An advanced strategy for comprehensive profiling of ADP-ribosylation sites using mass spectrometry-based proteomics

ABSTRACTADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for comprehensive profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. While ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered >11,000 unique ADP-ribosylated peptides mapping to >7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. High-resolution MS/MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADP-ribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADP-ribosylation linked to the mitochondrion.

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Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

10.1101/2020.01.27.921650 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sara C. Buch-Larsen ◽

Ivo A. Hendriks ◽

Jean M. Lodge ◽

Martin Rykær ◽

Benjamin Furtwängler ◽

...

Keyword(s):

Electron Transfer ◽

Electron Transfer Dissociation ◽

Post Translational Modification ◽

Physiological Regulation ◽

Physiological Conditions ◽

Adp Ribosylation ◽

Cellular Processes ◽

Wide Range ◽

Enrichment Strategy

SUMMARYADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine auto-modifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

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Integrative Analysis of four miRNA prognostic signatures of prostate cancer

10.21203/rs.3.rs-34448/v1 ◽

2020 ◽

Author(s):

Chen Chi ◽

Xianwu Chen ◽

Liping Yao ◽

Min Li ◽

Lanting Xiang ◽

...

Keyword(s):

Prostate Cancer ◽

Overall Survival ◽

Target Genes ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Differentially Expressed ◽

Protein Protein Interaction ◽

Cellular Processes ◽

Relationship Of

Abstract Background Prostate cancer (PCa) is the most common urological cancer among men, having a poor prognosis, which is hard to accurately evaluate based on the present methods. MicroRNAs (miRNAs), a class of internal non-coding small RNA, can involve in the regulation of tumor biological function. So far, many researchers have tried to explore the relationship of malignant progress of PCa with miRNA, while there are just limited studies conducting the comprehensive analysis of miRNA in PCa clinical significance. Methods The data of miRNA and mRNA expressions in PCa were downloaded from TCGA database, and were performed the overall survival (OS) analysis using Survival package of R software to harvest the differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs). The bioinformatics tools such as TargetScan, miRDB, and miRanda were also conducted to forecast the desired target genes related with prognostic DEMs. In addition, both GO and KEGG analyses were used to uncover the fundamental signaling pathways and cellular processes in PCa as well as the protein-protein interaction (PPI) network was constructed through STRING and Cytoscape software. Results Firstly, 4 DEMs (miR-19a-3p, miR-144-3p, miR-223-5p, and miR-483-3p) were found having significantly associated with overall survival in PCa. Based on the criteria with FDR < 0.05 and |log2FC| > 1, 33 genes were screened out as DEGs. Besides, the functional enrichment analysis revealed that these DEGs of 4 miRNAs may participate in cancer-related pathways like FoxO and PI3K-Akt signaling pathway. Lastly, the low expression of CD177 may be potentially associated with poor survival of patients in PCa. Conclusion This study systematically analyzed multiple PCa prognostic DEMs (miR-19a-3p, miR-144-3p, miR-223-5p, and miR-483-3p), and verified a novel DEG signature (CD177) that can be used to effectively assess the prognosis of PCa patients.

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Co-expression analysis reveals interpretable gene modules controlled by trans-acting genetic variants

eLife ◽

10.7554/elife.58705 ◽

2020 ◽

Vol 9 ◽

Author(s):

Liis Kolberg ◽

Nurlan Kerimov ◽

Hedi Peterson ◽

Kaur Alasoo

Keyword(s):

Gene Expression ◽

Multiple Testing ◽

Disease Onset ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Cell Types ◽

Functional Enrichment ◽

Eqtl Analysis ◽

Cellular Processes ◽

Causal Processes

Understanding the causal processes that contribute to disease onset and progression is essential for developing novel therapies. Although trans-acting expression quantitative trait loci (trans-eQTLs) can directly reveal cellular processes modulated by disease variants, detecting trans-eQTLs remains challenging due to their small effect sizes. Here, we analysed gene expression and genotype data from six blood cell types from 226 to 710 individuals. We used co-expression modules inferred from gene expression data with five methods as traits in trans-eQTL analysis to limit multiple testing and improve interpretability. In addition to replicating three established associations, we discovered a novel trans-eQTL near SLC39A8 regulating a module of metallothionein genes in LPS-stimulated monocytes. Interestingly, this effect was mediated by a transient cis-eQTL present only in early LPS response and lost before the trans effect appeared. Our analyses highlight how co-expression combined with functional enrichment analysis improves the identification and prioritisation of trans-eQTLs when applied to emerging cell-type-specific datasets.

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UbiNet 2.0: a verified, classified, annotated and updated database of E3 ubiquitin ligase–substrate interactions

Database ◽

10.1093/database/baab010 ◽

2021 ◽

Vol 2021 ◽

Author(s):

Zhongyan Li ◽

Siyu Chen ◽

Jhih-Hua Jhong ◽

Yuxuan Pang ◽

Kai-Yao Huang ◽

...

Keyword(s):

Substrate Specificity ◽

Protein Interactions ◽

Regulatory Networks ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Ubiquitin Proteasome System ◽

Functional Enrichment ◽

Post Translational Modification ◽

Substrate Interactions ◽

E3 Ligases

Abstract Ubiquitination is an important post-translational modification, which controls protein turnover by labeling malfunctional and redundant proteins for proteasomal degradation, and also serves intriguing non-proteolytic regulatory functions. E3 ubiquitin ligases, whose substrate specificity determines the recognition of target proteins of ubiquitination, play crucial roles in ubiquitin–proteasome system. UbiNet 2.0 is an updated version of the database UbiNet. It contains 3332 experimentally verified E3–substrate interactions (ESIs) in 54 organisms and rich annotations useful for investigating the regulation of ubiquitination and the substrate specificity of E3 ligases. Based on the accumulated ESIs data, the recognition motifs in substrates for each E3 were also identified and a functional enrichment analysis was conducted on the collected substrates. To facilitate the research on ESIs with different categories of E3 ligases, UbiNet 2.0 performed strictly evidence-based classification of the E3 ligases in the database based on their mechanisms of ubiquitin transfer and substrate specificity. The platform also provides users with an interactive tool that can visualize the ubiquitination network of a group of self-defined proteins, displaying ESIs and protein–protein interactions in a graphical manner. The tool can facilitate the exploration of inner regulatory relationships mediated by ubiquitination among proteins of interest. In summary, UbiNet 2.0 is a user-friendly web-based platform that provides comprehensive as well as updated information about experimentally validated ESIs and a visualized tool for the construction of ubiquitination regulatory networks available at http://awi.cuhk.edu.cn/~ubinet/index.php.

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Systematic analysis of the expression and prognosis relevance of FBXO family reveals the significance of FBXO1 in human breast cancer

10.21203/rs.3.rs-86575/v1 ◽

2020 ◽

Author(s):

Yaqian Liu ◽

Bo Pan ◽

Weikun Qu ◽

Yilong Cao ◽

Jun Li ◽

...

Keyword(s):

Breast Cancer ◽

Prognostic Factors ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Systematic Analysis ◽

Cellular Processes ◽

Kaplan Meier ◽

Disease Indicators ◽

Kaplan Meier Plotter

Abstract Background: Breast cancer (BC) remains a prevalent and common form of cancer with high heterogeneity. Making efforts to explore novel molecular biomarkers and serve as potential disease indicators, which is essential to effectively enhance the prognosis and individualized treatment of BC. FBXO proteins act as the core component of E3 ubiquitin ligase, which play essential regulators roles in multiple cellular processes. Recently, research has indicated that FBXOs also play significant roles in cancer development. However, the molecular functions of these family members in BC have not been fully elucidated.Methods: In this research, we investigated the expression data, survival relevance and mutation situation of 10 FBXO members (FBXO1, 2, 5, 6, 16, 17, 22, 28, 31 and 45) in patients with BC from the Oncomine, GEPIA, HPA, Kaplan-Meier Plotter, UALCAN and cBioPortal databases. The high transcriptional levels of FBXO1 in different subtypes of BC were verified by immunohistochemical staining and the specific mutations of FBXO1 were obtained from COSMIC database. Top 10 genes with the highest correlation to FBXO1 were identified through cBioPortal and COXPRESdb tools. Additionally, functional enrichment analysis, PPI network and survival relevance of FBXO1 and co-expressed genes in BC were obtained from DAVID, STRING, UCSC Xena, GEPIA, bc-GenExMiner and Kaplan-Meier Plotter databases.Results: We found that FBXO2, FBXO6, FBXO16 and FBXO17 were potential favorable prognostic factors for BC. FBXO1, FBXO5, FBXO22, FBXO28, FBXO31 and FBXO45 may be the independent poor prognostic factors for BC. All of them were correlated to clinicopathological staging. Moreover, we identified that FBXO1 was an excellent molecular biomarker and therapeutic target for BC. Conclusion: This study implies that FBXO1, FBXO2, FBXO5, FBXO6, FBXO16, FBXO17, FBXO22, FBXO28, FBXO31 and FBXO45 genes are potential clinical targets and prognostic biomarkers for patients with BC.

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Phosphorylation Targets of DNA-PK and Their Role in HIV-1 Replication

Cells ◽

10.3390/cells9081907 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1907

Author(s):

Andrey Anisenko ◽

Marina Kan ◽

Olga Shadrina ◽

Anna Brattseva ◽

Marina Gottikh

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Dna Double Strand Breaks ◽

Cellular Processes ◽

Hiv 1

The DNA dependent protein kinase (DNA-PK) is a trimeric nuclear complex consisting of a large protein kinase and the Ku heterodimer. The kinase activity of DNA-PK is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). We also showed that the kinase activity of DNA-PK is essential for post-integrational DNA repair in the case of HIV-1 infection. Besides, DNA-PK is known to participate in such cellular processes as protection of mammalian telomeres, transcription, and some others where the need for its phosphorylating activity is not clearly elucidated. We carried out a systematic search and analysis of DNA-PK targets described in the literature and identified 67 unique DNA-PK targets phosphorylated in response to various in vitro and/or in vivo stimuli. A functional enrichment analysis of DNA-PK targets and determination of protein–protein associations among them were performed. For 27 proteins from these 67 DNA-PK targets, their participation in the HIV-1 life cycle was demonstrated. This information may be useful for studying the functioning of DNA-PK in various cellular processes, as well as in various stages of HIV-1 replication.

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Transcriptomic Analysis of Respiratory Tissue and Cell Line Models to Examine Glycosylation Machinery during SARS-CoV-2 Infection

Viruses ◽

10.3390/v13010082 ◽

2021 ◽

Vol 13 (1) ◽

pp. 82

Author(s):

Anup Oommen ◽

Stephen Cunningham ◽

Lokesh Joshi

Keyword(s):

Immune Response ◽

Biological Significance ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Vital Role ◽

Biomedical Literature ◽

Transcriptomic Analysis ◽

Functional Enrichment ◽

Post Translational Modification ◽

And Function

Glycosylation, being the most abundant post-translational modification, plays a profound role affecting expression, localization and function of proteins and macromolecules in immune response to infection. Presented are the findings of a transcriptomic analysis performed using high-throughput functional genomics data from public repository to examine the altered transcription of the human glycosylation machinery in response to SARS-CoV-2 stimulus and infection. In addition to the conventional in silico functional enrichment analysis methods we also present results from the manual analysis of biomedical literature databases to bring about the biological significance of glycans and glycan-binding proteins in modulating the host immune response during SARS-CoV-2 infection. Our analysis revealed key immunomodulatory lectins, proteoglycans and glycan epitopes implicated in exerting both negative and positive downstream inflammatory signaling pathways, in addition to its vital role as adhesion receptors for SARS-CoV-2 pathogen. A hypothetical correlation of the differentially expressed human glycogenes with the altered host inflammatory response and the cytokine storm-generated in response to SARS-CoV-2 pathogen is proposed. These markers can provide novel insights into the diverse roles and functioning of glycosylation pathways modulated by SARS-CoV-2, provide avenues of stratification, treatment, and targeted approaches for COVID-19 immunity and other viral infectious agents.

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CRAFT: a bioinformatics software for custom prediction of circular RNA functions

10.1101/2021.11.17.468947 ◽

2021 ◽

Author(s):

Anna Dal Molin ◽

Enrico Gaffo ◽

Valeria Difilippo ◽

Alessia Buratin ◽

Caterina Tretti Parenzan ◽

...

Keyword(s):

Regulatory Networks ◽

Rna Binding ◽

Rna Binding Proteins ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Circular Rna ◽

Functional Enrichment ◽

Circular Rnas ◽

Cellular Processes ◽

Functional Investigation

Circular RNAs (circRNAs), transcripts generated by backsplicing, are particularly stable and pleiotropic molecules, whose dysregulation drives human diseases and cancer by modulating gene expression and signaling pathways. CircRNAs can regulate cellular processes by different mechanisms, including interaction with microRNAs (miRNAs) and RNA-binding proteins (RBP), and encoding specific peptides. The prediction of circRNA functions is instrumental to interpret their impact in diseases, and to prioritize circRNAs for functional investigation. Currently, circRNA functional predictions are provided by web databases that do not allow custom analyses, while self-standing circRNA prediction tools are mostly limited to predict only one type of function, mainly focusing on the miRNA sponge activity of circRNAs. To solve these issues, we developed CRAFT (CircRNA Function prediction Tool), a freely available computational pipeline that predicts circRNA sequence and molecular interactions with miRNAs and RBP, along with their coding potential. Analysis of a set of circRNAs with known functions has been used to appraise CRAFT predictions and to optimize its setting. CRAFT provides a comprehensive graphical visualization of the results, links to several knowledge databases, and extensive functional enrichment analysis. Moreover, it originally combines the predictions for different circRNAs. CRAFT is a useful tool to help the user explore the potential regulatory networks involving the circRNAs of interest and generate hypotheses about the cooperation of circRNAs into the modulation of biological processes.

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Systems Biology Approaches Reveal a Multi-stress Responsive WRKY Transcription Factor and Stress Associated Gene Co-expression Networks in Chickpea

Current Bioinformatics ◽

10.2174/1574893614666190204152500 ◽

2019 ◽

Vol 14 (7) ◽

pp. 591-601 ◽

Cited By ~ 1

Author(s):

Aravind K. Konda ◽

Parasappa R. Sabale ◽

Khela R. Soren ◽

Shanmugavadivel P. Subramaniam ◽

Pallavi Singh ◽

...

Keyword(s):

Expression Pattern ◽

Gene Networks ◽

Molecular Mechanisms ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Wrky Transcription Factor ◽

Functional Enrichment ◽

Differentially Expressed ◽

Callose Deposition ◽

Significant Probability

Background: Chickpea is a nutritional rich premier pulse crop but its production encounters setbacks due to various stresses and understanding of molecular mechanisms can be ascribed foremost importance. Objective: The investigation was carried out to identify the differentially expressed WRKY TFs in chickpea in response to herbicide stress and decipher their interacting partners. Methods: For this purpose, transcriptome wide identification of WRKY TFs in chickpea was done. Behavior of the differentially expressed TFs was compared between other stress conditions. Orthology based cofunctional gene networks were derived from Arabidopsis. Gene ontology and functional enrichment analysis was performed using Blast2GO and STRING software. Gene Coexpression Network (GCN) was constructed in chickpea using publicly available transcriptome data. Expression pattern of the identified gene network was studied in chickpea-Fusarium interactions. Results: A unique WRKY TF (Ca_08086) was found to be significantly (q value = 0.02) upregulated not only under herbicide stress but also in other stresses. Co-functional network of 14 genes, namely Ca_08086, Ca_19657, Ca_01317, Ca_20172, Ca_12226, Ca_15326, Ca_04218, Ca_07256, Ca_14620, Ca_12474, Ca_11595, Ca_15291, Ca_11762 and Ca_03543 were identified. GCN revealed 95 hub genes based on the significant probability scores. Functional annotation indicated role in callose deposition and response to chitin. Interestingly, contrasting expression pattern of the 14 network genes was observed in wilt resistant and susceptible chickpea genotypes, infected with Fusarium. Conclusion: This is the first report of identification of a multi-stress responsive WRKY TF and its associated GCN in chickpea.

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Structural variations in papaya genomes

BMC Genomics ◽

10.1186/s12864-021-07665-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zhenyang Liao ◽

Xunxiao Zhang ◽

Shengcheng Zhang ◽

Zhicong Lin ◽

Xingtan Zhang ◽

...

Keyword(s):

Agronomic Traits ◽

Enrichment Analysis ◽

Copy Number Variant ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Structural Variations ◽

Reproduction Traits ◽

Depth Study ◽

Environmental Adaptability ◽

The Impact

Abstract Background Structural variations (SVs) are a type of mutations that have not been widely detected in plant genomes and studies in animals have shown their role in the process of domestication. An in-depth study of SVs will help us to further understand the impact of SVs on the phenotype and environmental adaptability during papaya domestication and provide genomic resources for the development of molecular markers. Results We detected a total of 8083 SVs, including 5260 deletions, 552 tandem duplications and 2271 insertions with deletion being the predominant, indicating the universality of deletion in the evolution of papaya genome. The distribution of these SVs is non-random in each chromosome. A total of 1794 genes overlaps with SV, of which 1350 genes are expressed in at least one tissue. The weighted correlation network analysis (WGCNA) of these expressed genes reveals co-expression relationship between SVs-genes and different tissues, and functional enrichment analysis shows their role in biological growth and environmental responses. We also identified some domesticated SVs genes related to environmental adaptability, sexual reproduction, and important agronomic traits during the domestication of papaya. Analysis of artificially selected copy number variant genes (CNV-genes) also revealed genes associated with plant growth and environmental stress. Conclusions SVs played an indispensable role in the process of papaya domestication, especially in the reproduction traits of hermaphrodite plants. The detection of genome-wide SVs and CNV-genes between cultivated gynodioecious populations and wild dioecious populations provides a reference for further understanding of the evolution process from male to hermaphrodite in papaya.

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