Real-time analysis of the biomolecular interaction between gelsolin and Aß1-42 monomer and its implication for Alzheimer's disease.

Biomolecular interaction acts a pivotal part in understanding the mechanisms underlying the development of Alzheimer's disease (AD). Herein, we built a biosensing platform to explore the interaction between gelsolin (GSN) and different ß-amyloid protein 1-42 (Aß1-42) species, including Aß1-42 monomer (m-Aß), Aß1-42 oligomers with both low and high levels of aggregation (LLo-Aß and HLo-Aß) via dual polarization interferometry (DPI). Real-time molecular interaction process and kinetic analysis showed that m-Aß had the strongest affinity and specificity with GSN compared with LLo-Aß and HLo-Aß. The impact of GSN on inhibiting aggregation of Aß1-42 and solubilizing Aß1-42 aggregates was evaluated by circular dichroism (CD) spectroscopy. The maintenance of random coil structure of m-Aß and the reversal of ß-sheet structure in HLo-Aß were observed, demonstrating the beneficial effects of GSN on preventing Aß from aggregation. In addition, the structure of m-Aß/GSN complex was analyzed in detail by molecular dynamics (MD) simulation and molecular docking. The specific binding sites and crucial intermolecular forces were identified, which are believed to stabilize m-Aß in its soluble state and to inhibit the fibrilization of Aß1-42. Combined theoretical simulations and experiment results, we speculate that the success of GSN sequestration mechanism and the balance of GSN levels in cerebrospinal fluid and plasma of AD subjects may contribute to a delay in AD progression. This research not only unveils the molecular basis of the interaction between GSN and Aß1-42, but also provides clues to understanding the crucial functions of GSN in AD and drives the development of AD drugs and therapeutic approaches.

The actin-binding protein palladin associates with the respiratory syncytial virus matrix protein.

The respiratory syncytial virus (RSV) matrix (M) protein plays an important role in infection as it can interact with viral components as well as the host cell actin microfilaments. The M-actin interaction may play a role in facilitating the transportation of virion components to the apical surface, where RSV is released. We show that M protein's association with actin is facilitated by palladin, an actin-binding protein. Cells were infected with RSV or transfected to express full-length M as a green fluorescent protein (GFP)-tagged protein, followed by removal of nuclear and cytosolic proteins to enrich for cytoskeleton and its associated proteins. M protein was present in inclusion bodies tethered to microfilaments in infected cells. In transfected cells, GFP-M was presented close to microfilaments, without association, suggesting the possible involvement of an additional protein in this interaction. As palladin can bind to proteins that also bind actin, we investigated its interaction with M. Cells were co-transfected to express GFP-M and palladin as an mCherry fluorescent-tagged protein, followed by cytoskeleton enrichment. M and palladin were observed to colocalize towards microfilaments, suggesting that palladin is involved in the M-actin interaction. In co-immunoprecipitation studies, M was found to associate with two isoforms of palladin, of 140 and 37 kDa. Interestingly, siRNA downregulation of palladin resulted in reduced titer of released RSV, while cell associated RSV titer increased, suggesting a role for palladin in virus release. Together, our data show that the M-actin interaction mediated by palladin is important for RSV budding and release.IMPORTANCERespiratory syncytial virus is responsible for severe lower respiratory tract infections in young children under 5 years old, the elderly, and the immunosuppressed. The interaction of the respiratory syncytial virus matrix protein with the host actin cytoskeleton is important in infection but has not been investigated in depth. In this study, we show that the respiratory syncytial virus matrix protein associates with actin microfilaments and the actin-binding protein palladin, suggesting a role for palladin in respiratory syncytial virus release. This study provides new insight into the role of the actin cytoskeleton in respiratory syncytial virus infection, a key host-RSV interaction in assembly. Understanding the mechanism by which the RSV M protein and actin interact will ultimately provide a basis for the development of therapeutics targeted at RSV infections.

Large-scale map of RNA-binding protein interactomes across the mRNA life cycle.

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.

Identification and biophysical characterization of epitope atlas of Porcine Reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) have been a critical threat to swine health since 1987 due to its high mutation rate and substantial economic loss over half a billion dollar in USA. The rapid mutation rate of PRRSV presents a significant challenge in developing an effective vaccine. Even though surveillance and intervention studies have recently (2019) unveiled utilization of PRRSV glycoprotein 5 (GP5; encoded by ORF5 gene) to induce immunogenic reaction and production of neutralizing antibodies in porcine populations, the future viral generations can accrue escape mutations. In this study we identify 63 porcine-PRRSV protein-protein interactions which play primary or ancillary roles in viral entry and infection. Using genome-proteome annotation, protein structure prediction, multiple docking experiments, and binding energy calculations, we identified a list of 75 epitope locations on PRRSV proteins crucial for infection. Additionally, using machine learning-based diffusion model, we designed 56 stable immunogen peptides that contain one or more of these epitopes with their native tertiary structures stabilized through optimized N- and C-terminus flank sequences and interspersed with appropriate linker regions. Our workflow successfully identified numerous known interactions and predicted several novel PRRSV-porcine interactions. By leveraging the structural and sequence insights, this study paves the way for more effective, high-avidity, multi-valent PRRSV vaccines, and leveraging neural networks for immunogen design.

Protein-protein interactions in cGAS-STING pathway: a medicinal chemistry perspective.

Protein-protein interactions (PPIs) play pivotal roles in biological processes and are closely linked with human diseases. Research on small molecule inhibitors targeting PPIs provides valuable insights and guidance for novel drug development. The cGAS-STING pathway plays a crucial role in regulating human innate immunity and is implicated in various pathological conditions. Therefore, modulators of the cGAS-STING pathway have garnered extensive attention. Given that this pathway involves multiple PPIs, modulating PPIs associated with the cGAS-STING pathway has emerged as a promising strategy for modulating this pathway. In this review, we summarize an overview of recent advancements in medicinal chemistry insights into cGAS-STING PPI-based modulators and propose alternative strategies for further drug discovery based on the cGAS-STING pathway.

[Box: see text].

Engineering conditional protein-protein interactions for dynamic cellular control.

Conditional protein-protein interactions enable dynamic regulation of cellular activity and are an attractive approach to probe native protein interactions, improve metabolic engineering of microbial factories, and develop smart therapeutics. Conditional protein-protein interactions have been engineered to respond to various chemical, light, and nucleic acid-based stimuli. These interactions have been applied to assemble protein fragments, build protein scaffolds, and spatially organize proteins in many microbial and higher-order hosts. To foster the development of novel conditional protein-protein interactions that respond to new inputs or can be utilized in alternative settings, we provide an overview of the process of designing new engineered protein interactions while showcasing many recently developed computational tools that may accelerate protein engineering in this space.

Hetero-oligomeric interaction as a new regulatory mechanism for protein arginine methyltransferases.

Protein arginine methylation is a versatile post-translational protein modification that has notable cellular roles such as transcriptional activation or repression, cell signaling, cell cycle regulation, and DNA damage response. However, in spite of their extensive significance in the biological system, there is still a significant gap in understanding of the entire function of the protein arginine methyltransferases (PRMTs). It has been well-established that PRMTs form homo-oligomeric complexes to be catalytically active, but in recent years, several studies have showcased evidence that different members of PRMTs can have cross-talk with one another to form hetero-oligomeric complexes. Additionally, these heteromeric complexes have distinct roles separate from their homomeric counterparts. Here, we review and highlight the discovery of the heterodimerization of PRMTs and discuss the biological implications of these hetero-oligomeric interactions.

Protein assemblies in the Arabidopsis thaliana chloroplast compartment.

Introduction: Equipped with a photosynthetic apparatus that uses the energy of solar radiation to fuel biosynthesis of organic compounds, chloroplasts are the metabolic factories of mature leaf cells. The first steps of energy conversion are catalyzed by a collection of protein complexes, which can dynamically interact with each other for optimizing metabolic efficiency under changing environmental conditions. Materials and methods: For a deeper insight into the organization of protein assemblies and their roles in chloroplast adaption to changing environmental conditions, an improved complexome profiling protocol employing a MS-cleavable cross-linker is used to stabilize labile protein assemblies during the organelle isolation procedure. Results and discussion: Changes in protein:protein interaction patterns of chloroplast proteins in response to four different light intensities are reported. High molecular mass assemblies of central chloroplast electron transfer chain components as well as the PSII repair machinery react to different light intensities. In addition, the chloroplast encoded RNA-polymerase complex was found to migrate at a molecular mass of ~8 MDa, well above its previously reported molecular mass. Complexome profiling data produced during the course of this study can be interrogated by interested readers via a web-based online resource (https://complexomemap.de/projectsinteraction-chloroplasts).

Identification of hub genes and potential networks by centrality network analysis of PCR amplified Fusarium oxysporum f. sp. lycopersici EF1α gene.

BACKGROUND: Fusarium wilt is a devastating soil-borne fungal disease of tomato across the world. Conventional method of disease prevention including usage of common pesticides and methods like soil solarisation are usually ineffective in the treatment of this disease. Therefore, there is an urgent need to identify virulence related genes in the pathogen which can be targeted for fungicide development. RESULTS: Pathogenicity testing and phylogenetic classification of the pathogen used in this study confirmed it as Fusarium oxysporum f. sp. lycopersici (Fol) strain. A recent discovery indicates that EF1α, a protein with conserved structural similarity across several fungal genera, has a role in the pathogenicity of Magnaporthe oryzae, the rice blast fungus. Therefore, in this study we have done structural and functional classification of EF1α to understand its role in pathogenicity of Fol. The protein model of Fol EF1α was created using the template crystal structure of the yeast elongation factor complex EEF1A:EEF1BA which showed maximum similarity with the target protein. Using the STRING online database, the interactive information among the hub genes of EF1α was identified and the protein-protein interaction network was recognized using the Cytoscape software. On combining the results of functional analysis, MCODE, CytoNCA and CytoHubba 4 hub genes including Fol EF1α were selected for further investigation. The three interactors of Fol EF1α showed maximum similarity with homologous proteins found in Neurospora crassa complexed with the known fungicide, cycloheximide. Through the sequence similarity and PDB database analysis, homologs of Fol EF1α were found: EEF1A:EEF1BA in complex with GDPNP in yeast and EF1α in complex with GDP in Sulfolobus solfataricus. The STITCH database analysis suggested that EF1α and its other interacting partners interact with guanosine diphosphate (GDPNP) and guanosine triphosphate (GTP). CONCLUSIONS: Our study offers a framework for recognition of several hub genes network in Fusarium wilt that can be used as novel targets for fungicide development. The involvement of EF1α in nucleocytoplasmic transport pathway suggests that it plays role in GTP binding and thus apart from its use as a biomarker, it may be further exploited as an effective target for fungicide development. Since, the three other proteins that were found to be tightly associated Fol EF1α have shown maximum similarity with homologous proteins of Neurospora crassa that form complex with fungicide- Cycloheximide. Therefore, we suggest that cycloheximide can also be used against Fusarium wilt disease in tomato. The active site cavity of Fol EF1α can also be determined for computational screening of fungicides using the homologous proteins observed in yeast and Sulfolobus solfataricus. On this basis, we also suggest that the other closely associated genes that have been identified through STITCH analysis, they can also be targeted for fungicide development.

Structural proteomics defines a sequential priming mechanism for the progesterone receptor.

The progesterone receptor (PR) is a steroid-responsive nuclear receptor, expressed as two isoforms: PR-A and PR-B. The isoforms display distinct expression patterns and biological actions in reproductive target tissues and disruption of PR-A:PR-B signaling is associated with breast cancer development potentially by altering interactions with oncogenic co-regulatory protein (CoRs). However, the molecular details of isoform-specific PR-CoR interactions that influence progesterone signaling remain poorly understood. We employed structural mass spectrometry in this study to investigate the sequential binding mechanism of purified full-length PR and full-length CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes with target DNA. Our findings reveal selective CoR NR-box binding by PR and novel interaction surfaces between PR, SRC3, and p300, which change during complex assembly. This provides a structural model for a sequential priming mechanism that activates PR. Comparisons of PR bound to progesterone agonist versus antagonist challenges the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and elucidate the mechanisms behind the interactions of these proteins, both in active and inactive conformations.