Investigate Protein Interactions Research Articles

RAB32 Ser71Arg in autosomal dominant Parkinson's disease: linkage, association, and functional analyses

Parkinson's disease is a progressive neurodegenerative disorder with multifactorial causes, among which genetic risk factors play a part. The RAB GTPases are regulators and substrates of LRRK2, and variants in the LRRK2 gene are important risk factors for Parkinson's disease. We aimed to explore genetic variability in RAB GTPases within cases of familial Parkinson's disease. We did whole-exome sequencing in probands from families in Canada and Tunisia with Parkinson's disease without a genetic cause, who were recruited from the Centre for Applied Neurogenetics (Vancouver, BC, Canada), an international consortium that includes people with Parkinson's disease from 36 sites in 24 countries. 61 RAB GTPases were genetically screened, and candidate variants were genotyped in relatives of the probands to assess disease segregation by linkage analysis. Genotyping was also done to assess variant frequencies in individuals with idiopathic Parkinson's disease and controls, matched for age and sex, who were also from the Centre for Applied Neurogenetics but unrelated to the probands or each other. All participants were aged 18 years or older. The sequencing and genotyping findings were validated by case-control association analyses using bioinformatic data obtained from publicly available clinicogenomic databases (AMP-PD, GP2, and 100 000 Genomes Project) and a private German clinical diagnostic database (University of Tübingen). Clinical and pathological findings were summarised and haplotypes were determined. In-vitro studies were done to investigate protein interactions and enzyme activities. Between June 1, 2010, and May 31, 2017, 130 probands from Canada and Tunisia (47 [36%] female and 83 [64%] male; mean age 72·7 years [SD 11·7; range 38-96]; 109 White European ancestry, 18 north African, two east Asian, and one Hispanic] underwent whole-exome sequencing. 15 variants in RAB GTPase genes were identified, of which the RAB32 variant c.213C>G (Ser71Arg) cosegregated with autosomal dominant Parkinson's disease in three families (nine affected individuals; non-parametric linkage Z score=1·95; p=0·03). 2604 unrelated individuals with Parkinson's disease and 344 matched controls were additionally genotyped, and five more people originating from five countries (Canada, Italy, Poland, Turkey, and Tunisia) were identified with the RAB32 variant. From the database searches, in which 6043 individuals with Parkinson's disease and 62 549 controls were included, another eight individuals were identified with the RAB32 variant from four countries (Canada, Germany, UK, and USA). Overall, the association of RAB32 c.213C>G (Ser71Arg) with Parkinson's disease was significant (odds ratio [OR] 13·17, 95% CI 2·15-87·23; p=0·0055; I2=99·96%). In the people who had the variant, Parkinson's disease presented at age 54·6 years (SD 12·75, range 31-81, n=16), and two-thirds had a family history of parkinsonism. RAB32 Ser71Arg heterozygotes shared a common haplotype, although penetrance was incomplete. Findings in one individual at autopsy showed sparse neurofibrillary tangle pathology in the midbrain and thalamus, without Lewy body pathology. In functional studies, RAB32 Arg71 activated LRRK2 kinase to a level greater than RAB32 Ser71. RAB32 Ser71Arg is a novel genetic risk factor for Parkinson's disease, with reduced penetrance. The variant was found in individuals with Parkinson's disease from multiple ethnic groups, with the same haplotype. In-vitro assays show that RAB32 Arg71 activates LRRK2 kinase, which indicates that genetically distinct causes of familial parkinsonism share the same mechanism. The discovery of RAB32 Ser71Arg also suggests several genetically inherited causes of Parkinson's disease originated to control intracellular immunity. This shared aetiology should be considered in future translational research, while the global epidemiology of RAB32 Ser71Arg needs to be assessed to inform genetic counselling. National Institutes of Health, the Canada Excellence Research Chairs program, Aligning Science Across Parkinson's, the Michael J Fox Foundation for Parkinson's Research, and the UK Medical Research Council.

Abstract 2269: Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer

Abstract PIK3CA mutations occur in ~15% of triple-negative breast cancers (TNBCs) and aberrantly activate the PI3K-AKT pathway, resulting in uncontrolled cell proliferation. Alpelisib is approved for hormone receptor-positive (HR+), HER2-negative BC patients with a PIK3CA mutation; however, not all PIK3CA-mutated (MUT), HR+ patients respond to alpelisib and others acquire resistance following exposure, suggesting adaptive mechanisms of PI3K activation. It has been proven that in TNBC, there exists a cohort of mutated genes which activate PI3K-AKT signaling. We hypothesize that testing alpelisib in combination with repurposed approved drugs, targeting alternative mechanisms of PI3K-AKT pathway activation, may be used to treat a cohort of TNBC patients. TNBC patient genetic data from the MSK (N=168), METABRIC (N=209), and TCGA (N=171) datasets were analyzed using cBioPortal to identify frequently occurring mutations. Differential expression analyses based on mutational status were performed on CCLE cell line (N=705) microarray and TCGA BC patient (N=981) RNA-sequencing data using R. Dysregulated genes were functionally analyzed using DAVID Ontology and those associated with poorer survival in TNBC were identified using KM-Plotter. STRING database was used to investigate protein interactions. The antiproliferative effects of targeted drugs tested alone and in combination in a panel of 6 TNBC cell lines were determined using acid phosphatase assays. Reverse-phase protein array analysis was carried out on protein lysates from cells following 24 hour treatment with drugs used alone and in combination.TP53 is the most frequently mutated gene in TNBC and mutations were associated with significant genetic dysregulation. A comprehensive analysis of upregulated genes in TP53-MUT TNBC led to the identification of 5 potential therapeutic targets - MET, S100A9, LCN2, CA9, TACSTD2, and KLK5 - based on their association with poorer survival in TNBC, protein interactions with TP53 and PIK3CA, and availability of targeted drugs. MET was selected for in vitro analysis based on its strong correlation with PI3K-AKT signaling. The presence of a TP53 mutation was associated with decreased antiproliferative effects in TNBC cell lines treated with alpelisib. Treatment with alpelisib in combination with a MET inhibitor, crizotinib, demonstrated greater antiproliferative effects in TP53-MUT/PIK3CA-MUT cell lines when compared to TP53 or PIK3CA MUT only models.TP53 mutations are associated with genetic dysregulation in TNBC and may confer resistance to alpelisib. Targeting upregulated proteins in a cohort of TP53-MUT/PIK3CA-MUT patients may improve sensitivity to alpelisib via dual inhibition of PI3K-AKT pathway activation. Protein analysis will uncover the mechanisms of synergy observed between alpelisib and crizotinib, and the therapeutic potential of the combination will be experimentally investigated in vivo. Citation Format: Grace Colley, Alexander Eustace, Dalal AlSultan. Dual targeting of the PI3K-AKT pathway in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2269.

A novel cell biological tool to explain mechanics and dynamics in fission yeast.

The Rho guanosine triphosphatase hydrolase enzyme (GTPase) is required for the control of the actin cytoskeleton, but its activation in vivo condition is unknown. The study's goal was to find a new synthetic nanobody VHH (P-36 tagged with mNeonGreen) that interacts strongly with the Rho GTPase. We present the first novel synthetic nanobody, VHH (P-36 tagged with mNeonGreen), tested in fission yeast cells and found to have a particular interaction with Rho1GTPase. Plasmids were constructed by using of certain enzymes to digest the pDUAL-pef1a vector plasmid to produce a protein that was encoded by cloned genes. A varied VHH library was created synthetically, then transformed into yeast cells, and positive clones were chosen using chemical agents. To investigate protein interactions and cellular reactions, several studies were carried out, such as live cell imaging, growth curve analysis, coimmunoprecipitation, structural analysis, and cell therapies. Prism and RStudio were used for the statistical analysis. The presence of VHH (P-36) has no effect on the growth pattern making it an appropriate model for studying cytokinesis in vivo. According to a computational biological study, its affinity to interact with Rho1GTPase with all the complementarity-determining region (CDR) regions found on VHH (P-36) is extremely strong. We were able to track its subcellular target by localization using a fluorescent confocal microscope, ensuring the maintenance of cell polarity and morphology. Spheroplast analysis revealed a circular-shaped cell with an even distribution of Rho1 tagged VHH (P-36), indicating that the interaction occurs near the plasma membrane. The introduction of latrunculin-A (Lat-A) disrupted Rho GTPase localization, demonstrating the control over actin production, and the cell did not show evidence of mitotic phase commencement while Lat-A was present. Finally, this important biological tool can aid in our understanding of the mechanics and dynamics of cytokinesis in relation to Rho1GTPase.

Interactome Analysis Reveals a Link of the Novel ALMS1-CEP70 Complex to Centrosomal Clusters

Alström syndrome (ALMS) is a very rare autosomal-recessive disorder, causing a broad range of clinical defects most notably retinal degeneration, type 2 diabetes, and truncal obesity. The ALMS1 gene encodes a complex and huge ∼0.5MDa protein, which has hampered analysis in the past. The ALMS1 protein is localized to the centrioles and the basal body of cilia and is involved in signaling processes, for example, TGF-β signaling. However, the exact molecular function of ALMS1 at the basal body remains elusive and controversial. We recently demonstrated that protein complex analysis utilizing endogenously tagged cells provides an excellent tool to investigate protein interactions of ciliary proteins. Here, CRISPR/Cas9-mediated endogenously tagged ALMS1 cells were used for affinity-based protein complex analysis. Centrosomal and microtubule-associated proteins were identified, which are potential regulators of ALMS1 function, such as the centrosomal protein 70kDa (CEP70). Candidate proteins were further investigated in ALMS1-deficient hTERT-RPE1 cells. Loss of ALMS1 led to shortened cilia with no change in structural protein localization, for example, acetylated and ɣ-tubulin, Centrin-3, or the novel interactor CEP70. Conversely, reduction of CEP70 resulted in decreased ALMS1 at the ciliary basal body. Complex analysis of CEP70 revealed domain-specific ALMS1 interaction involving the TPR-containing C-terminal (TRP-CT) fragment of CEP70. In addition to ALMS1, several ciliary proteins, including CEP135, were found to specifically bind to the TPR-CT domain. Data are available via ProteomeXchange with the identifier PXD046401. Protein interactors identified in this study provide candidate lists that help to understand ALMS1 and CEP70 function in cilia-related protein modification, cell death, and disease-related mechanisms.

Potential Therapeutic Mechanism of Radix Angelicae Biseratae and Dipsaci Radix Herb Pair against Osteoarthritis: Based on Network Pharmacology and Molecular Docking.

A major contributor to older disability is osteoarthritis. Radix Angelicae Biseratae (known as Duhuo in China, DH, the dried rhizome of Angelica pubescens) and Dipsaci Radix (known as Xuduan in China, XD, the dried rhizome of Dipsacus asper Wall) herb pair (DXHP) is widely used to treat osteoarthritis, but the underlying molecular mechanisms still have not been revealed. This research aimed to illustrate the therapeutic mechanism of DXHP against osteoarthritis through the techniques of network pharmacology and molecular docking. Gene targets for osteoarthritis and active ingredients for DXHP were screened based on the pharmacology public database and the gene-disease target database. The software program Cytoscape was used to visualize the active chemical target-disease gene network. The STRING biological information website was used to investigate protein interactions. On the Metascape bioinformatics website, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were carried out. The molecular docking of the important chemicals and primary targets identified by the aforementioned screening was performed using Autodock software. Twenty-six active substances from the DXHP that had strong connections to 138 osteoarthritis-related targets were screened out. According to network analysis, TNF, GAPDH, IL-6, AKT-1, IL-1B, and VEGFA are prospective therapeutic targets, while osthole, cauloside A, ammidin, angelicone, beta-sitosterol, and asperosaponin VI may be significant active components. 1705 biological processes (BP), 155 molecular functions (MF), and 89 cellular components (CC) were identified by GO analysis. KEGG analysis indicated that IL-17, NF-kappa B, HIF-1, MAPK, and AGE-RAGE signaling pathways are potentially involved. Molecular docking showed that cauloside A, osthole, and β-sitosterol have excellent binding activity with main targets. This study comprehensively illuminated the active ingredients, potential targets, primary pharmacological effects, and relevant mechanisms of the DXHP in the treatment of OA. These findings provide fresh thoughts into the therapeutic mechanisms of the main active ingredients of DXHP and provide a reference for further exploration and clinical applications of DXHP.

Use of legume flours and fiber for tailoring structure and texture of pea protein-based extruded meat alternatives.

The overall objective of this study was to understand texturization of pea protein isolate (PPI) using low moisture extrusion, and investigate protein interactions, functionality, and cross-linking with the inclusion of different levels of pea fiber (5-15%) and different types of starch-containing legume flours (20% chickpea flour or pea flour). PPI/ legume flour raw formulations had 18-27% lower water absorption capacity (WAC) as compared to the PPI control. However, WAC increased by 8-16% with the addition of pea fiber to a PPI/ legume flour control. Rapid Visco Analysis trends mirrored these results with peak viscosity shifting to higher temperatures with the addition of legume flour and lower temperatures with the addition of pea fiber. The role of starch in interfering with protein hydrophilic interactions and that of fiber in decoupling this effect were discussed. These interactions determined extruded textured protein properties, with more layering and denser products (174-229% higher bulk density as compared to control) observed with the addition of legume flours leading to lower water hydration capacity (WHC), as opposed to more cellular and porous microstructure (55-58% lower bulk density as compared to control) with the addition of fiber. Bulk density and WHC trends due to these porosity and layering effects impacted the instrumental texture characteristics of ground hydrated product, including hardness that increased from 475g to 837-2334g with the higher layering caused by starch, but decreased from 1295g to 534-1050g due to the porosity induced by fiber. To summarize, the use of legume flours and fiber can allow flexibility in targeting specific qualities while reducing costs and increasing sustainability of plant-based meats. PRACTICAL APPLICATION: Health, environment, and animal welfare concerns are creating a growing movement toward plant-based meat. Pea protein isolate and concentrate have become popular ingredients for texturized plant protein. Understanding of the role of starch and fiber in the structuring of textured pea protein could lead to use of legume flours and co-products of protein isolation to reduce cost and increase sustainability and nutrition of meat alternatives while targeting desired textural attributes.

A proximity labeling method for protein-protein interactions on cell membrane.

Antibodies targeting specific antigens are widely utilized in biological research to investigate protein interactions or to quantify target antigens. Here, we introduce antigen–antibody proximity labeling (AAPL), a novel method to map the antigen interaction sites as well as interactors of antibody-targeted proteins. As a proof of concept, AAPL was demonstrated using sodium/potassium transporting ATPase (ATP1A1) and epidermal growth factor receptor 2 (ERBB2)-specific antibodies that were modified with an Fe(iii) catalytic probe. Once bound to their target proteins, Fe(iii)-induced catalytic oxidation occurred in proximity of the antigen's epitope. Oxidative proteomic analysis was then used to determine the degree of oxidation, the site of oxidation within the targeted antigen, and the interacting proteins that were in close proximity to the targeted antigen. An AAPL score was generated for each protein yielding the specificity of the oxidation and proximity of the interacting protein to the target antigen. As a final demonstration of its utility, the AAPL approach was applied to map the interactors of liver–intestine-cadherin (CDH17) in colon cancer cells.

Systematic characterization of extracellular glycoproteins using mass spectrometry.

Surface and secreted glycoproteins are essential to cells and regulate many extracellular events. Because of the diversity of glycans, the low abundance of many glycoproteins, and the complexity of biological samples, a system-wide investigation of extracellular glycoproteins is a daunting task. With the development of modern mass spectrometry (MS)-based proteomics, comprehensive analysis of different protein modifications including glycosylation has advanced dramatically. This review focuses on the investigation of extracellular glycoproteins using MS-based proteomics. We first discuss the methods for selectively enriching surface glycoproteins and investigating protein interactions on the cell surface, followed by the application of MS-based proteomics for surface glycoprotein dynamics analysis and biomarker discovery. We then summarize the methods to comprehensively study secreted glycoproteins by integrating various enrichment approaches with MS-based proteomics and their applications for global analysis of secreted glycoproteins in different biological samples. Collectively, MS significantly expands our knowledge of extracellular glycoproteins and enables us to identify extracellular glycoproteins as potential biomarkers for disease detection and drug targets for disease treatment.

Photoactivatable Glycolipid Probes for Identifying Mycolate-Protein Interactions in Live Mycobacteria.

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

A Phosphorylation Dependent Regulatory Switch at the Gα12 N‐terminus Modulates Cell Growth Signaling

The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins (G proteins) is composed of Gα12 and Gα13, which play important intracellular signaling roles including activation of serum response factor (SRF). SRF activates transcription of serum response element (SRE) regulated genes involved in cell growth and tumorigenic events. Although crystal structures for Gα12 and Gα13 have been reported, very little is understood about the structure and function of these proteins at the N‐terminus. Previous reports have suggested serine phosphorylation occurs near the N‐terminus of Gα12. To identify the target serine in Gα12 and its possible role in SRF signaling, a series of point mutations were engineered to either abolish or mimic serine phosphorylation. Replacing a Gα12‐specific N‐terminal serine with the phosphomimic aspartic acid disrupted Gα12 signaling to SRF, as measured by firefly luciferase assays. Signaling by Gα13 was unaffected by phosphomimics of potentially homologous serines, suggesting a Gα12‐specific mechanism. Insertion of the Gα12 N‐terminus harboring the phosphomimic into Gα13 bestowed negative regulation of SRF signaling upon this chimeric protein. These results point to a kinase‐dependent switching mechanism at the N‐terminus of Gα12 that governs its growth signaling. Currently, we are investigating protein interactions affected by the phosphomimic and screening potential kinases for phosphorylation of Gα12.Support or Funding InformationNC GlaxoSmithKline Foundation; UNC Lineberger Comprehensive Cancer Center; NC Biotechnology Center

Using X-ray footprinting to investigate protein interactions and conformation

Using X-ray footprinting to investigate protein interactions and conformation

Workflows and considerations for investigating protein interactions of viral DNA sensors.

DNA sensors are a core component of innate immunity in mammalian cells. In response to pathogen infection, these specialized proteins sense pathogenic DNA from bacteria or viruses and initiate immune signaling cascades. These defense mechanisms rely on the rapid formation and temporal regulation of protein-protein interactions. Similarly, protein interactions underlie virus immune evasion mechanisms, as proteins from diverse viruses associate with and inhibit DNA sensors. Here, we describe experimental protocols for identifying protein interactions of DNA sensors, and discuss considerations for optimal isolation of protein complexes when targeting either endogenous or tagged proteins. Additionally, as viral infections and immune responses are known to induce prominent changes in cellular protein abundances, we provide a workflow for investigating these protein associations in the context of proteome alterations.

Photocrosslinking Approach to Investigate Protein Interactions in the BCL-2 Family.

The Bcl-2 family of proteins regulates mitochondrial outer membrane permeability thereby making life or death decisions for cells. Most of Bcl-2 proteins contain hydrophobic regions that are embedded in intracellular membranes such as mitochondria. These membrane proteins are difficult to express and purify thereby preluding biochemical and biophysical characterizations. Here, we describe a photocrosslinking approach based on in vitro synthesis of Bcl-2 proteins with photoreactive amino acid analogs incorporated at specific locations. These photoreactive proteins are reconstituted into liposomal membranes with defined phospholipids or mitochondrial membranes isolated from animals, and their interactions with other Bcl-2 proteins are detected by photocrosslinking.

Abstract 1367: BRCA1 and BARD1 protein interactions that are required for DNA repair function

Abstract Breast and ovarian cancers are prevalent among women, and hereditary breast and ovarian cancers (HBOCs) have been associated with germline mutations in genes such as BRCA1 and BARD1. BRCA1 and BARD1 form an obligate heterodimer, and the BRCA1/BARD1 complex is required for tumor suppression functions. Our lab has tested hundreds of BARD1 and BRCA1 variants and identified many that are deficient in homologous recombination, which we have shown to accurately predict cancer predisposition in the clinic. Several of these functionally defective BRCA1 mutants map to a pocket of amino acids on the surface of the protein that does not have any known binding partners. Repair-deficient mutations are also present on the surface of the BARD1 protein in domains that are not known to be associated with DNA repair. We hypothesize that the DNA repair deficiencies mediated by these BRCA1 and BARD1 mutants are due to differences in protein binding when compared to wild-type protein. We have found that BRCA1 mutants in this protein pocket do not phosphorylate and do not localize to the nucleus following DNA damage, both of which are characteristic of the DNA damage response. However, BARD1 repair-deficient mutants still bind phosphorylated BRCA1, indicating that their deficiencies are not due to loss of BRCA1 function. To investigate protein interaction differences we are creating fusion proteins of wild-type and mutant BRCA1 with BioID2, which will biotinylate proteins that bind to BRCA1. We will then be able to identify, via avidin purification and mass spectrometry, protein interactions that are present in the wild-type but absent in the mutant BRCA1. Novel proteins that we identify will be tested for DNA repair and tumor suppressor function. This information will allow us to better understand both BARD1 and BRCA1 function and the mechanism of homologous recombination. Citation Format: Aleksandra Adamovich, Margaret Wingo, Tapahsama Banerjee, Miranda Gardner, Michael Freitas, Jeffrey Parvin. BRCA1 and BARD1 protein interactions that are required for DNA repair function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1367.

Isolation of Phagosomes Containing Bovine Photoreceptor Outer Segments from Retinal Pigment Epithelial Cells by Magnetic Selection

Daily phagocytosis of the distal tips of photoreceptor cells by the retinal pigment epithelium (RPE) is known to be an essential mechanism in the maintenance of the retina. The outermost portions of the photoreceptor cells, termed outer segments, are ingested and subsequently processed by the RPE. Previous studies involving the isolation of phagosomes have utilized latex beads to induce phagocytosis in macrophages, which lacks specificity due to the large foreign body being ingested. Therefore, the purpose of this study was to develop a standard protocol for the isolation of outer segment enriched phagosomes from RPE cells. To generate phagosomes with the selected cargo, outer segments isolated from bovine retinas were covalently labelled with magnetite particles and then fed to RPE‐J cells. After feeding, the cells were washed and gently lysed with a Dounce homogenizer. The phagosomes containing the magnetite bound outer segments were collected via magnetic rack isolation and stored in a homogenization solution. Immunoblots using antibodies against Mertk, N and C‐terminal rhodopsin, and cathepsin D confirmed the presence of outer segment‐containing phagosomes of varying maturity in the isolated fraction. The protocol we have developed has successfully isolated outer segment enriched phagosomes from RPE‐J cells by magnetic selection. This study differentiates itself from other studies that employ latex beads to isolate phagosomes through density gradient centrifugation. The isolation of these phagosomes will potentially play an important role in investigating protein interactions that impact RPE membrane trafficking and maturation.Support or Funding InformationFlorida Southern College Faculty‐Student Collaborative Research GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies.

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.

Redox-Inactive Peptide Disrupting Trx1-Ask1 Interaction for Selective Activation of Stress Signaling.

Thioredoxin 1 (Trx1) and glutaredoxin 1 (Grx1) are two ubiquitous redox enzymes that are central for redox homeostasis but also are implicated in many other processes, including stress sensing, inflammation, and apoptosis. In addition to their enzymatic redox activity, a growing body of evidence shows that Trx1 and Grx1 play regulatory roles via protein-protein interactions with specific proteins, including Ask1. The currently available inhibitors of Trx1 and Grx1 are thiol-reactive electrophiles or disulfides that may suffer from low selectivity because of their thiol reactivity. In this report, we used a phage peptide library to identify a 7-mer peptide, 2GTP1, that binds to both Trx1 and Grx1. We further showed that a cell-permeable derivative of 2GTP1, TAT-2GTP1, disrupts the Trx1-Ask1 interaction, which induces Ask1 phosphorylation with subsequent activation of JNK, stabilization of p53, and reduced viability of cancer cells. Notably, as opposed to a disulfide-derived Trx1 inhibitor (PX-12), TAT-2GTP1 was selective for activating the Ask1 pathway without affecting other stress signaling pathways, such as endoplasmic reticulum stress and AMPK activation. Overall, 2GTP1 will serve as a useful probe for investigating protein interactions of Trx1.

SwitchSENSE: A new technology to study protein-RNA interactions.

Characterization of RNA-binding protein interactions with RNA became inevitable to properly understand the cellular mechanisms involved in gene expression regulation. Structural investigations bring information at the atomic level on these interactions and complementary methods such as Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) are commonly used to quantify the affinity of these RNA-protein complexes and evaluate the effect of mutations affecting these interactions. The switchSENSE technology has recently been developed and already successfully used to investigate protein interactions with different types of binding partners (DNA, protein/peptide or even small molecules). In this study, we show that this method is also well suited to study RNA binding proteins (RBPs). We could successfully investigate the binding to RNA of three different RBPs (Fox-1, SRSF1 and Tra2-β1) and obtained KD values very close to the ones determined previously by SPR or ITC for these complexes. These results show that the switchSENSE technology can be used as an alternative method to study protein-RNA interactions with KD values in the low micromolar (10-6) to nanomolar (10-7-10-9) and probably picomolar (10-10-10-12) range. The absence of labelling requirement for the analyte molecules and the use of very low amounts of protein and RNA molecules make the switchSENSE approach very attractive compared to other methods. Finally, we discuss about the potential of this approach in obtaining more sophisticated information such as structural conformational changes upon RBP binding to RNA.

A novel DFP tripeptide motif interacts with the coagulation factor XI apple 2 domain

AbstractFactor XI (FXI) is the zymogen of FXIa, which cleaves FIX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and interacts with multiple proteins via its 4 apple domains in the “saucer section” of the enzyme; however, to date, no complex crystal structure has been described. To investigate protein interactions of FXI, a large random peptide library consisting of 106 to 107 peptides was screened for FXI binding, which identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins, including the known ligand high-molecular-weight kininogen (HK), as well as the extracellular matrix proteins laminin and collagen V. To define the binding site on FXI, we determined the crystal structure of FXI in complex with the HK-derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain, and central to the interaction, the DFP phenylalanine side-chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin-derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket, although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain.

Proteomic and Microscopic Strategies towards the Analysis of the Cytoskeletal Networks in Major Neuropsychiatric Disorders.

Mental health disorders have become worldwide health priorities. It is estimated that in the next 20 years they will account for a 16 trillion United State dollars (US$) loss. Up to now, the underlying pathophysiology of psychiatric disorders remains elusive. Altered cytoskeleton proteins expression that may influence the assembly, organization and maintenance of cytoskeletal integrity has been reported in major depressive disorders, schizophrenia and to some extent bipolar disorders. The use of quantitative proteomics, dynamic microscopy and super-resolution microscopy to investigate disease-specific protein signatures holds great promise to improve our understanding of these disorders. In this review, we present the currently available quantitative proteomic approaches use in neurology, gel-based, stable isotope-labelling and label-free methodologies and evaluate their strengths and limitations. We also reported on enrichment/subfractionation methods that target the cytoskeleton associated proteins and discuss the need of alternative methods for further characterization of the neurocytoskeletal proteome. Finally, we present live cell imaging approaches and emerging dynamic microscopy technology that will provide the tools necessary to investigate protein interactions and their dynamics in the whole cells. While these areas of research are still in their infancy, they offer huge potential towards the understanding of the neuronal network stability and its modification across neuropsychiatric disorders.