scholarly journals Quantitative isotope-labeled crosslinker proteomics reveals developmental variation in protein interactions and posttranslational modifications in Diaphorinacitri, the citrus greening insect vector

2021 ◽  
Author(s):  
John S Ramsey ◽  
Xuefei Zhong ◽  
Surya Saha ◽  
Juan D Chavez ◽  
Richard Johnson ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Chromatin Structure ◽  
Life Stage ◽  
Bacterial Pathogen ◽  
Protein Quantification ◽  
Citrus Greening ◽  
Insect Vector ◽  
Tail Region

Acquisition of the citrus greening bacterial pathogen, Candidatus Liberibacter asiaticus (CLas) by Asian citrus psyllid (Diaphorinacitri) nymphs is required efficient tree-to-tree transmission during the adult stage. Quantitative isotope-labeled protein interaction reporter (PIR) cross-linkers were used in parallel with protein quantification using spectral counting to quantify protein interactions within microbe-enriched cellular fractions of nymph and adult D. citri. Over 100 unique crosslinks were found between five insect histone proteins, and over 30% of these were more abundant in nymph compared to adult insects. Strikingly, some cross-links detected in D. citri proteins are conserved in cross-linking studies on human cells, suggesting these protein interaction topologies were present in the common ancestor (~750MYA) or are subject to convergent evolution. Analysis of posttranslational modifications of crosslinked histones revealed the presence of acetylated and methylated lysine residues, which may impact psyllid chromatin structure and gene expression. Histone H3 peptides acetylated in the N terminal tail region were found to be more abundant in nymph compared to adult insects in two orthogonal proteomics methods. The insect life stage-specific histone posttranslational modifications and protein interactions represent physical evidence that metamorphosis is associated with changes in chromatin structure that regulate genome-wide transcriptional reprogramming.

scholarly journals Protein interaction networks at the host–microbe interface in Diaphorina citri , the insect vector of the citrus greening pathogen

2017 ◽  
Vol 4 (2) ◽  
pp. 160545 ◽  
Author(s):  
J. S. Ramsey ◽  
J. D. Chavez ◽  
R. Johnson ◽  
S. Hosseinzadeh ◽  
J. E. Mahoney ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Bacterial Pathogen ◽  
Pathogen Transmission ◽  
Protein Interaction Networks ◽  
Interaction Networks ◽  
Differentially Expressed ◽  
Citrus Greening ◽  
Insect Vector ◽  
Diaphorina Citri ◽  
Citrus Greening Disease

The Asian citrus psyllid ( Diaphorina citri) is the insect vector responsible for the worldwide spread of ‘ Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host–microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host–microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.

scholarly journals Straightforward Protein-Protein Interaction Interface Mapping via Random Mutagenesis and Mammalian Protein Protein Interaction Trap (MAPPIT)

2019 ◽  
Vol 20 (9) ◽  
pp. 2058 ◽  
Author(s):  
Laurens Vyncke ◽  
Delphine Masschaele ◽  
Jan Tavernier ◽  
Frank Peelman
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Random Mutagenesis ◽  
Interaction Mechanism ◽  
Protein Protein Interaction ◽  
Interaction Sites ◽  
Multiple Partners ◽  
Interaction Trap ◽  
Mammalian Protein

The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available.

An efficient transient assays system using Agrobacterium-mediated transformation of onion (Allium cepa) epidermal cells

2020 ◽  
Vol 80 (03) ◽  
Author(s):  
Yu-Miao Zhang ◽  
Jun Wang ◽  
Tao Wu
Keyword(s):  
Subcellular Localization ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Epidermal Cells ◽  
Cyclin Dependent Kinase ◽  
Protein Subcellular Localization ◽  
Protein Protein Interactions ◽  
Efficient System ◽  
Protein Protein Interaction ◽  
Onion Epidermal Cells

In this study, the Agrobacterium infection medium, infection duration, detergent, and cell density were optimized. The sorghum-based infection medium (SbIM), 10-20 min infection time, addition of 0.01% Silwet L-77, and Agrobacterium optical density at 600 nm (OD600), improved the competence of onion epidermal cells to support Agrobacterium infection at >90% efficiency. Cyclin-dependent kinase D-2 (CDKD-2) and cytochrome c-type biogenesis protein (CYCH), protein-protein interactions were localized. The optimized procedure is a quick and efficient system for examining protein subcellular localization and protein-protein interaction.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

scholarly journals Mutations and Protein Interaction Landscape Reveal Key Cellular Events Perturbed in Upper Motor Neurons with HSP and PLS

2021 ◽  
Vol 11 (5) ◽  
pp. 578
Author(s):  
Oge Gozutok ◽  
Benjamin Ryan Helmold ◽  
P. Hande Ozdinler
Keyword(s):  
Motor Neurons ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Cortical Neurons ◽  
Therapeutic Interventions ◽  
Functional Identification ◽  
Protein Protein Interaction ◽  
Cellular Events ◽  
Interaction Domains ◽  
Upper Motor Neurons

Hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS) are rare motor neuron diseases, which affect mostly the upper motor neurons (UMNs) in patients. The UMNs display early vulnerability and progressive degeneration, while other cortical neurons mostly remain functional. Identification of numerous mutations either directly linked or associated with HSP and PLS begins to reveal the genetic component of UMN diseases. Since each of these mutations are identified on genes that code for a protein, and because cellular functions mostly depend on protein-protein interactions, we hypothesized that the mutations detected in patients and the alterations in protein interaction domains would hold the key to unravel the underlying causes of their vulnerability. In an effort to bring a mechanistic insight, we utilized computational analyses to identify interaction partners of proteins and developed the protein-protein interaction landscape with respect to HSP and PLS. Protein-protein interaction domains, upstream regulators and canonical pathways begin to highlight key cellular events. Here we report that proteins involved in maintaining lipid homeostasis and cytoarchitectural dynamics and their interactions are of great importance for UMN health and stability. Their perturbation may result in neuronal vulnerability, and thus maintaining their balance could offer therapeutic interventions.

scholarly journals The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

2018 ◽  
Vol 39 (3) ◽  
Author(s):  
Kyle T. Helzer ◽  
Mary Szatkowski Ozers ◽  
Mark B. Meyer ◽  
Nancy A. Benkusky ◽  
Natalia Solodin ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Protein Function ◽  
Estrogen Receptor Α ◽  
Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

scholarly journals Deciphering and engineering chromodomain-methyllysine peptide recognition

2018 ◽  
Vol 4 (11) ◽  
pp. eaau1447 ◽  
Author(s):  
Ryan Hard ◽  
Nan Li ◽  
Wei He ◽  
Brian Ross ◽  
Gary C. H. Mo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Regulation Of Gene Expression ◽  
Live Cells ◽  
Lysine Methylation ◽  
Dynamic Regulation ◽  
Peptide Recognition ◽  
Protein Functions ◽  
Domain Peptide

Posttranslational modifications (PTMs) play critical roles in regulating protein functions and mediating protein-protein interactions. An important PTM is lysine methylation that orchestrates chromatin modifications and regulates functions of non-histone proteins. Methyllysine peptides are bound by modular domains, of which chromodomains are representative. Here, we conducted the first large-scale study of chromodomains in the human proteome interacting with both histone and non-histone methyllysine peptides. We observed significant degenerate binding between chromodomains and histone peptides, i.e., different histone sites can be recognized by the same set of chromodomains, and different chromodomains can share similar binding profiles to individual histone sites. Such degenerate binding is not dictated by amino acid sequence or PTM motif but rather rooted in the physiochemical properties defined by the PTMs on the histone peptides. This molecular mechanism is confirmed by the accurate prediction of the binding specificity using a computational model that captures the structural and energetic patterns of the domain-peptide interaction. To further illustrate the power and accuracy of our model, we used it to effectively engineer an exceptionally strong H3K9me3-binding chromodomain and to label H3K9me3 in live cells. This study presents a systematic approach to deciphering domain-peptide recognition and reveals a general principle by which histone modifications are interpreted by reader proteins, leading to dynamic regulation of gene expression and other biological processes.

scholarly journals The Role of Posttranslational Modifications in DNA Repair

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Miaomiao Bai ◽  
Dongdong Ti ◽  
Qian Mei ◽  
Jiejie Liu ◽  
Xin Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Genome Stability ◽  
Protein Localization ◽  
Complex Structure ◽  
Protein Protein Interactions ◽  
Regulate Protein

The human body is a complex structure of cells, which are exposed to many types of stress. Cells must utilize various mechanisms to protect their DNA from damage caused by metabolic and external sources to maintain genomic integrity and homeostasis and to prevent the development of cancer. DNA damage inevitably occurs regardless of physiological or abnormal conditions. In response to DNA damage, signaling pathways are activated to repair the damaged DNA or to induce cell apoptosis. During the process, posttranslational modifications (PTMs) can be used to modulate enzymatic activities and regulate protein stability, protein localization, and protein-protein interactions. Thus, PTMs in DNA repair should be studied. In this review, we will focus on the current understanding of the phosphorylation, poly(ADP-ribosyl)ation, ubiquitination, SUMOylation, acetylation, and methylation of six typical PTMs and summarize PTMs of the key proteins in DNA repair, providing important insight into the role of PTMs in the maintenance of genome stability and contributing to reveal new and selective therapeutic approaches to target cancers.

scholarly journals Improving Classification of Protein Interaction Articles Using Context Similarity-Based Feature Selection

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Yifei Chen ◽  
Yuxing Sun ◽  
Bing-Qing Han
Keyword(s):  
Feature Selection ◽  
Protein Interaction ◽  
Text Classification ◽  
Protein Interactions ◽  
Reduction Rate ◽  
Importance Measure ◽  
Context Information ◽  
Selection Methods ◽  
Term Frequency ◽  
Context Similarity

Protein interaction article classification is a text classification task in the biological domain to determine which articles describe protein-protein interactions. Since the feature space in text classification is high-dimensional, feature selection is widely used for reducing the dimensionality of features to speed up computation without sacrificing classification performance. Many existing feature selection methods are based on the statistical measure of document frequency and term frequency. One potential drawback of these methods is that they treat features separately. Hence, first we design a similarity measure between the context information to take word cooccurrences and phrase chunks around the features into account. Then we introduce the similarity of context information to the importance measure of the features to substitute the document and term frequency. Hence we propose new context similarity-based feature selection methods. Their performance is evaluated on two protein interaction article collections and compared against the frequency-based methods. The experimental results reveal that the context similarity-based methods perform better in terms of theF1measure and the dimension reduction rate. Benefiting from the context information surrounding the features, the proposed methods can select distinctive features effectively for protein interaction article classification.

Furan warheads for covalent trapping of weak protein-protein interactions: cross-linking of thymosin β4 to actin

2021 ◽  
Author(s):  
Laia Miret Casals ◽  
Willem Vannecke ◽  
Kurt Hoogewijs ◽  
Gianluca Arauz ◽  
Marina Gay ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
3D Structure ◽  
Cross Linking ◽  
Thymosin Β4 ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Protein Protein Interaction ◽  
Covalent Trapping

We describe furan as a triggerable ‘warhead’ for site-specific cross-linking using the actin and thymosin β4 (Tβ4)-complex as model of a weak and dynamic protein-protein interaction with known 3D structure...

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