bioRxiv Science⌕ Search

Biology subjects

Ito, Y.

Publications and source records attributed to Ito, Y..

At least 19 recordsLinked to original sources

Prior experience establishes a proactive cortical filter that blocks redundant associations

Prior experience allows selective learning by filtering out redundant cues, as exemplified by blocking, in which prior learning about one cue prevents a novel but redundant cue from being learned during subsequent compound conditioning. Top-down cortico-cortical pathways have been implicated in sensory filtering, but whether, how, and when they contribute to blocking remain unknown. Here we establish a head-fixed blocking paradigm in mice in which prior auditory cue-reward learning prevents a redundant somatosensory cue-reward association during subsequent compound conditioning. We found that a projection from anterior cingulate cortex (ACC) to primary somatosensory cortex (S1) facilitates blocking. Prior auditory learning establishes ACC-driven inhibitory control over S1 via parvalbumin neurons, before explicit somatosensory experience, thereby suppressing burst responses to the somatosensory cue, limiting network plasticity, and promoting blocking. These findings propose a proactive cortical filter that preconfigures inhibitory sensory bias to constrain future learning about redundant cues. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/736748v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@6e1c4org.highwire.dtl.DTLVardef@1d01b49org.highwire.dtl.DTLVardef@1af67fdorg.highwire.dtl.DTLVardef@d42d8d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

Personalized reference genome-based pipeline reveals comprehensive haplotype-resolved views of cancer genomes

Cancer genome analysis relies on standard human reference genomes, but detecting somatic alterations in highly repetitive or individual-specific regions remains challenging. We developed the Personalized Reference genome-based Cancer Genome Analysis Pipeline (PRCGAP, https://github.com/yos-sk/PRCGAP), to our knowledge, the first comprehensive pipeline integrating haplotype-resolved analyses of somatic point mutations, structural variants, copy number, and DNA methylation on personalized diploid reference genomes, with each variant phased and annotated by genomic features. We applied PRCGAP to eight tumor-normal cell line pairs and three newly collected pediatric B-cell acute lymphoblastic leukemia (B-ALL) clinical samples. PRCGAP recovered most standard reference-based variants (86.7-96.4% across variant types), validated by orthogonal short-read sequencing, while additionally detecting variants that standard references missed (23.4% of SNVs and 32.6% of SVs). Notably, PRCGAP uncovered variants in centromeric and telomeric regions, and somatic single-nucleotide variants were enriched in centromere dip regions, the putative kinetochore formation sites. Using PRCGAP outputs, we identified L1 retrotransposition source sites absent from standard references and showed that a B-ALL IGH::DUX4 fusion arose from a DUX4 pseudogene within an internal D4Z4 repeat unit, rather than the canonical full-length DUX4 gene. Within a single haplotype-resolved framework, PRCGAP opens regions of the cancer genome that are inaccessible to standard references.

bioinformatics↗

Turn-taking-like temporal coordination of ultrasonic vocalizations during close-range social interactions in Mongolian gerbils (Meriones unguiculatus)

Vocal communication depends not only on the acoustic features and rate of vocalizations, but also on their timing relative to the vocalizations of others. In this study, we examined whether ultrasonic vocalizations (USVs) produced by adult female Mongolian gerbils during freely moving social interactions are temporally related to the vocal timing of a partner. Using USVCAM, we assigned each USV to an individual caller and evaluated whether brief USVs exhibit turn-taking-like temporal organization by analyzing response latency after partner calls, call overlap rate, and circular-shift surrogate data. The offset-to-onset gaps of alternating vocalizations were concentrated within a short time range, with a median of 130 ms. In addition, the call overlap rate was significantly lower in the observed data than in the circularly shifted surrogate data, indicating that USVs occurred with short latencies after partner calls while being less likely to overlap with ongoing partner calls. Furthermore, USV counts were reduced in unoperated Ctrl animals that interacted with devocalized Mute animals. These findings suggest that gerbil USVs may be coordinated in relation to partner vocal timing and vocal input. This study provides a basis for understanding rodent USVs not merely as individual vocal outputs, but as temporally organized dyadic social signals.

animal behavior and cognition↗

Spatial proteomics reveals prefrontal circuit diversity in socioemotional behaviour

Understanding how molecular diversity across long-range neural circuits governs brain function remains a central challenge in neuroscience. Here, we developed a projection-specific spatial proteomics approach and revealed robust presynaptic molecular divergence across six projection-defined pathways of the medial prefrontal cortex (mPFC), including efferent projections to the basolateral amygdala (BLA), nucleus accumbens (NAc), thalamus (Thal), hypothalamus (HT) and cortex (CTX), as well as the afferent projection from the BLA to the mPFC. Among these pathways, we identify BLTP2 (KIAA0100), a previously uncharacterized transmembrane protein, as highly enriched in the projection from the mPFC to the BLA. BLTP2 localizes to excitatory presynaptic terminals and is enriched at synapses that are activated during memory formation. Loss of BLTP2 impairs synaptic structure and transmission, and reduces activity-dependent remodelling, resulting in selective deficits in contextual fear memory, anxiety-related behavior, and social behavior. Mechanistically, BLTP2 promotes presynaptic assembly by recruiting Neurexin 1. These findings reveal projection-specific presynaptic molecular diversity and provide mechanistic insights into circuit-level vulnerabilities in neuropsychiatric disorders.

neuroscience↗

MDA5 multimerization on LINE RNA drives pathogenic extracellular immune complexes in autoimmunity

Autoantibodies are hallmarks of many autoimmune diseases, but their potential pathogenic roles, particularly for those targeting intracellular proteins, remain unclear. Anti-MDA5-positive dermatomyositis (anti-MDA5 DM) is characterized by autoantibodies against the intracellular protein MDA51,2, a conserved innate immune receptor that recognizes viral dsRNA by forming filaments3. Here, using four patient-derived monoclonal autoantibodies (mAbs), we reconstitute and define the molecular architecture, biogenesis, and immunological activity of pathogenic MDA5 immune complexes. Our cryo-EM analysis revealed that these mAbs bind dsRNA-scaffolded MDA5 filaments in at least two distinct binding modes, each exhibiting striking epitope convergence using germline-encoded residues. Extracellular immune complexes formed between mAbs and filamentous, but not monomeric, MDA5 potently activate multiple innate immune pathways, with the magnitude of activation determined by antibody binding mode and immune complex stoichiometry. Antibody bivalency further crosslinks MDA5 filaments into higher-order aggregates with heightened immunostimulatory activity, demonstrating an active role of autoantibodies in shaping immune complex architecture. Analysis of patient plasma reveals elevated levels of extracellular MDA5 filaments and identifies LINE retroelement-derived dsRNA as a structural scaffold. Notably, MDA5 immune complexes induce endogenous LINE dsRNA expression, likely promoting additional MDA5 filament formation and extracellular release through inflammatory cell death. These data thus support a self-amplifying inflammatory cycle as a pathogenic mechanism for anti-MDA5 DM. Collectively, our study defines a broadly applicable architectural principle, in which higher-order organization and binding modes of autoantibodies--beyond antibody affinity or nucleic acid presence alone--govern innate immune activation.

biochemistry↗

Chromosome-scale genome assembly and annotation of the two-spotted cricket Gryllus bimaculatus (Orthoptera: Gryllidae)

The two-spotted cricket, Gryllus bimaculatus, is a key hemimetabolous model organism for developmental biology, neuroscience, and regeneration. The existing reference genome is, however, highly fragmented into 47,877 scaffolds, hampering chromosome-scale analyses for these fields. Here, we report a high-quality, chromosome-scale genome assembly for the white-eyed mutant strain of this cricket, generated using a combination of Nanopore and PacBio HiFi long reads, integrated with Hi-C data. The final 1.62 Gbp assembly achieves a scaffold N50 of 107.4 Mbp, a significant improvement in contiguity over the previous 6.3 Mbp N50. We anchored 94.45% of the assembly into 15 pseudomolecules, consistent with the known karyotype (n = 15). The genome completeness (BUSCO v6.0.0 insecta_odb12) reached 98.1%. We also updated the annotation, identifying 14,964 protein-coding genes. This gene set shows markedly improved completeness (BUSCO v6.0.0 insecta_odb12: 95.7%) compared with the previous annotation (81.2%) and successfully recovers all nine essential neuropeptide genes previously reported as missing from the draft assembly. This chromosome-scale genomic resource provides an essential foundation for comparative and functional genomics in G. bimaculatus.

genomics↗

Structural basis for color tuning and passive ion conductance in red-shifted pump-fold channelrhodopsin ChR024

ChR024 is a cation-conducting channelrhodopsin with a red-shifted absorption spectrum, recently discovered from a previously unidentified clade through machine-learning-guided gene mining. Due to its unique position in the phylogenetic tree and distinctive spectral properties, it serves as an important model for elucidating rhodopsin evolution and for engineering optogenetic tools with enhanced performance, but its structure and function remain poorly understood. Here, we present cryo-electron microscopy structures of ChR024 in detergent micelles and lipid nanodiscs at resolutions of 3.22 and 2.45 [A], respectively. The structures reveal an architecture strikingly similar to that of ion-pumping rhodopsins, even more so than other pump-like channelrhodopsins such as ChRmine. Structural, theoretical, and spectroscopic analyses uncover a unique color-tuning mechanism, in which charged residues close and distant from the retinal chromophore cooperatively modulate the pKa of the Schiff base counterion and thereby determine the absorption maximum wavelength. Finally, comparative structural analysis of channel- and pump-type rhodopsins, combined with electrophysiology, provides insights into the molecular boundary between these two functional classes and demonstrates the conversion of an outward proton pump into a light-gated channel. Together, these findings illuminate diverse mechanisms of color tuning and functional specification within the rhodopsin family, paving the way for the rational engineering of next-generation optogenetic tools.

biophysics↗

Pathological Angiogenesis Precedes the Onset of Aortic Dissection

Aortic dissection (AD) is a severe, life-threatening disease that occurs abruptly1. Although remodeling and inherent vulnerability of the aortic media, as observed in heritable connective tissue disorders such as Marfan syndrome, have been implicated in the onset of AD, the precise pathological mechanisms underlying the non-heritable form, which accounts for most cases, remains largely unknown2. Here, using hyperacute AD patient-derived samples, we show that pathological angiogenesis within the aortic media precedes the onset of dissection. Integrating single-cell transcriptomic analysis of aortic medial endothelial cells with high-resolution imaging revealed temporal changes preceding dissection. Hypoxic alterations in the media triggered osteochondrogenic changes in smooth muscle cells, promoted hydroxyapatite deposition, and induced angiogenesis via VEGF secretion from AD-specific CD14+CD68+CD163+MRC1(CD206)neg-low macrophages. Atomic force microscopy revealed that these processes culminated in the formation of a soft capillary layer, potentially concentrating stress on this region and contributing to AD onset. H&E staining of archived specimens revealed that non-heritable AD can be classified into angiogenic or non-angiogenic subtypes, with more than half exhibiting the former. In contrast, acute AD cases associated with Marfan syndrome typically exhibit a non-angiogenic pattern, suggesting distinct underlying mechanisms. This study reports angiogenesis as a key pathological driver in non-heritable AD and highlight potential targets for early diagnosis, prevention, and treatment.

pathology↗

Rare k-mers reveal centromere haplogroups underlying human diversity and cancer translocations

Centromeres are among the most diverse and dynamically evolving regions of the human genome and are commonly affected in various human cancers. However, organized into highly repetitive -satellite higher-order repeats (HORs), human centromere sequences have long resisted detailed genomic analysis. Although the development of long-read sequencing platforms has enabled the analysis of complete centromere sequences, their application to a large set of samples is still largely limited, preventing our understanding of centromere variation and haplotype structures across large human populations and the structural basis of centromere-involving translocations in cancer. Here we show that rare k-mers present in centromeric regions can serve as effective markers for dissecting the complexity of centromere structure, particularly that of active -satellite HOR arrays (aHOR arrays), across human populations and for understanding centromere-involving abnormalities in cancer. Based on rare k-mer-based clustering, centromere aHOR arrays are clustered into discrete haplogroups (aHOR-HGs) with distinct structural features. These k-mers were also used to develop a framework that enables the inference of haplogroups in a given sample based on short-read whole genome sequencing (WGS) data (ascairn). By applying ascairn to large-scale human population datasets (n > 3,300), we revealed the diversity of aHOR-HGs and their geographic histories across populations. The rare k-mer-based approach was also applied to investigate the structure of 1p/19q co-deletion, a highly recurrent centromere-involving translocation in IDH-mutated oligodendrogliomas. Analyzing short-read WGS data from 142 cases with 1p/19q co-deletion using rare k-mers, we showed that breakpoints of 1p/19q co-deletion were mapped to aHOR arrays in chromosomes 1 (D1Z7) and 19 (D19Z3), which was validated by long-read sequencing of two 1p/19q co-deletion-positive cases. Notably, the translocation preferentially involved haplogroups composed of haplotypes containing larger regions susceptible to rearrangement. These results highlight the role of rare k-mers in dissecting the complexity of centromere sequences and their evolutionary history as well as understanding centromere-involving abnormalities associated with human diseases.

genomics↗

The constitutive oncogenic and signaling activities of phospha-tidylinositol 3-kinase (PI3K) isoforms p110β and p110δ

AbstractThe p110{beta} and p110{delta} isoforms of the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) show enhanced oncogenic and signaling activities as compared with the p110 protein. The adapter binding domains (ABDs) of p110{beta} and p110{delta} contain an isoform-specific PXXP mo- tif. Mutations of this PXXP to AXXA diminish the oncogenic and signaling activities. This loss of function can be compensated by placing a gain-of-function mutation in the helical domain of the P/A mutants. The P/A mutants still associate with the regulatory p85 subunit, but the affinity of this interaction is decreased. p110 with the ABD of either p110{beta} or p110{delta} shows an increase of oncogenic and signaling activities, whereas p110{beta} or p110{delta} with the ABD of p110 have greatly reduced oncogenic and signaling activities. Introducing the PXXP motif in the ABD of p110 re- sults in a significant gain of function. We conclude that the PXXP motif in the ABD of p110{beta} and p110{delta} is essential for the elevated oncogenic and signaling activities of these isoforms. We pro- pose that the PXXP motif in the ABD of p110{beta} and p110{delta} affects the interaction with the iSH2 domain of p85, shifting the regulatory SH2 domains into less effective inhibitory conformations.

cancer biology↗

The metabolic ability of swallowtails results in the production of bioactive substances from plant components

Host plant selection may depend on the metabolic system in herbivorous insects. Although oligophagous insects take up specific host plant components, how host plant components and their biological activities are altered through their metabolic systems remains unknown. Here, by examining gene expression of metabolic enzymes and components in the larval frass, we investigated the metabolic ability of Papilio memnon larvae fed with Citrus x paradisi (grapefruit) against host plant components. The gene expression levels of some metabolic enzymes were fluctuated between the larval midgut and the larval fat bodies. Furthermore, the chloroform extract from the larval frass, but not that from grapefruit leaves, inhibited cell viability of human pancreatic cancer cell line, MIA PaCa2. Finally, we identified two chlorophyll catabolites, pheophorbide-a and pyropheophorbide-a, in the larval frass extract. Pyropheophorbide-a reduces cell viability of and induces morphological changes in cells of MIA PaCa2; in addition, pheophorbide-a and pyropheophorbide-a inhibit the aggregation of amyloid {beta}-protein (human, 1-42). Therefore, the chemical structure and biological activity of host plant components are affected by the P. memnon metabolic system. Our findings may contribute to the development of a bioprocess for the production of pheophorbide-a and pyrophephorbide-a from chlorophyll via the metabolic functions of P. memnon larvae.

biochemistry↗

Wnt target IQGAP3 promotes Wnt signaling via disrupting Axin1-CK1α interaction.

The scaffold protein IQGAP3 is highly upregulated in most epithelial cancers. While recent studies have highlighted its pivotal roles in cancer cell proliferation and metastasis, a deeper mechanistic understanding of IQGAP3 is currently lacking. We have here used TurboID to map IQGAP3 proximity partners and identified the Wnt signaling members Axin1 and CK1 as IQGAP3-interacting proteins. Our functional studies demonstrated that overexpression of IQGAP3 increases {beta}-catenin levels, while IQGAP3 depletion reduces {beta}-catenin levels in gastric cancer cells. Mechanistically, IQGAP3 disrupts Axin1-CK1 interaction, thereby inhibiting {beta}-catenin phosphorylation and ultimately leading to its accumulation. Importantly, we discovered that IQGAP3 itself is regulated by Wnt signaling, suggesting its involvement in a positive feedback loop in Wnt/{beta}-catenin signaling through interactions with Axin1 and CK1. These findings identify IQGAP3 as a novel mediator of {beta}-catenin stabilization and underscore its potential as a target for cancer therapy.

cancer biology↗

MEGA-FISH: Multi-omics Extensible GPU-Accelerated FISH Processing Framework for Huge-Scale Spatial Omics

Spatial omics enables comprehensive mapping of cell types and states in their spatial context, providing profound insights into cellular communication and tissue organization. However, analyzing large tissue sections, especially crucial for clinical applications, remains a significant challenge due to the computational demands of current image processing methods. To overcome these limitations, we developed MEGA-FISH, a flexible, GPU-accelerated Python framework optimized for large-scale spatial omics image analysis. Benchmarking on simulated and tissue images demonstrated that MEGA-FISH achieved high accuracy in spot detection while significantly reducing processing times compared with established tools. The frameworks adaptable computational capabilities optimize resource allocation (e.g., GPU or multi-core CPU) for diverse tasks, and its scalable architecture enables integration with advanced imaging and segmentation techniques. By bridging cutting-edge imaging methods and single-cell analysis, MEGA-FISH provides an efficient platform for multi-modal analysis and advances research and clinical applications of spatial omics at organ and organism scales.

bioinformatics↗

Developing simple DNA extraction and PCR-RFLP for MALE STERILITY 4 (MS4) gene in Cryptomeria japonica D. Don: Toward an environmentally friendly protocol

This study presents the development of a simple DNA extraction method and a novel PCR-RFLP protocol for genotyping the MALE STERILITY 4 (MS4) gene in Cryptomeria japonica as a proof of concept. Traditional CTAB-based DNA extraction methods, while effective, involve hazardous chemicals and require high-speed centrifugation, which are impractical in many field settings. Our approach utilizes a household dish detergent-based buffer, sodium chloride, and polyvinylpyrrolidone K-30 to extract DNA from C. japonica needle leaf tissues--without the need for liquid nitrogen. The simplicity of this method makes it more accessible and environmentally friendly. The extracted DNA was successfully used in PCR-RFLP analysis, targeting a single nucleotide polymorphism in the MS4 gene, demonstrating its efficacy for genotyping. The PCR-RFLP markers reliably discriminated between individual genotypes, confirming the practical application of our simple extraction method, even for conifers containing inhibitory substances. This technique is particularly advantageous for use in arboretums and field stations, where the use of hazardous chemicals, specialized equipment, and a draft chamber is limited. Our study contributes to genetic resource management by providing an easy, reliable, and safer method for DNA extraction and genetic analysis.

genetics↗

Comparative genomic analysis of Latilactobacillus sakei strains provides new insights into their association with different niche adaptations

Latilactobacillus sakei, a lactic acid bacterium in diverse environments such as fermented foods, meat, and the human gastrointestinal tract, exhibits significant genetic diversity and niche-specific adaptations. This study conducts a comprehensive comparative genomic analysis of 30 complete L. sakei genomes to uncover the genetic mechanisms underlying these adaptations. Phylogenetic analysis divided the species into three distinct clades that did not correlate with the source of isolation and did not suggest any niche-specific evolutionary direction. The pan-genome analysis revealed a substantial core genome alongside a diverse genetic repertoire, indicating both high genetic conservation and adaptability. Predicted growth rates based on codon use bias analysis suggest that L. sakei strains have an overall faster growth rate and may be able to efficiently dominant in competitive environments. Plasmid analysis revealed a variety of plasmids carrying genes essential for carbohydrate metabolism, enhancing L. sakeis ability to thrive in various fermentation substrates. It was also found that the number of genes belonging to the GH1 family among sugar metabolism-related genes present on chromosomes and plasmids varies between strains, and that AA1, which is involved in alcohol oxidation, has been acquired from plasmids. BLAST analysis revealed that some strains have environmental adaptation gene clusters of cell surface polysaccharides that may mediate attachment to food and mucosa. These findings not only underscore the genetic and functional diversity of L. sakei but also highlight its potential as a potent starter culture in fermentation and as a probiotic. The knowledge gleaned from this study lays a solid foundation for future research aimed at harnessing the genetic traits of L. sakei strains for industrial and health-related applications.

microbiology↗

Comparative genomic analysis of multidrug-resistant clinical isolates of Mycobacterium abscessus revealed worldwide horizontal plasmid transfer

Genetic epidemiological analysis of mobile genetic elements, such as plasmids, has rarely been carried out in Mycobacterium abscessus, regardless of its usefulness in speculating the past contact between bacteria. In this study, through whole genome sequencing analysis of clinical isolates of M. abscessus sequentially collected from the same patient, we identified a single nucleotide variant that could cause antibiotic resistance, and three plasmids (pMAB625-1, pMAB625-2 and pMAB625-3) not found in the type strain. We investigated the distribution of plasmids previously identified in M. abscessus, including pMAB625 plasmids, in 462 clinical isolates worldwide in silico. pMAB625-3 was detected in the largest number of isolates. Furthermore, phylogenetic tree analysis revealed that these plasmids transferred beyond regions and subspecies and acquired unique mutations. These results indicate that transmission of plasmids increases the genomic diversity in M. abscessus, and plasmid epidemiology is useful for estimating the past contact between bacteria.

genomics↗

Bioinformatic evaluation of the potential oral-gut translocation of periodontal pathogens in patients with colorectal polyps

ObjectiveThis study aimed to characterize the profiles of the oral and gut microbiota of patients with colorectal polyps using 16S rRNA gene sequencing and bioinformatic approaches. BackgroundPrevious studies have shown microbial translocation from the oral cavity to the gut, implying pathogenic impacts on gastroesophageal disease, including colorectal cancer (CRC). However, its details remain unclear. MethodsTwenty patients scheduled for endoscopic colorectal polypectomy were enrolled in this study. Oral samples (saliva and subgingival dental plaque) and intestinal samples (feces and swab of intestinal mucosa) were collected during preoperative and 6-month-postoperative reassessment periods. After sequencing the V3-V4 region of the bacterial 16S rRNA gene, several bioinformatic analyses (bacterial composition, diversity, core microbiome, and shared ASV) were performed on pre- and postoperative samples for each subject. ResultsThe bacterial composition was dominated by Bacteroides, Streptococcus, Fusobacterium, Veillonella, and Prevotella_7 in all four samples. Beta diversity analysis using weighted UniFrac distance distinctly segregated the samples between oral and intestinal environments in the principal coordinate analysis plot. Core microbiome analysis revealed that Streptococcus and Porphyromonas were dominantly shared in intra-oral environments. Additionally, alongside Streptococcus, periodontitis-related bacteria, such as Veillonella, Fusobacterium, Porphyromonas, Prevotella_7, Haemophilus, and Prevotella, were identified as shared genera between oral and intestinal environments. Finally, shared ASV analysis demonstrated that Streptococcus was shared in the oral and intestinal environments of most patients, while periodontal pathogens were shared in some patients. ConclusionsThe core microbiome and shared ASV analyses revealed that several genes are shared between oral and intestinal environments in patients with colorectal polyps, indicating the oral-gut translocation of periodontitis-related bacteria. Further large-scale studies are needed to elucidate their involvement in CRC.

bioinformatics↗

Different molecular recognition by three domains of the full-length GRB2 to SOS1 proline-rich motifs and EGFR phosphorylated sites

The adaptor protein human GRB2 plays crucial roles in mediating signal transduction from cell membrane receptors to RAS and its downstream proteins by recruiting SOS1. Recent studies have revealed that GRB2 also serves as a scaffold for liquid-liquid phase separation (LLPS) with SOS1 and transmembrane receptors, which is thought to regulate the magnitude of cell signalling pathways. In this study, we employed solution NMR spectroscopy to investigate the interactions of the full-length GRB2 with proline-rich motifs (PRMs) derived from ten potential GRB2-binding sites in SOS1, as well as a peptide from a phosphorylation site of EGFR. Our findings indicate that the binding affinity of the two SH3 domains of GRB2 for PRMs differs by a factor of ten to twenty, with the N-terminal SH3 domain (NSH3) exhibiting a markedly higher affinity. The interactions of PRMs with the SH3 domains affected not only the regions surrounding the PRM binding sites on the SH3 domains but also the linker area connecting the three domains and parts of the SH2 domain. Analysis of the interaction between the phosphorylated EGFR binding site and the SH2 domain revealed chemical shift perturbations in regions distal from the known binding site of SH2. Moreover, we observed that the inter-domain interactions of the two SH3 domains with the SH2 domain of GRB2 are asymmetric. These findings suggest that the local binding of PRMs and phosphorylated EGFR to GRB2 impacts the overall structure of the GRB2 molecule, including domain orientation and dimerisation, which may contribute to LLPS formation.

biophysics↗