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

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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Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury

Cell Death and Disease ◽

10.1038/s41419-020-03379-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Anna M. Kip ◽

Zita Soons ◽

Ronny Mohren ◽

Annet A. M. Duivenvoorden ◽

Anjali A. J. Röth ◽

...

Keyword(s):

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Profile Analysis ◽

Ischemia Reperfusion ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Intestinal Organoids

AbstractIntestinal ischemia-reperfusion (IR) injury is associated with high mortality rates, which have not improved in the past decades despite advanced insight in its pathophysiology using in vivo animal and human models. The inability to translate previous findings to effective therapies emphasizes the need for a physiologically relevant in vitro model to thoroughly investigate mechanisms of IR-induced epithelial injury and test potential therapies. In this study, we demonstrate the use of human small intestinal organoids to model IR injury by exposing organoids to hypoxia and reoxygenation (HR). A mass-spectrometry-based proteomics approach was applied to characterize organoid differentiation and decipher protein dynamics and molecular mechanisms of IR injury in crypt-like and villus-like human intestinal organoids. We showed successful separation of organoids exhibiting a crypt-like proliferative phenotype, and organoids exhibiting a villus-like phenotype, enriched for enterocytes and goblet cells. Functional enrichment analysis of significantly changing proteins during HR revealed that processes related to mitochondrial metabolism and organization, other metabolic processes, and the immune response were altered in both organoid phenotypes. Changes in protein metabolism, as well as mitophagy pathway and protection against oxidative stress were more pronounced in crypt-like organoids, whereas cellular stress and cell death associated protein changes were more pronounced in villus-like organoids. Profile analysis highlighted several interesting proteins showing a consistent temporal profile during HR in organoids from different origin, such as NDRG1, SDF4 or DMBT1. This study demonstrates that the HR response in human intestinal organoids recapitulates properties of the in vivo IR response. Our findings provide a framework for further investigations to elucidate underlying mechanisms of IR injury in crypt and/or villus separately, and a model to test therapeutics to prevent IR injury.

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IMMU-28. IMMUNOGENOMIC ANALYSIS REVEALS LGALS1 CONTRIBUTES TO THE IMMUNE HETEROGENEITY AND IMMUNOSUPPRESSION IN GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.382 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii365-iii365

Author(s):

Qun Chen ◽

Jinquan Cai ◽

Bo Han ◽

Xiangqi Meng

Keyword(s):

Prognostic Significance ◽

Suppressor Cells ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Genomic Variation ◽

Functional Enrichment ◽

Immunosuppressive Microenvironment

Abstract Mutualistic and dynamic communication between tumour cells and the surrounding microenvironment accelerates the initiation, progression, chemoresistance and immune evasion of glioblastoma (GBM). However, the immunosuppressive mechanisms of GBM has not been thoroughly elucidated to date. We enrolled six microenvironmental signatures to identify glioma microenvironmental genes. The functional enrichment analysis such as ssGSEA, ESTIMATE algorithm, Gene Ontology, Pathway analysis is conducted to discover the potential function of microenvironmental genes. In vivo and in vitro experiments are used to verify the immunologic function of LGALS1 in GBM. We screen eight glioma microenvironmental genes from glioma databases, and discover a key immunosuppressive gene (LGALS1 encoding Galectin-1) exhibiting obviously prognostic significance among glioma microenvironmental genes. Gliomas with different LGALS1 expression have specific genomic variation spectrums. Immunosuppression is a predominate characteristic in GBMs with high expression of LGALS1. Knockdown of LGALS1 remodels the GBM immunosuppressive microenvironment by down regulating M2 macrophages and myeloid-derived suppressor cells (MDSCs), and inhibiting immunosuppressive cytokines. Our results thus implied an important role of microenvironmental regulation in glioma malignancy and provided evidences of LGALS1 contributing to immunosuppressive environment in glioma and that targeting LGALS1 could remodel immunosuppressive microenvironment of glioma.

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Plant Physiology, 2021 Functional redox links between lumen thiol oxidoreductase1 and serine/threonine-protein kinase STN7

Plant Physiology ◽

10.1093/plphys/kiab091 ◽

2021 ◽

Cited By ~ 1

Author(s):

Jianghao Wu ◽

Liwei Rong ◽

Weijun Lin ◽

Lingxi Kong ◽

Dengjie Wei ◽

...

Keyword(s):

Protein Kinase ◽

Disulfide Bonds ◽

Kinase Activity ◽

Light Harvesting ◽

Photosynthetic Electron Transport ◽

State Transitions ◽

Light Harvesting Complex ◽

Light Harvesting Complex Ii

Abstract In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

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Benchmarking substrate-based kinase activity inference using phosphoproteomic data

10.1101/080978 ◽

2016 ◽

Author(s):

Claudia Hernandez-Armenta ◽

David Ochoa ◽

Emanuel Gonçalves ◽

Julio Saez-Rodriguez ◽

Pedro Beltrao

Keyword(s):

Kinase Activity ◽

Multiple Linear Regression Model ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Target Sequence ◽

Sequence Specificity ◽

Gold Standard Dataset ◽

Kolmogorov Smirnov

AbstractMotivationPhosphoproteomic experiments are increasingly used to study the changes in signalling occurring across different conditions. It has been proposed that changes in phosphorylation of kinase target sites can be used to infer when a kinase activity is under regulation. However, these approaches have not yet been benchmarked due to a lack of appropriate benchmarking strategies.ResultsWe curated public phosphoproteomic experiments to identify a gold standard dataset containing a total of 184 kinase-condition pairs where regulation is expected to occur. A list of kinase substrates was compiled and used to estimate changes in kinase activities using the following methods: Z-test, Kolmogorov Smirnov test, Wilcoxon rank sum test, gene set enrichment analysis (GSEA), and a multiple linear regression model (MLR). We also tested weighted variants of the Z-test, and GSEA that include information on kinase sequence specificity as proxy for affinity. Finally, we tested how the number of known substrates and the type of evidence (in vivo, in vitro or in silico) supporting these influence the predictions.ConclusionsMost models performed well with the Z-test and the GSEA performing best as determined by the area under the ROc curve (Mean AUC=0.722). Weighting kinase targets by the kinase target sequence preference improves the results only marginally. However, the number of known substrates and the evidence supporting the interactions has a strong effect on the predictions.

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Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2899 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2899-2908 ◽

Cited By ~ 153

Author(s):

A L Jackson ◽

P M Pahl ◽

K Harrison ◽

J Rosamond ◽

R A Sclafani

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Protein Interactions ◽

Kinase Activity ◽

Mitotic Cell ◽

Common Point ◽

Temperature Sensitive ◽

Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

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Cks1 Is Required for G1Cyclin–Cyclin-Dependent Kinase Activity in Budding Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.16.5858-5864.2000 ◽

2000 ◽

Vol 20 (16) ◽

pp. 5858-5864 ◽

Cited By ~ 39

Author(s):

Gregory J. Reynard ◽

William Reynolds ◽

Rati Verma ◽

Raymond J. Deshaies

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Budding Yeast ◽

Protein Kinase Activity ◽

Cyclin Dependent Kinase ◽

Multiple Substrates ◽

Cell Extracts

ABSTRACT p13suc1 (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G1 cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G1cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G1 cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G1 cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G1 phase in budding yeast.

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Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.7143 ◽

1995 ◽

Vol 15 (12) ◽

pp. 7143-7151 ◽

Cited By ~ 181

Author(s):

K S Lee ◽

Y L Yuan ◽

R Kuriyama ◽

R L Erikson

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Maximum Level ◽

Specific Protein ◽

Cyclin B ◽

Cdc2 Kinase ◽

M Phase ◽

Nih 3T3

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro.

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Nucleophosmin/B23 activates Aurora A at the centrosome through phosphorylation of serine 89

The Journal of Cell Biology ◽

10.1083/jcb.201107134 ◽

2012 ◽

Vol 197 (1) ◽

pp. 19-26 ◽

Cited By ~ 38

Author(s):

David Reboutier ◽

Marie-Bérengère Troadec ◽

Jean-Yves Cremet ◽

Kenji Fukasawa ◽

Claude Prigent

Keyword(s):

Protein Kinase ◽

Ribonucleic Acid ◽

Kinase Activity ◽

Spindle Assembly ◽

Aurora A ◽

Local Loss ◽

Cellular Processes ◽

G2 Phase ◽

Strong Activator

Aurora A (AurA) is a major mitotic protein kinase involved in centrosome maturation and spindle assembly. Nucleophosmin/B23 (NPM) is a pleiotropic nucleolar protein involved in a variety of cellular processes including centrosome maturation. In the present study, we report that NPM is a strong activator of AurA kinase activity. NPM and AurA coimmunoprecipitate and colocalize to centrosomes in G2 phase, where AurA becomes active. In contrast with previously characterized AurA activators, NPM does not trigger autophosphorylation of AurA on threonine 288. NPM induces phosphorylation of AurA on serine 89, and this phosphorylation is necessary for activation of AurA. These data were confirmed in vivo, as depletion of NPM by ribonucleic acid interference eliminated phosphorylation of CDC25B on S353 at the centrosome, indicating a local loss of AurA activity. Our data demonstrate that NPM is a strong activator of AurA kinase activity at the centrosome and support a novel mechanism of activation for AurA.

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Comprehensive Profile Analysis of Differentially Expressed circRNAs in Glucose Deprivation-Induced Human Nucleus Pulposus Cell Degeneration

BioMed Research International ◽

10.1155/2021/4770792 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Hongze Chang ◽

Hongzhang Wang ◽

Xiaolong Yang ◽

Kemin You ◽

Mingwei Jiang ◽

...

Keyword(s):

Intervertebral Disc ◽

Nucleus Pulposus ◽

Enrichment Analysis ◽

Nucleus Pulposus Cell ◽

Glucose Deprivation ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Differentially Expressed ◽

Cell Degeneration

Nucleus pulposus (NP) is the core substance to maintain the homeostasis of intervertebral disc and stability of biomechanics. The insufficient supply of nutrition (especially glucose) is an important factor that leads to the degeneration of NP cells. circRNAs play an important role in the process of intervertebral disc degeneration (IDD) by regulating the functions of NP cells. However, glucose deprivation-related circRNAs and their functions in IDD have not been reported. In this study, the differentially expressed circRNAs in NP cells after 0, 6, 12, and 24 h of glucose deprivation culture were detected by a microarray assay. Besides, time series clustering analysis by STEM software obtained the differentially up- and downregulated circRNAs during glucose deficiency. Then, the main functions and pathways of up- and downregulated circRNAs were predicted by the functional enrichment analysis. By constructing the circRNA-miRNA regulatory network, the potential mechanisms of the most differentially expressed circRNAs were predicted. In addition, according to in vitro validation, circ_0075062 was upregulated in degenerating NP tissues and glucose deprivation-induced NP cell degeneration. Based on Sanger sequencing and RNase tolerance assay, circ_0075062 was the circular transcript. Interfering with circ_0075062 expression could potentially alleviate the imbalance of extracellular matrix (ECM) synthesis and degradation in the NP cells induced by glucose deprivation. Together, these findings help us gain a comprehensive understanding of the underlying mechanisms of IDD, and circ_0075062 may be a promising therapeutic target of IDD.

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Identify Molecular Mechanisms of Jiangzhi Decoction on Nonalcoholic Fatty Liver Disease by Network Pharmacology Analysis and Experimental Validation

BioMed Research International ◽

10.1155/2020/8829346 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16

Author(s):

Lei Wang ◽

Yin Zhi ◽

Ying Ye ◽

Miao Zhang ◽

Xing Ma ◽

...

Keyword(s):

Liver Disease ◽

Fatty Liver Disease ◽

Molecular Mechanisms ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Active Ingredients ◽

Nonalcoholic Fatty Liver ◽

The Core

Background. Jiangzhi Decoction (JZD), a traditional herb mixture, has shown significant clinical efficacy against nonalcoholic fatty liver disease (NAFLD). However, its multicomponent and multitarget characteristics bring difficulty in deciphering its pharmacological mechanisms. Our study is aimed at identifying the core molecular mechanisms of JZD against NAFLD. Methods. The active ingredients were searched from Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Traditional Chinese Medicine Integrated Database (TCMID). The targets of those ingredients were identified using ChemMapper database based on 3D structure similarity. NAFLD-related genes were searched from DisGeNET database and Gene Expression Omnibus (GEO) database. Then, we performed protein-protein interaction (PPI) analysis, functional enrichment analysis, and constructed pathway networks of “herbs-active ingredients-candidate targets” and identified the core molecular mechanisms and key active ingredients in the network. Also, molecular docking was carried out to predict the ligands of candidate targets using SwissDock. Finally, the human hepatic L02 cell line was used to establish the NAFLD model in vitro. The effect and key molecules were validated by Oil Red O staining, biochemical assays, and quantitative real-time PCR (qRT-PCR). Results. We found 147 active ingredients in JZD, 1285 targets of active ingredients, 401 NAFLD-related genes, and 59 overlapped candidate targets of JZD against NAFLD. 22 core targets were obtained by PPI analysis. Finally, nuclear receptor transcription and lipid metabolism regulation were found as the core molecular mechanisms of JZD against NAFLD by functional enrichment analysis. The candidate targets PPARα and LXRα were both docked with hyperin as the most favorable interaction, and HNF4α was docked with linolenic acid ethyl ester. According to in vitro experiments, it was found that JZD had an inhibitory effect on lipid accumulation and regulatory effects on cholesterol and triglycerides. Compared with OA group, the mRNA expression levels of PPARα and HNF4α were significantly upregulated in JZD group ( P < 0.05 ), and LXRα was significantly downregulated ( P < 0.001 ). Conclusion. JZD might alleviate hepatocyte steatosis by regulating some key molecules related to nuclear receptor transcription and lipid metabolism, such as PPARα, LXRα, and HNF4α. Our study will provide the scientific evidences of the clinical efficacy of JZD against NAFLD.

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