Effects Of Post-translational Modifications Research Articles

Post-translational modifications in kidney diseases and associated cardiovascular risk.

Patients with chronic kidney disease (CKD) are at an increased cardiovascular risk compared with the general population, which is driven, at least in part, by mechanisms that are uniquely associated with kidney disease. In CKD, increased levels of oxidative stress and uraemic retention solutes, including urea and advanced glycation end products, enhance non-enzymatic post-translational modification events, such as protein oxidation, glycation, carbamylation and guanidinylation. Alterations in enzymatic post-translational modifications such as glycosylation, ubiquitination, acetylation and methylation are also detected in CKD. Post-translational modifications can alter the structure and function of proteins and lipoprotein particles, thereby affecting cellular processes. In CKD, evidence suggests that post-translationally modified proteins can contribute to inflammation, oxidative stress and fibrosis,and induce vascular damage or prothrombotic effects, which might contribute to CKD progression and/or increase cardiovascular risk in patients with CKD. Consequently, post-translational protein modifications prevalent in CKD might be useful as diagnostic biomarkers and indicators of disease activity that could be used to guide and evaluate therapeutic interventions, in addition to providing potential novel therapeutic targets.

The PTM profiling of CTCF reveals the regulation of 3D chromatin structure by O-GlcNAcylation

CCCTC-binding factor (CTCF), a ubiquitously expressed and highly conserved protein, is known to play a critical role in chromatin structure. Post-translational modifications (PTMs) diversify the functions of protein to regulate numerous cellular processes. However, the effects of PTMs on the genome-wide binding of CTCF and the organization of three-dimensional (3D) chromatin structure have not been fully understood. In this study, we uncovered the PTM profiling of CTCF and demonstrated that CTCF can be O-GlcNAcylated and arginine methylated. Functionally, we demonstrated that O-GlcNAcylation inhibits CTCF binding to chromatin. Meanwhile, deficiency of CTCF O-GlcNAcylation results in the disruption of loop domains and the alteration of chromatin loops associated with cellular development. Furthermore, the deficiency of CTCF O-GlcNAcylation increases the expression of developmental genes and negatively regulates maintenance and establishment of stem cell pluripotency. In conclusion, these results provide key insights into the role of PTMs for the 3D chromatin structure.

Role of the histone variant H2A.Z.1 in memory, transcription, and alternative splicing is mediated by lysine modification

Creating long-lasting memories requires learning-induced changes in gene expression, which are impacted by epigenetic modifications of DNA and associated histone proteins. Post-translational modifications (PTMs) of histones are key regulators of transcription, with different PTMs producing unique effects on gene activity and behavior. Although recent studies implicate histone variants as novel regulators of memory, effects of PTMs on the function of histone variants are rarely considered. We previously showed that the histone variant H2A.Z suppresses memory, but it is unclear if this role is impacted by H2A.Z acetylation, a PTM that is typically associated with positive effects on transcription and memory. To answer this question, we used a mutation approach to manipulate acetylation on H2A.Z without impacting acetylation of other histone types. Specifically, we used adeno-associated virus (AAV) constructs to overexpress mutated H2A.Z.1 isoforms that either mimic acetylation (acetyl-mimic) by replacing lysines 4, 7 and 11 with glutamine (KQ), or H2A.Z.1 with impaired acetylation (acetyl-defective) by replacing the same lysines with alanine (KA). Expressing the H2A.Z.1 acetyl-mimic (H2A.Z.1KQ) improved memory under weak learning conditions, whereas expressing the acetyl-defective H2A.Z.1KA generally impaired memory, indicating that the effect of H2A.Z.1 on memory depends on its acetylation status. RNA sequencing showed that H2A.Z.1KQ and H2A.Z.1KA uniquely impact the expression of different classes of genes in both females and males. Specifically, H2A.Z.1KA preferentially impacts genes involved in synaptic function, suggesting that acetyl-defective H2A.Z.1 impairs memory by altering synaptic regulation. Finally, we describe, for the first time, that H2A.Z is also involved in alternative splicing of neuronal genes, whereby H2A.Z depletion, as well as expression of H2A.Z.1 lysine mutants influence transcription and splicing of different gene targets, suggesting that H2A.Z.1 can impact behavior through effects on both splicing and gene expression. This is the first study to demonstrate that direct manipulation of H2A.Z post-translational modifications regulates memory, whereby acetylation adds another regulatory layer by which histone variants can fine tune higher brain functions through effects on gene expression and splicing.

Abstract A022: Poly (ADP-Ribose) glycohydrolase (PARG) removes repressive Poly-ADP Ribose marks from DNA polymerase theta (polq) to stimulate DNA double-strand breaks repair in myeloid malignancies

Abstract Poly (ADP-ribose) polymerase 1 (PARP1) dependent poly-(ADP)-ribosylation (PARylation) of proteins is one of the major post-translational modification events involved in the DNA damage response (DDR). PARylation is tightly controlled by the glycohydrolase activity of poly (ADP-ribose) glycohydrolase (PARG). Thus, interplay between PARP1 and PARG is thought to regulate the PARylation status of relevant DDR proteins and multiple studies demonstrated that both PARP1 and PARG contribute to DDR. Here, we investigated whether the interplay of PARP1 and PARG is important for the regulation of TMEJ and the specific activities of Polq in myeloid malignancies harboring OTKs. We found that upon DNA damage OTKs exerted temporal effect on Polq, PARP1 and PARG detection in chromatin fractions. While both Polq and PARG displayed continuous time-dependent accumulation during 120 min. after irradiation, PARP1 levels sharply increased at 20 min. and dissipated at 120 min. post-irradiation confocal microscopy detected spatiotemporal changes of Polq - PARP1 – PARG colocalization and PARylation of Polq. Polq - PARP1 interaction and PARylation of Polq was detected early followed by dissipation of that interaction and enhanced colocalization of Polq and PARG and dePARylation of Polq. At the time when Polq was PARylated by PARP1, it was not detected at DSBs marked by gH2AX whereas PARG-mediated dePARylation of Polq was associated with its colocalization with DSBs. We find that PARP1 binds to and directly PEGylates Polq in vitro and in cells. Biochemical assays revealed that PARP1 recruits Polq to the vicinity of DNA damage via PARylation-dependent formation of biomolecular condensates containing Polq. However, PARylated Polq is unable to perform TMEJ due to its inability to bind directly to DNA. Hence, PARG is needed to reactivate Polq DNA binding and its TMEJ activity by removing repressive PAR marks on Polq. In support of this, cellular studies show that PARP1-PARG – regulated spatiotemporal recruitment of Polq to DSBs is essential for TMEJ activity. In conclusion, using intracellular and biochemical approaches we show here that both PARP1 and PARG are required for TMEJ despite their counteracting enzymatic activities. We also pinpointed a unique dynamic spatiotemporal interplay between Polq, PARP1 and PARG to regulate TMEJ activity in OTK-positive myeloid malignant cells. Our studies support a two-step mechanism of a PARP1-PARG regulatory axis of Polq and TMEJ. In the first step, the rate of PARP1-Polq spatiotemporal recruitment to DNA damage foci supports a rapid step whereby PARP1 PARylates Polq and facilitates its recruitment to the vicinity of DNA damage in an inactive state. The rate of PARG spatiotemporal recruitment supports a second step whereby subsequent recruitment of PARG corresponds to dissipation of PARP1 and PAR at DNA damage sites, PARG-mediated removal of PAR repressive marks on Polq, and activation of TMEJ. Citation Format: Umeshkumar Vekariya, Leonid Minakhin, Mrityunjay Tyagi, Tatiana Kent, Gurushankar Chandramouly, Katherine Sullivan-Reed, Jessica Atkins, Margaret Nieborowska-Skorska, Anna-Mariya Kukuyan, Richard Pomerantz, Tomasz Skorski. Poly (ADP-Ribose) glycohydrolase (PARG) removes repressive Poly-ADP Ribose marks from DNA polymerase theta (polq) to stimulate DNA double-strand breaks repair in myeloid malignancies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A022.

Unraveling the complex relationship between mRNA and protein abundances: a machine learning-based approach for imputing protein levels from RNA-seq data.

The correlation between messenger RNA (mRNA) and protein abundances has long been debated. RNA sequencing (RNA-seq), a high-throughput, commonly used method for analyzing transcriptional dynamics, leaves questions about whether we can translate RNA-seq-identified gene signatures directly to protein changes. In this study, we utilized a set of 17 widely assessed immune and wound healing mediators in the context of canine volumetric muscle loss to investigate the correlation of mRNA and protein abundances. Our data reveal an overall agreement between mRNA and protein levels on these 17 mediators when examining samples from the same experimental condition (e.g. the same biopsy). However, we observed a lack of correlation between mRNA and protein levels for individual genes under different conditions, underscoring the challenges in converting transcriptional changes into protein changes. To address this discrepancy, we developed a machine learning model to predict protein abundances from RNA-seq data, achieving high accuracy. Our approach also effectively corrected multiple extreme outliers measured by antibody-based protein assays. Additionally, this model has the potential to detect post-translational modification events, as shown by accurately estimating activated transforming growth factor β1 levels. This study presents a promising approach for converting RNA-seq data into protein abundance and its biological significance.

Poly(ADP-Ribose) Glycohydrolase (PARG) Removes Repressive Poly-ADP Ribose Marks from DNA Polymerase Theta (PolΘ) to Stimulate DNA Double-Strand Breaks Repair in Myeloid Malignancies

Myeloid malignant cells (AML, MPN, CML) expressing oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1, respectively] accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We reported before that formaldehyde generated by altered serine/one-carbon cycle metabolism in malignant cells harboring OTKs contributed to accumulation of toxic DNA-protein crosslinks (DPCs) which were converted to highly lethal DNA double-strand breaks (DSBs). To counteract the toxicity of DSBs, OTKs enhanced the expression of DNA polymerase theta (PolΘ, encoded by POLQ gene), a unique DNA helicase-DNA polymerase fusion protein that promotes error-prone repair of DSBs by a mechanism referred to as PolΘ-mediated DNA end-joining (TMEJ). PolΘ plays an essential role in the initiation and maintenance of hematological malignancies harboring OTKs. Although the cellular activities of PolΘ have been widely studied, little is known about how PolΘ is regulated at the molecular level. For example, whether post-translational modifications of PolΘ are important for its TMEJ activity and regulation is unknown. Poly(ADP-ribose) polymerase 1 (PARP1) dependent poly-(ADP)-ribosylation (PARylation) of proteins is one of the major post-translational modification events involved in the DNA damage response (DDR). PARP1 - the founding member of the poly(ADP-ribose) polymerase family -transfers adenosine diphosphate (ADP)-ribose from nicotinamide adenine dinucleotide (NAD +) to substrate proteins enabling their PARylation. PARylation is tightly controlled by the glycohydrolase activity of poly(ADP-ribose) glycohydrolase (PARG). Thus, interplay between PARP1 and PARG is thought to regulate the PARylation status of relevant DDR proteins and multiple studies demonstrated that both PARP1 and PARG contribute to DDR. Here, we investigated whether the interplay of PARP1 and PARG is important for the regulation of TMEJ and the specific activities of PolΘ in myeloid malignancies harboring OTKs. We found that upon DNA damage OTKs exerted temporal effect on PolΘ, PARP1 and PARG detection in chromatin fractions. While both PolΘ and PARG displayed continuous time-dependent accumulation during 120 min. after irradiation, PARP1 levels sharply increased at 20 min. and dissipated at 120 min. We find that PARP1 binds to and directly PARylates PolΘ in vitro and in cells. However, PARylated PolΘ is unable to perform TMEJ in vitro due to its inability to bind DNA despite its PARylation dependent recruitment to DNA. Hence, PARG is needed to reactivate PolΘ DNA binding and its TMEJ activity by removing repressive PAR marks on PolΘ. In support of this, cellular studies show that PARG is essential for TMEJ and support a two-step mechanism of a PARP1-PARG regulatory axis of PolΘ and TMEJ. In the first step, the rate of PARP1-PolΘ spatiotemporal recruitment to DNA damage foci supports a rapid step whereby PARP1 PARylates PolΘ and facilitates its recruitment to the vicinity of DNA damage in an inactive state. The rate of PARG spatiotemporal recruitment supports a second step whereby subsequent recruitment of PARG corresponds to dissipation of PARP1 and PAR at DNA damage sites, dePARylation of PolΘ, and activation of TMEJ. These studies elucidate an unprecedented mechanism of PARP1-PARG activation of TMEJ and reveal the molecular basis by which PARP1 and PARG inhibition suppresses TMEJ. In conclusion, using intracellular and biochemical approaches we show here that both PARP1 and PARG are required for TMEJ despite their counteracting enzymatic activities. We also pinpointed a unique dynamic spatiotemporal interplay between PolΘ, PARP1 and PARG to regulate TMEJ activity. During the initial stage, PARP1 activated by DNA damage PARylates PolΘ and facilitates its recruitment to the vicinity of DNA damage. PARG removal of PAR from PolΘ reactivated its interaction with DNA resulting in robust TMEJ activity in vitro and in cells. Thus, DNA/chromatin binding and TMEJ activity of PolΘ were restored upon PARG-mediated removal of PAR repressive marks on PolΘ. This process seems to play a critical role in protecting OTK-positive hematopoietic malignant cells from genotoxic effect of formaldehyde generated by altered serine/one-carbon cycle metabolism.

The effects of post-translational modifications on alpha-synuclein aggregation and immune cell activation in Parkinson’s disease

Post-translational modifications (PTMs) of alpha-synuclein (α-syn) can alter protein aggregation propensity to affect α-syn oligomer and fibril formation. The inflammatory response in Parkinson’s disease (PD) is mediated by microglia, astrocytes, T cells, B cells, macrophages, and neutrophils, which respond to α-syn aggregates in an attempt to clear synucleinopathy and restore brain homeostasis. This review focuses on the effects of PTMs on α-syn aggregation and cell-specific immune responses to α-syn aggregates in the context of PD.

Post-translational modifications and their implications in cancer.

Post-translational modifications (PTMs) are crucial regulatory mechanisms that alter the properties of a protein by covalently attaching a modified chemical group to some of its amino acid residues. PTMs modulate essential physiological processes such as signal transduction, metabolism, protein localization, and turnover and have clinical relevance in cancer and age-related pathologies. Majority of proteins undergo post-translational modifications, irrespective of their occurrence in or after protein biosynthesis. Post-translational modifications link to amino acid termini or side chains, causing the protein backbone to get cleaved, spliced, or cyclized, to name a few. These chemical modifications expand the diversity of the proteome and regulate protein activity, structure, locations, functions, and protein-protein interactions (PPIs). This ability to modify the physical and chemical properties and functions of proteins render PTMs vital. To date, over 200 different protein modifications have been reported, owing to advanced detection technologies. Some of these modifications include phosphorylation, glycosylation, methylation, acetylation, and ubiquitination. Here, we discuss about the existing as well as some novel post-translational protein modifications, with their implications in aberrant states, which will help us better understand the modified sites in different proteins and the effect of PTMs on protein functions in core biological processes and progression in cancer.

N-Glycosylation of LRP6 by B3GnT2 Promotes Wnt/β-Catenin Signalling.

Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/β-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/β-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events.

Pharmacological inhibition of the SKP2/p300 signaling axis restricts castration-resistant prostate cancer

SKP2, an F-box protein of the SCF type of the E3 ubiquitin ligase complex, plays an important function in driving tumorigenesis through the destruction of numerous tumor-suppressive proteins. Besides its critical role in cell cycle regulation, proto-oncogenic functions of SKP2 have also been shown in a cell cycle regulation-independent manner. Therefore, uncovering novel physiological upstream regulators of SKP2 signaling pathways would be essential to retard aggressive malignancies. Here, we report that elevation of SKP2 and EP300 transcriptomic expression is a hallmark of castration-resistant prostate cancer. We also found that SKP2 acetylation is likely a critical driven event in castration-resistant prostate cancer cells. Mechanistically, SKP2-acetylation is mediated by the p300 acetyltransferase enzyme for post-translational modification (PTM) event that is induced upon stimulation with dihydrotestosterone (DHT) in prostate cancer cells. Moreover, ectopic expression of acetylation-mimetic K68/71Q mutant of SKP2 in LNCaP cells could confer resistance to androgen withdrawal-induced growth arrest and promotes prostate cancer stem cell (CSC)-like traits including survival, proliferation, stemness formation, lactate production, migration, and invasion. Furthermore, inhibition of p300-mediated SKP2 acetylation or SKP2-mediated p27-degradation by pharmacological inhibition of p300 or SKP2 could attenuate epithelial-mesenchymal transition (EMT) and the proto-oncogenic activities of the SKP2/p300 and androgen receptor (AR) signaling pathways. Therefore, our study identifies the SKP2/p300 axis as a possible molecular mechanism driving castration-resistant prostate cancers, which provides pharmaceutical insight into inactivation of the SKP2/p300 axis for restriction of CSC-like properties, thereby benefiting clinical diagnosis and cancer therapy.

The C-terminus of the cargo receptor Erv14 affects COPII vesicle formation and cargo delivery.

The endoplasmic reticulum (ER) is the start site of the secretory pathway, where newly synthesized secreted and membrane proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Little is known about how post-translational modification events regulate packaging of cargo into COPII vesicles. The Saccharomyces cerevisiae protein Erv14, also known as cornichon, belongs to a conserved family of cargo receptors required for the selection and ER export of transmembrane proteins. In this work, we show the importance of a phosphorylation consensus site (S134) at the C-terminus of Erv14. Mimicking phosphorylation of S134 (S134D) prevents the incorporation of Erv14 into COPII vesicles, delays cell growth, exacerbates growth of sec mutants, modifies ER structure and affects localization of several plasma membrane transporters. In contrast, the dephosphorylated mimic (S134A) had less deleterious effects, but still modifies ER structure and slows cell growth. Our results suggest that a possible cycle of phosphorylation and dephosphorylation is important for the correct functioning of Erv14.

Succinylation and redox status in cancer cells.

Succinylation is a post-translational modification (PTM) event that associates metabolic reprogramming with various pathological disorders including cancers via transferring a succinyl group to a residue of the target protein in an enzymic or non-enzymic manner. With our incremental knowledge on the roles of PTM played in tumor initiation and progression, relatively little has been focused on succinylation and its clinical implications. By delineating the associations of succinylation with cancer hallmarks, we identify the, in general, promotive roles of succinylation in manifesting cancer hallmarks, and conceptualize two working modes of succinylation in driving oncogenic signaling, i.e., via altering the structure and charge of target proteins towards enhanced stability and activity. We also characterize succinylation as a reflection of cellular redox homeostatic status and metabolic state, and bring forth the possible use of hyper-succinylated genome for early cancer diagnosis or disease progression indication. In addition, we propose redox modulation tools such as cold atmospheric plasma as a promising intervention approach against tumor cells and cancer stemness via targeting the redox homeostatic environment cells established under a pathological condition such as hypoxia. Taken together, we emphasize the central role of succinylation in bridging the gap between cellular metabolism and redox status, and its clinical relevance as a mark for cancer diagnosis as well as a target in onco-therapeutics.

Abstract 5027: PTMcosmos: A web portal of post-translational modifications and proteogenomic resources in cancer

Abstract PTMcosmos is a comprehensive database with an interactive web portal designed to catalog and visualize post-translational modifications (PTMs) in humans. It contains 469,183 experimentally-validated PTM sites and their supporting evidence from UniProt Knowledge Base, PhosphoSitePlus, and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). PTMcosmos summarizes the entire spectrum of CPTAC proteomics data on human cancer patients, including protein and PTM peptide abundance data from 10 different cancer types. Additionally, PTMcosmos contains cancer somatic mutations from The Cancer Genome Atlas (TCGA), thus allowing for the collective integration and analysis of different data types. In PTMcosmos, we have built an ensemble of interactive visualization tools that allow researchers to investigate altered PTM functions due to genetic alterations in close proximity. The database is live at https://ptmcosmos.wustl.edu. We used PTMcosmos to investigate PTM regulation across cancer types. First, we examined the differential abundance of the PTM sites of cancer driver genes, focusing primarily on phosphorylation of the tumor suppressor retinoblastoma protein (encoded by RB1) and acetylation of the histone acetyltransferase E1A Binding Protein P300 (EP300) across cancer types. We analyzed the association between these PTM events and downstream targets, as well as with tumor subtypes, significantly mutated gene (SMG) mutation status, and clinical features. Second, we investigated the association of the protein abundance of cancer driver genes with ubiquitylsites in lung squamous cell carcinoma (LSCC) to nominate potentially novel modes of regulation of these proteins’ activities. We further analyzed the tumor subtype specificity and tumor-normal abundance changes of these ubiquitylsites and their corresponding substrate proteins, identifying several EGFR ubiquitylsites which may regulate EGFR abundance in LSCC. Finally, we identified the linear and spatial clustering of mutations and PTM sites, identifying multiple mutation-PTM clusters in cancer related genes, including TP53, PIK3CA, CTNNB1, EGFR, and IDH1. We envision that PTMcosmos will serve both the CPTAC consortium and the wider research community to better understand the role of PTMs in cancer. Citation Format: Liang-Bo Wang, Akshay Govindan, Song Cao, Li Ding. PTMcosmos: A web portal of post-translational modifications and proteogenomic resources in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5027.

A Review On Huntington Protein: Insight Into Protein Aggregation and Therapeutic Interventions.

Huntington's disease (HD) is a distressing, innate neurodegenerative disease that descends from CAG repeat expansion in the huntingtin gene causing behavioral changes, motor dysfunction, and dementia in children and adults. Mutation in huntingtin (HTT) protein has been suggested to cause neuron loss in the cortex and striatum through various mechanisms, including abnormal regulation of transcription, proteasomal dysfunction, posttranslational modification, and other events regulating toxicity. Pathogenesis of HD involves cleavage of the huntingtin protein followed by the neuronal accumulation of its aggregated form. Several research groups made possible efforts to reduce huntingtin gene expression, protein accumulation, and protein aggregation using inhibitors and molecular chaperones as developing drugs against HD. Herein, we review the mechanism proposed towards the formation of HTT protein aggregation and the impact of therapeutic strategies for the treatment of HD.

Aβ42 Expressing Drosophila melanogaster Model for Alzheimer's Disease: Quantitative Proteomics Identifies Altered Protein Dynamics of Relevance to Neurodegeneration.

Production and deposition of β-amyloid peptides (Aβ) are among the major hallmarks of the pathogenesis of Alzheimer's disease (AD). Mapping the altered protein dynamics associated with Aβ accumulation and neuronal damage may open up new avenues to innovation for drug target discovery in AD. Using quantitative proteomics, we report new findings from the amyloid beta-peptide with 42 amino acids (Aβ42) expressing Drosophila melanogaster model for AD compared to that of the wild-type flies. We identified 302,241 peptide-spectrum matches with 25,641 nonredundant peptides corresponding to 7959 D. melanogaster proteins. Furthermore, we unraveled 538 significantly altered proteins in Aβ42 expressing flies. These differentially expressed proteins were enriched for biological processes associated with neuronal damage leading to AD progression. We also identified 463 unique post-translational modification events mapping to 202 proteins from the same dataset. Among these, 303 modified peptides corresponding to 246 proteins were also altered in the AD model. These modified proteins are known to be involved in the disruption of molecular functions maintaining neuronal plasticity. This study provides new molecular leads on altered protein dynamics relevant to neurodegeneration, neuroplasticity, and AD progression induced by Aβ42 toxicity. These proteins may prove useful to discover new drugs in an AD model of D. melanogaster and evaluate their efficacy and mode of molecular action in the future.

Improving the Genome Annotation of Rhizoctonia solani Using Proteogenomics

Background Rhizoctonia solani is a pathogenic fungus that causes serious diseases in many crops, including rice, wheat, and soybeans. In crop production, it is very important to understand the pathogenicity of this fungus, which is still elusive. It might be helpful to comprehensively understand its genomic information using different genome annotation strategies.MethodsAiming to improve the genome annotation of R. solani, we performed a proteogenomic study based on the existing data. Based on our study, a total of 1060 newly identified genes, 36 revised genes, 139 single amino acid variants (SAAVs), 8 alternative splicing genes, and diverse post-translational modifications (PTMs) events were identified in R. solani AG3. Further functional annotation on these 1060 newly identified genes was performed through homology analysis with its 5 closest relative fungi.ResultsBased on this, 2 novel candidate pathogenic genes, which might be associated with pathogen-host interaction, were discovered. In addition, in order to increase the reliability and novelty of the newly identified genes in R. solani AG3, 1060 newly identified genes were compared with the newly published available R. solani genome sequences of AG1, AG2, AG4, AG5, AG6, and AG8. There are 490 homologous sequences. We combined the proteogenomic results with the genome alignment results and finally identified 570 novel genes in R. solani.ConclusionThese findings extended R. solani genome annotation and provided a wealth of resources for research on R. solani.

QPTMplants: an integrative database of quantitative post-translational modifications in plants.

As a crucial molecular mechanism, post-translational modifications (PTMs) play critical roles in a wide range of biological processes in plants. Recent advances in mass spectrometry-based proteomic technologies have greatly accelerated the profiling and quantification of plant PTM events. Although several databases have been constructed to store plant PTM data, a resource including more plant species and more PTM types with quantitative dynamics still remains to be developed. In this paper, we present an integrative database of quantitative PTMs in plants named qPTMplants (http://qptmplants.omicsbio.info), which hosts 1 242 365 experimentally identified PTM events for 429 821 nonredundant sites on 123 551 proteins under 583 conditions for 23 PTM types in 43 plant species from 293 published studies, with 620 509 quantification events for 136 700 PTM sites on 55 361 proteins under 354 conditions. Moreover, the experimental details, such as conditions, samples, instruments and methods, were manually curated, while a variety of annotations, including the sequence and structural characteristics, were integrated into qPTMplants. Then, various search and browse functions were implemented to access the qPTMplants data in a user-friendly manner. Overall, we anticipate that the qPTMplants database will be a valuable resource for further research on PTMs in plants.

Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation.

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington’s disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.

Regulation of Dual Activity of Ascorbate Peroxidase 1 From Arabidopsis thaliana by Conformational Changes and Posttranslational Modifications.

Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS)-scavenging enzyme, which catalyzes the removal of hydrogen peroxide (H2O2) to prevent oxidative damage. The peroxidase activity of APX is regulated by posttranslational modifications (PTMs), such as S-nitrosylation, tyrosine nitration, and S-sulfhydration. In addition, it has been recently reported that APX functions as a molecular chaperone, protecting rice against heat stress. In this study, we attempted to identify the various functions of APX in Arabidopsis and the effects of PTMs on these functions. Cytosol type APX1 from Arabidopsis thaliana (AtAPX1) exists in multimeric forms ranging from dimeric to high-molecular-weight (HMW) complexes. Similar to the rice APX2, AtAPX1 plays a dual role behaving both as a regular peroxidase and a chaperone molecule. The dual activity of AtAPX1 was strongly related to its structural status. The main dimeric form of the AtAPX1 protein showed the highest peroxidase activity, whereas the HMW form exhibited the highest chaperone activity. Moreover, in vivo studies indicated that the structure of AtAPX1 was regulated by heat and salt stresses, with both involved in the association and dissociation of complexes, respectively. Additionally, we investigated the effects of S-nitrosylation, S-sulfhydration, and tyrosine nitration on the protein structure and functions using gel analysis and enzymatic activity assays. S-nitrosylation and S-sulfhydration positively regulated the peroxidase activity, whereas tyrosine nitration had a negative impact. However, no effects were observed on the chaperone function and the oligomeric status of AtAPX1. Our results will facilitate the understanding of the role and regulation of APX under abiotic stress and posttranslational modifications.

Modulating Microtubules: A Molecular Perspective on the Effects of Tail Modifications

Microtubules (MTs), an essential component of the eukaryotic cytoskeleton, are a lattice of polymerized tubulin dimers and are crucial for various cellular processes. The genetic and chemical diversity of tubulin and their disordered tails gives rise to a “tubulin code”. The functional role of tubulin post-translational modifications (PTMs), which contribute to the chemical diversity of the tubulin code, is gradually being unraveled. However, variation in the length and spatial organization of tubulin poly-modifications leads to an enormous combinatorial PTM space, which is difficult to study experimentally. Hence, the impact of the combinatorial tubulin PTM space on the biophysical properties of tubulin tails and their interactions with other proteins remains elusive.Here, we combine all-atom and coarse-grained molecular dynamics simulations to elucidate the biophysical implications of the large combinatorial tubulin PTM space in the context of an MT lattice. We find that tail–body interactions are more dominant in the tubulin dimer than in an MT lattice, and are more significant for the tails of α compared with β tubulin. In addition, polyglutamylation, but not polyglycylation, expands the dimensions of the tubulin tails. Polyglutamylation also leads to a decrease in the diffusion rate of MT-associated protein EB1 on MTs, while polyglycylation often increases diffusion rate. These observations are generally not sensitive to the organization of the polymodifications. The effect of PTMs on MT charge density and tail dynamics are also discussed. Overall, this study presents a molecular quantification of the biophysical properties of tubulin tails and their polymodifications, and provides predictions on the functional importance of tubulin PTMs.