decrease in survival for patients with high HDAC7 mRNA expression. In addition, GlioVis webtool revealed that HDAC7 expression is significantly higher in GBM tumors relative to non‐tumor samples. Differential gene expression analysis of RNASeq performed on HDAC7 siRNA KD GSCs versus control, revealed 4963 differentially expressed genes with the Gene Ontology enrichment showing significant enrichment for cell cycle and cell division suppression in HDAC7 KD GSCs. Additionally, 653 genes that were downregulated in HDAC7 KD GSCS were found to play significant role in stemness processes of embryonic stem cells and embryonal carcinoma stem cells. Mass spectrometry on GSCs following HDAC7 siRNA KD, revealed non‐significant de‐acetylation changes on global histone marks suggesting that HDAC7 has minimal or no histone deacetylation activity. Finally, RIME revealed novel protein partners in the proximity of 2.5 A° to HDAC7 in the GSCs nucleus. The newly discovered protein complex of HDAC7 suggests that HDAC7 regulates heterochromatin formation and spreading, which mechanistically control transcriptional programs of GSCs.ConclusionOur data suggest that HDAC7 plays a pivotal role in GBM. It regulates expression of transcripts that define cancer stemness, while inhibition of HDAC7 results in global increase in heterochromatin and transcriptional repression in GSCs. This study supports HDAC7 as a novel druggable target for GBM in a preclinical setting.

Global post-translational modification profiling of HIV-1-infected cells reveals mechanisms of host cellular pathway remodeling.

SUMMARYViruses must effectively remodel host cellular pathways to replicate and evade immune defenses, and they must do so with limited genomic coding capacity. Targeting post-translational modification (PTM) pathways provides a mechanism by which viruses can broadly and rapidly transform a hostile host environment into a hospitable one. We use mass spectrometry-based proteomics to quantify changes in protein abundance and two PTM types—phosphorylation and ubiquitination—in response to HIV-1 infection with viruses harboring targeted deletions of a subset of HIV-1 genes. PTM analysis reveals a requirement for Aurora kinase activity in HIV-1 infection and identified putative substrates of a phosphatase that is degraded during infection. Finally, we demonstrate that the HIV-1 Vpr protein inhibits histone H1 ubiquitination, leading to defects in DNA repair.

Organ-Based Proteome and Post-Translational Modification Profiling of a Widely Cultivated Tropical Water Fish, Labeo rohita.

Proteomics has enormous applications in human and animal research. However, proteomic studies in fisheries science are quite scanty particularly for economically important species. Few proteomic studies have been carried out in model fish species, but comprehensive proteomics of aquaculture species are still scarce. This study aimed to perform a comprehensive organ-based protein profiling of important tissue samples for one of the most important aquaculture species,Labeo rohita.Deep proteomic profiling of 17 histologically normal tissues, blood plasma, and embryo provided mass-spectrometric evidence for 8498 proteins at 1% false discovery rate that make up about 26% of the total annotated protein-coding sequences in Rohu. Tissue-wise expression analysis was performed, and the presence of several biologically important proteins was also verified using a targeted proteomic approach. We identified the global post-translational modifications (PTMs) in terms of acetylation (N-terminus and lysine), methylation (N-terminus, lysine, and arginine), and phosphorylation (serine, threonine, and tyrosine) to present a comprehensive proteome resource. An interactive web-based portal has been developed for an overall landscape of protein expression across the studied tissues of Labeo rohita (www.fishprot.org). This draft proteome map of Labeo rohita would advance basic and applied research in aquaculture to meet the most critical challenge of providing food and nutritional security to an increasing world population.

Effects of Rigosertib (RIGO) Alone or in Combination with Azacitidine or Vorinostat on Epigenetic Reprogramming of CD34+ Cells in the Myelodysplastic Syndrome

Rigosertib (RIG) which functions as a RAS-mimetic, binds to the RAS-binding domain (RBD) impacting many pathways that involve RBD-mediated interactions, including the RAS-Raf and AKT/PI3-kinase pathways (Divakar et al., Cell 2016). Recently, we demonstrated that RIG also functions as an epigenetic modifier in MDS. RIG alone or in combination with Azacitidine (AZA) exerted epigenetic effects on the global histone post-translational modifications (PTMs), chromatin remodelers, HDACs, and DNMTs and caused enhanced apoptosis in MDS (MDS-L) and AML (BW-90) cell lines as well as in bone marrow cells (BM) derived from pts with MDS (Chaurasia et al ASCO 2016). In clinical trials, the combination of RIG and AZA demonstrated an overall response rate of 76% in all MDS patients; 62% response in pts who had failed prior hypomethyating agent (HMA) (Navada et al ASH 2016) and 85% in HMA naive pts. The growing evidence suggests that combined therapy may be more effective than either agent alone. To further study these effects, we explored activity of RIG in combination with Vorinostat (VOR), a histone deacetylase inhibitor, on the cell lines. Combined treatment in sequential order VOR/RIG and RIG/VOR potentiates far greater effects on the global PTMs, HDACs, DNMTs, chromatin remodelers, cell cycle checkpoint proteins and PI3/AKT pathway than RIG/AZA or AZA/RIG.RIG in combination with AZA or VOR (AZA/RIG, RIG/AZA, VOR/RIG or RIG/VOR) demonstrated differential effects on the association of RNA polymerase II (Pol II) with active histone marks (H3K4me3 and H3K4me2) in MDS-L and BW-90 cells. An overall decrease in association of Pol II/H3K4me2 was observed with the combinations (AZA/RIG, VOR/RIG or vice versa) compared to control in MDS-L and BW-90, 10-33% (ANOVA, p=0.0006), 9-20% (ANOVA, p=0.0004), respectively. Significant differences were observed in association of H3K4me3/Pol II in BW-90 cells (7-30%; ANOVA, p<0.0001). Similarly, in BM from a pt with MDS after 1 cycle of RIG and AZA treatment demonstrated a decrease in association of Pol II with H3K4me2 (67%) and H3K4me3 (28%).Treatment of MDS-L cells with RIG alone or in combination with AZA failed to induce expansion of CD34+ cells, but the RIG/VOR combination induced 1.9-fold expansion of CD34+ cells (ANOVA p=0.002). Individually AZA or VOR treatment led to an enhanced expansion of CD34+ cells, 3.8 and 4.0 fold, respectively (ANOVA, p=0.004). RIG alone or with AZA yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSPCs) (ANOVA, p=0.006). However, a marked decrease in ALDH activity was observed in AZA or VOR or RIG/VOR, that was inversely proportion to the expansion of CD34+ cells.In a single MDS pt treated with RIG/AZA, expansion of primitive HPSCs expressing low levels of CD34 was observed. A highly expressed subpopulation of HPSCs, which co-existed prior to treatment of the pt and disappeared upon treatment, suggests either an induced cell death or commitment of HSPCs towards improved lineage differentiation in conjunction with clinical response.In order to evaluate effects of RIG on expansion of CD34+ cells, we examined the pluripotent genes (SOX2, OCT4 NANOG and ZIC3) in the BM from MDS pts, prior and after RIG/AZA treatment. RNA expression levels of SOX2, OCT4, NANOG and ZIC3 were elevated post treatment (≥2 fold). In MDS-L cells the pluripotency genes were highly upregulated in the presence of RIG or RIG/AZA or RIG/VOR (1.7-34 folds). These findings indicate that expression of pluripotency genes is a consequence of epigenetic reprogramming that favors expansion of more primitive HSPCs in a pt, while increase in ALDH activity was inversely proportional to the enhanced expansion of CD34+ cells in MDS-L suggesting a more primitive state of HSPC.The epigenetic events modulated by RIG in combination with either AZA or VOR led to global histone PTMs, differential Pol II association with active histone marks, epigenetic reprogramming of pluripotency genes and expansion of primitive HSPC with downregulation of the PI3K/AKT pathway and cell cycle checkpoint proteins. Collectively, these studies indicate that epigenetic effects of RIG on chromatin alterations lead to HSPC reprogramming that may lead to improved hematopoietic function and response in the clinical setting. These preclinical models suggest potential novel clinical strategies to improve outcomes for patients with higher-risk MDS. DisclosuresReddy:Onconova Therapeutics: Equity Ownership.

O‐GlcNAc controls mitochondrial quality control mechanisms and mitochondrial stress response proteins

The accumulation of dysfunctional mitochondria is closely associated with Alzheimer Disease (AD). Mitochondrial dysfunction is an early feature of the disease and impaired turnover of damaged mitochondria increases cellular toxicity leading to neuronal damage. Importantly, AD exhibited a marked reduction in global post-translational modification β-N-acetylglucosamine (O-GlcNAc) level in post-mortem human brain tissue with AD and triple transgenic mouse model of AD (3xTg-AD). O-GlcNAc is a ubiquitous single sugar modification found on nuclear, cytoplasmic, and mitochondrial proteins. Cells maintain a homeostatic level of O-GlcNAc by cycling the addition and removal of the sugar by O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) respectively. Previously, we reported that sustained alterations in O-GlcNAcylation by pharmacologically or genetically manipulation impaired mitochondrial function. Here, we have established an essential role for O-GlcNAc in inducing the expression of several mitochondrial quality control mechanisms using cell lines and in vitro models of AD (differentiated organoids isolated from AD and control patients). We established that sustained alterations in O-GlcNAcylation by pharmacological inhibiting OGA induces the expression of two mitochondrial stress response proteins Activating Transcription Factor 4 (ATF4) and heat shock protein family A (GRP75). ATF4 is a key transcription factor to initiate mitophagy (mitochondria-selective autophagy). Consistence with the ATF4 induction, several mitophagy proteins are increased in the sustained alterations in O-GlcNAcylation. This post-translational modification plays crucial role in AD and restoring mitochondrial quality control mechanisms is important to prevent the accumulation of dysfunctional mitochondria.

Global analysis of HBV-mediated host proteome and ubiquitylome change in HepG2.2.15 human hepatoblastoma cell line

Hepatitis B virus (HBV) infection remains a major health issue worldwide and the leading cause of cirrhosis and hepatocellular carcinoma (HCC). It has been reported previously that HBV invasion can extensively alter transcriptome, the proteome of exosomes and host cell lipid rafts. The impact of HBV on host proteins through regulating their global post-translational modifications (PTMs), however, is not well studied. Viruses have been reported to exploit cellular processes by enhancing or inhibiting the ubiquitination of specific substrates. Nevertheless, host cell physiology in terms of global proteome and ubiquitylome has not been addressed yet. Here by using HBV-integrated HepG2.2.15 model cell line we first report that HBV significantly modify the host global ubiquitylome. As currently the most widely used HBV cell culture model, HepG2.2.15 can be cultivated for multiple generations for protein labeling, and can replicate HBV, express HBV proteins and secrete complete HBV Dane particles, which makes it a suitable cell line for ubiquitylome analysis to study HBV replication, hepatocyte immune response and HBV-related HCC progression. Our previous experimental results showed that the total ubiquitination level of HepG2.2.15 cell line was significantly higher than that of the corresponding parental HepG2 cell line. By performing a Ubiscan quantification analysis based on stable isotope labeling of amino acids in cell culture (SILAC) of HepG2.2.15 and HepG2 cell lines, we identified a total of 7188 proteins and the protein levels of nearly 19% of them were changed over 2-folds. We further identified 3798 ubiquitinated Lys sites in 1476 host proteins with altered ubiquitination in response to HBV. Our results also showed that the global proteome and ubiquitylome were negatively correlated, indicating that ubiquitination might be involved in the degradation of host proteins upon HBV integration. We first demonstrated the ubiquitination change of VAMP3, VAMP8, DNAJB6, RAB8A, LYN, VDAC2, OTULIN, SLC1A4, SLC1A5, HGS and TOLLIP. In addition, we described 5 novel host factors SLC1A4, SLC1A5, EIF4A1, TOLLIP and BRCC36 that efficiently reduced the amounts of secreted HBsAg and HBeAg. Overall, the HBV-mediated host proteome and ubiquitylome change we reported will provide a valuable resource for further investigation of HBV pathogenesis and host-virus interaction networks.

Discovery of Post-Translational Modifications in Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

Integration of liquid chromatography mass spectrometry with a heavy peptide response curve accurately measures unprocessed C-terminal lysine during peptide mapping analysis of therapeutic antibodies in a single run

Therapeutic monoclonal and bispecific antibodies are susceptible to modification after protein biosynthesis. These post-translational modifications (PTMs) not only contribute to mass and charge heterogeneity, but they can also negatively impact the molecule’s activity, half-life, and immunogenicity. Therefore, identification and quantification of PTMs are critical to ensure the safety and efficacy of an antibody therapeutic as well as demonstrate product consistency and process control. Unprocessed C-terminal lysine on the heavy chain (HC) is a prevalent modification that contributes to this charge heterogeneity in antibodies. Peptide mapping through liquid chromatography tandem mass spectrometry (LC–MS2) enjoys higher selectivity and sensitivity for measuring this PTM relative to global PTM methods, but differences in the ionization efficiencies of the unprocessed C-terminal K peptide and the truncated C-terminal K peptide result in its overestimation. Consequently, large discrepancies in this PTM’s measured abundance may exist between different characterization assays used in regulatory filings, which can be further compounded by large variability when multiple mass spectrometers are used to quantify C-terminal K during a therapeutic’s lifespan. In this study, we propose a simple new method to quantify unprocessed C-terminal K in antibodies in a single LC–MS2 run that incorporates heavy isotopic standards for both the unprocessed and truncated C-terminal K peptide to build a response curve and correct for the disparity in ionization efficiency between these two different peptide sequences. The approach was evaluated across two different Orbitrap-based mass spectrometers using multiple monoclonal and bispecific therapeutic antibodies, resulting in accurate (<10% error, as determined with peptide standards) and precise C-terminal K quantification during peptide mapping analysis.

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients.

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.

Reshaping of the Arabidopsis thaliana Proteome Landscape and Co-regulation of Proteins in Development and Immunity

Proteome remodeling is a fundamental adaptive response, and proteins in complexes and functionally related proteins are often co-expressed. Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana tissues with around 10 000 proteins per tissue, and absolutely quantify (copy numbers per cell) nearly 16 000 proteins throughout the plant lifecycle. A proteome-wide survey of global post-translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue- and age-specific roles of entire signaling modules regulating transcription in photosynthesis, seed development, and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of cysteine-rich receptor-like kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were found to be co-expressed in a tissue- and age-specific manner, indicating functional promiscuity in the assembly of these less-studied protein complexes in Arabidopsis.Furthermore, we characterized detailed proteome remodeling in basal immunity by treating Arabidopsis seeldings with flg22. Through simultaneously monitoring phytohormone and transcript changes upon flg22 treatment, we obtained strong evidence of suppression of jasmonate (JA) and JA-isoleucine (JA-Ile) levels by deconjugation and hydroxylation by IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2), respectively, under the control of JASMONATE INSENSITIVE 1 (MYC2), suggesting an unrecognized role of a new JA regulatory switch in pattern-triggered immunity. Taken together, the datasets generated in this study present extensive coverage of the Arabidopsis proteome in various biological scenarios, providing a rich resource available to the whole plant science community.

Spritz: A Proteogenomic Database Engine.

Proteoforms are the workhorses of the cell, and subtle differences between their amino acid sequences or post-translational modifications (PTMs) can change their biological function. To most effectively identify and quantify proteoforms in genetically diverse samples by mass spectrometry (MS), it is advantageous to search the MS data against a sample-specific protein database that is tailored to the sample being analyzed, in that it contains the correct amino acid sequences and relevant PTMs for that sample. To this end, we have developed Spritz (https://smith-chem-wisc.github.io/Spritz/), an open-source software tool for generating protein databases annotated with sequence variations and PTMs. We provide a simple graphical user interface for Windows and scripts that can be run on any operating system. Spritz automatically sets up and executes approximately 20 tools, which enable the construction of a proteogenomic database from only raw RNA sequencing data. Sequence variations that are discovered in RNA sequencing data upon comparison to the Ensembl reference genome are annotated on proteins in these databases, and PTM annotations are transferred from UniProt. Modifications can also be discovered and added to the database using bottom-up mass spectrometry data and global PTM discovery in MetaMorpheus. We demonstrate that such sample-specific databases allow the identification of variant peptides, modified variant peptides, and variant proteoforms by searching bottom-up and top-down proteomic data from the Jurkat human T lymphocyte cell line and demonstrate the identification of phosphorylated variant sites with phosphoproteomic data from the U2OS human osteosarcoma cell line.

Proteomic variations of esophageal squamous cell carcinoma revealed by combining RNA-seq proteogenomics and G-PTM search strategy

BackgroundCancer that arises from epithelial cells of the esophagus is called esophagus squamous cell carcinoma (ESCC) and is mostly observed in developing nations. Evaluation of cancer genomes and its regulation into proteins plays a predominant role in understanding the cancer progressions. Mass-spectrometry-based proteomics is a consequential tool to estimate proteomic variation and posttranslational modifications (PTMs) from standard protein databases. Post-translational modifications play a crucial role in protein folding and PTMs can be accounted for as a biological signal to interpret the structural changes and transition order of proteins. Functional validation of cancer-related mutations can explain the effects of mutations on genes and the identification of Oncogenes and tumor suppressor genes. Therefore, we present a study on protein variations to interpret the structural changes and transition order of proteins in ESCC carcinogenesis. MethodologyWe are using a bottom-up proteomics approach with Galaxy-P framework and RNA sequence data analysis to generate the sample-specific databases containing details of RNA splicing and variant peptides. Once the database generated with information on variable modification, only the curated PTMs at specific positions are considered to perform spectral matching. Proteogenomics mapping was performed to identify protein variations in ESCC. ResultsRNA-sequence proteogenomics with G-PTM (Global Post-Translational Modification) searching strategy has revealed proteomic events including several peptides that contain single amino acid variations, novel splice junction peptides and posttranslationally modified peptides. Proteogenomic mapping exhibited the splice junction peptides mapped predominantly for Malic enzyme exon type (ME-3) and MCM7 protein-coding genes that promote cancer progression, found to be exhibited in ESCC samples. Approximately 25 ± types of PTM modifications were recorded, and Protein Phosphorylation was largely noted. ConclusionESCC cancer prognosis at the molecular level enables a better understanding of cancer carcinogenesis and protein modifications can be used as potential biomarkers.

IRF4 Is Reciprocally Dysregulated Via NF-Κb-Dependent Expression and Loss-of-Function Mutations in Multiple Myeloma

Introduction: Interferon regulatory factor 4 (IRF4) is a transcription factor involved in B cell activation and differentiation into plasma cells, as well as B-cell lineage neoplasia. Diverse genetic abnormalities and dysregulated expression of IRF4 have been documented in various hematologic malignancies, including multiple myeloma (MM). IRF4 is also identified as a major downstream target of CRBN/IKZF to regulate MYC expression, representing a primary mechanism underlying anti-MM action of IMiDs. However, it remains unclear how the expression and function of IRF4 are regulated in MM cells. Materials and Methods: DNAseq, whole exome RNAseq (WES), and label-free analyses of quantitative proteomics and post-translational modifications (PTMs) mapping were conducted to examine genetic aberrations, transcriptional dysregulation, or protein and PTM alterations in primary patient samples and drug-naive vs -resistant cell lines. Flow cytometry, qPCR, and Western blot analysis were utilized to monitor apoptosis or mRNA and protein levels of target genes. Stable transfection with wild-type or mutated IRF4 was carried out to evaluate its function. Analysis of the MM genome-wide GEP databases (R2: Genomics Analysis and Visualization Platform) was performed to validate the clinical significance of IRF4 and its target genes in MM patients. Results: MM cells that acquired bortezomib (btz) resistance (PS-R) exhibited a marked increase in protein levels of multiple key components of the canonical and non-canonical NF-κB signaling cascades, in association with increased NF-κB transcriptional activity. Pharmacological inhibition of NF-κB using several compounds (e.g., Bay 11-7082, parthenolide, triptolide) significantly increased btz sensitivity in PS-R cells and their parental U266 cells, as well as other MM cell lines. These events were accompanied by down-regulation of various genes, including NF-κB-dependent genes and those whose relationship with NF-κB remains uncertain. The latter included IRF4, MYC, MCL1, BIRC2/cIAP1, SQSTM1/p62, among others. Of note, overexpression of wild-type IRF4 sharply reduced sensitivity of drug-naive cells to btz alone or in combination with NF-κB inhibitors. However, IRF4 overexpression did not prevent inactivation of NF-κB and down-regulation of the NF-κB-dependent genes (e.g., Bcl-xL, TNFAIP3/A20), suggesting that IRF4 acts downstream of NF-κB. Surprisingly, IRF4 overexpression failed to restore expression of MYC, a well-established target of IRF4, while largely reversed expression of cIAP1 and Mcl-1 at both mRNA and protein levels. Moreover, inhibitors of either IAP (birinapant/TL32711) or Mcl-1 (S63845) overcame btz-resistance conferred by IRF4 overexpression. DNAseq unveiled multiple IRF4 SNPs located in introns and exons (both 3'-UTR and coding region) of chromosome 6. WES revealed that IRF4 was highly expressed in primary bone marrow and extramedullary samples of MM patients (2~10 folds over normal donors). But no difference at mRNA and protein levels of IRF4 was observed between drug-naive and -resistant cell lines. The global PTM mapping identified two lysine residues (K59 and K399) that were highly ubiquitinated in PS-R cells (p = 0.0447 and p = 0.0004, vs U266 cells). Interestingly, ectopic expression of IRF4 mutants (e.g., K59N, K123R) lost the cytoprotective capability of their wild-type counterpart, in association with failure to reverse down-regulation of cIAP1 and Mcl-1. This observation argues that these point mutations are loss-of-function, which might explain better prognosis of patients carrying IRF4 mutations (including K59N and K123R) than wild-type IRF4 as reported previously. Last, the MM GEP database analysis validated the correlation between IRF4 and Mcl-1 or cIAP expression, as well as the adverse effect of their dysregulation on overall survival of MM patients. Conclusion: IRF4 is highly expressed in MM cells, likely via an NF-κB-dependent mechanism. IRF4 acts downstream of the NF-κB signaling pathways, which in turn directly targets cIAP1 and Mcl-1, rather than MYC, to confer drug resistance. IRF4 could be mutated in MM patients, while the point mutations, at least at certain lysine sites, seem to be loss-of-function and thus predict better outcome. Together, these findings suggest that the oncogenic function of IRF4 might be governed at genetic, transcriptional, and PTM levels in MM cells. Disclosures Kumar: Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.