scholarly journals Characterization of the Key Region and Key Phosphorylation Sites of EcaICE1 for its Molecular Interaction with EcaHOS1 Protein in Eucalyptus camaldulensis

2020 ◽  
Author(s):  
Ling Cheng ◽  
Weihua Zhang ◽  
Jianlin Hu ◽  
Ziyang Zhang ◽  
Yan Liu ◽  
...  
Keyword(s):  
Cold Stress ◽  
Protein Interactions ◽  
Phosphorylation Site ◽  
Eucalyptus Camaldulensis ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
Amino Acid Region ◽  
Mda Content ◽  
Proteasome Pathway

Abstract Background ICE1 (inducer of CBF expression 1), a MYC-like bHLH transcriptional activator, plays an important role in plant under cold stress via regulating transcriptional expression of downstream cold-responsive genes. Ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of expression of CBFs and their downstream genes under cold stress response in Arabidopsis , but the knowledge about ubiquitination regulation of ICE1 by HOS1 is still unknown in woody plants.Results The complete EcaICE1 gene and a new E3 ubiquitin ligase gene EcaHOS1 were amplified from the tissue culture seedlings of Eucalyptus camaldulensis . Yeast two-hybrid (Y2H) and BiFC assay results showed that EcaICE1 can interact with EcaHOS1 protein in the nucleus, and further Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 143, 145, 158 and 184 within the key interaction region were the potential phosphorylation sites of EcaICE1 based on bioinformatics analysis, and that only the substitution of Serine (Ser) 158 by Alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 by Y2H and β-galactosidase assays using site-direct mutagenesis. Overexpression of EcaICE1 and its mutations in Arabidopsis could significantly increase POD and SOD activities with a reduction for MDA content and up-regulate four cold-responsive genes ( CBF3 , KIN1 , COR15 and COR47A ) in the transgenic lines.Conclusion We first reported that EcaICE1 could interact with EcaHOS1 protein in Eucalyptus , and identified Ser 158 of EcaICE1 as the key phosphorylation site for its interaction with EcaHOS1 protein.

scholarly journals Characterization of the Key Region and Phosphorylation Sites of EcaICE1 for its Molecular Interaction with EcaHOS1 Protein from Eucalyptus camaldulensis

2019 ◽  
Author(s):  
Ling Cheng ◽  
Weihua Zhang ◽  
Jianlin Hu ◽  
Ziyang Zhang ◽  
Yan Liu ◽  
...  
Keyword(s):  
Cold Stress ◽  
Protein Interactions ◽  
Phosphorylation Site ◽  
Eucalyptus Camaldulensis ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
Reading Frame ◽  
Amino Acid Region ◽  
Proteasome Pathway

AbstractICE1 (inducer of CBF expression 1), a MYC-like bHLH transcriptional activator, plays an important role in plant under cold stress via regulating transcriptional expression of downstream cold-responsive genes. Ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of expression of CBFs and their downstream genes under cold stress response in Arabidopsis, but the knowledge about ubiquitination regulation of ICE1 by HOS1 is still limited in woody plants. In this study, a complete open reading frame of EcaICE1 gene and EcaHOS1 was amplified from the tissue culture seedlings of Eucalyptus camaldulensis. Yeast two-hybrid (Y2H) and BiFC assay results showed that EcaICE1 can interact with EcaHOS1 protein in the nucleus, and further Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 143, 145, 158 and 184 within the key interaction region were the potential phosphorylation sites of EcaICE1 based on bioinformatics analysis, and that only the substitution of Serine (Ser) 158 by Alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 by Y2H and β-galactosidase assays using site-direct mutagenesis. In summary, we first reported that EcaICE1 could interact with EcaHOS1 protein in Eucalyptus, and identified Ser 158 of EcaICE1 as the key phosphorylation site for its interaction with EcaHOS1 protein.

scholarly journals A Novel Phosphorylation Site-Kinase Network-Based Method for the Accurate Prediction of Kinase-Substrate Relationships

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Minghui Wang ◽  
Tao Wang ◽  
Binghua Wang ◽  
Yu Liu ◽  
Ao Li
Keyword(s):  
Protein Interactions ◽  
Phosphorylation Site ◽  
Prediction Performance ◽  
Accurate Prediction ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
Biological Mechanisms ◽  
Kinase Substrate ◽  
Prediction Tools ◽  
Local Sequence

Protein phosphorylation is catalyzed by kinases which regulate many aspects that control death, movement, and cell growth. Identification of the phosphorylation site-specific kinase-substrate relationships (ssKSRs) is important for understanding cellular dynamics and provides a fundamental basis for further disease-related research and drug design. Although several computational methods have been developed, most of these methods mainly use local sequence of phosphorylation sites and protein-protein interactions (PPIs) to construct the prediction model. While phosphorylation presents very complicated processes and is usually involved in various biological mechanisms, the aforementioned information is not sufficient for accurate prediction. In this study, we propose a new and powerful computational approach named KSRPred for ssKSRs prediction, by introducing a novel phosphorylation site-kinase network (pSKN) profiles that can efficiently incorporate the relationships between various protein kinases and phosphorylation sites. The experimental results show that the pSKN profiles can efficiently improve the prediction performance in collaboration with local sequence and PPI information. Furthermore, we compare our method with the existing ssKSRs prediction tools and the results demonstrate that KSRPred can significantly improve the prediction performance compared with existing tools.

scholarly journals Using Multilayer Heterogeneous Networks to Infer Functions of Phosphorylated Sites

2020 ◽  
Author(s):  
Joanne Watson ◽  
Jean-Marc Schwartz ◽  
Chiara Francavilla
Keyword(s):  
Protein Interactions ◽  
Phosphorylation Site ◽  
Random Permutation ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
Functional Annotations ◽  
Cellular Processes ◽  
Context Specific ◽  
Pathway Databases ◽  
Permutation Analysis

AbstractMass spectrometry-based quantitative phosphoproteomics has become an essential approach in the study of cellular processes such as cell signaling. Commonly used methods to analyze phosphoproteomics datasets depend on generic, gene-centric annotations such as Gene Ontology terms and do not account for the function of a protein in a given phosphorylation state. Thus, analysis of phosphoproteomics data is hampered by a lack of phosphorylated site-specific annotations. Here, we propose a method that combines shotgun phosphoproteomics data, protein-protein interactions and functional annotations from ontologies or pathway databases into a heterogeneous multilayer network. Phosphorylation sites are then associated to potential functions using a random walk on heterogeneous network (RWHN) algorithm. We validated our approach using a dataset modelling the MAPK/ERK pathway and were able to associate differentially regulated sites on the same protein to their previously described functions. Random permutation analysis proved that these associations were not random and were determined by the network topology. We then applied the RWHN algorithm to two previously published datasets; the algorithm was able to reproduce the experimentally validated conclusions from the publications, and associate phosphorylation sites with both new and known functions based on their regulatory patterns. The approach described here provides a robust, phosphorylation site-centric method to analyzing phosphoproteomics data and identifying potential context-specific functions for sites with similar phosphorylation profiles.

scholarly journals In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome

2019 ◽  
Author(s):  
Michael Charles Lanz ◽  
Kumar Yugandhar ◽  
Shagun Gupta ◽  
Ethan Sanford ◽  
Vitor Faça ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cell Biology ◽  
Budding Yeast ◽  
Protein Structures ◽  
Phosphorylation Site ◽  
Eukaryotic Cell ◽  
Phosphorylation Sites ◽  
Protein Protein Interactions ◽  
3 Dimensional ◽  
Regulate Protein

AbstractPhosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here we present a comprehensive phosphoproteomic dataset for budding yeast, comprised of over 30,000 high confidence phosphorylation sites identified by mass spectrometry. This single dataset nearly doubles the size of the known phosphoproteome in budding yeast and defines a set of cell cycle-regulated phosphorylation events. With the goal of enhancing the identification of functional phosphorylation events, we performed computational positioning of phosphorylation sites on available 3D protein structures and systematically identified events predicted to regulate protein complex architecture. Results reveal a large number of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic “clashes” predicted to disrupt the interaction. Phosphorylation site mutants experimentally validate our predictions and support a role for phosphorylation in negatively regulating protein-protein interactions. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

scholarly journals Genetic and Molecular Characterization of the Caenorhabditis elegans Gene, mel-26, a Postmeiotic Negative Regulator of MEI-1, a Meiotic-Specific Spindle Component

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 119-128
Author(s):  
M Rhys Dow ◽  
Paul E Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Interactions ◽  
Negative Regulator ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Gain Of Function ◽  
Recessive Mutations ◽  
Female Germline

Abstract We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

Proteomic Characterization of Protein-Protein Interactions

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Veenstra TD ◽  
Keyword(s):  
Protein Interactions ◽  
Disease Diagnosis ◽  
Cell System ◽  
Protein Protein Interactions ◽  
Novel Technologies ◽  
Cell Functions ◽  
Single Protein ◽  
A Cell ◽  
Molecular Components

Identifying all the molecular components within a living cell is the first step into understanding how it functions. To further understand how a cell functions requires identifying the interactions that occur between these components. This fact is especially relevant for proteins. No protein within a human cell functions on its own without interacting with another biomolecule - usually another protein. While Protein-Protein Interactions (PPI) have historically been determined by examining a single protein per study, novel technologies developed over the past couple of decades are enabling high-throughput methods that aim to describe entire protein networks within cells. In this review, some of the technologies that have led to these developments are described along with applications of these techniques. Ultimately the goal of these technologies is to map out the entire circuitry of PPI within human cells to be able to predict the global consequences of perturbations to the cell system. This predictive capability will have major impacts on the future of both disease diagnosis and treatment.

scholarly journals Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

2021 ◽  
Author(s):  
Syed N Shah
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Chromatin Assembly ◽  
Histone Chaperones ◽  
Protein Protein Interactions ◽  
Selective Forces ◽  
Assembly Proteins

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

scholarly journals Characterization of COMMD protein–protein interactions in NF-κB signalling

2006 ◽  
Vol 398 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Prim de Bie ◽  
Bart van de Sluis ◽  
Ezra Burstein ◽  
Karen J. Duran ◽  
Ruud Berger ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Copper Metabolism ◽  
Nuclear Factor Κb ◽  
Inhibitory Effect ◽  
Protein Family ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Necrosis Factor

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-κB (nuclear factor κB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-κB signalling through characterization of protein–protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-κB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-κB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-κB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IκBα (inhibitory κBα), indicating that both proteins inhibit NF-κB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein–protein interactions and provide new mechanistic insight into the function of this protein family in NF-κB signalling.

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