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At least 19 recordsLinked to original sources

PubMed2026

Post-transcriptional regulation of Profilin-2 by microRNAs and RNA-binding proteins forms a critical regulatory node for early embryonic cell fate decisions.

Post-transcriptional control by RNA binding proteins (RBPs) and microRNAs play central roles in mRNA stability and translation, yet how RBPs and microRNAs coordinate in developmental time to regulate cell fate remains poorly understood. Here, we demonstrate that post-transcriptional regulation of the Profilin 2 (Pfn2) transcript is essential for differentiation of embryonic stem cells (ESCs) into the primary germ layer lineages. The Pfn2 3'untranslated region has both an Iron Regulatory Protein binding site (IRE) and a nearby binding site for ESC enriched microRNAs. Deletion of this microRNA site leads to increased PFN2 and reduced FGF signaling during pluripotency transition prior to germ layer formation. In contrast, deletion of the IRE leads to decreased PFN2, a Wnt signaling defect, reduced nuclear beta-catenin, and a subsequent block in mesendodermal lineages during early germ layer formation. We further find that loss of the IRE site results in a cell autonomous defect in Wnt signaling and mesendodermal differentiation. The IRE site acts to stabilize beta-catenin, as disruption of the site leads to reduced nuclear beta-catenin levels. Together, these findings reveal the Pfn2 microRNA-IRE regulatory axis as a critical post-transcriptional regulatory node governing the switch from pluripotency to somatic differentiation.

MicroRNAs↗

PubMed2025

Site-Specific Profiling of RNA-Binding Proteins Enabled by Isotopic Signature-Enhanced Mass Spectrometry.

RNA-binding proteins (RBPs) ubiquitously regulate RNA throughout their lifespan, being extensively involved in cellular metabolism and genetic evolution. Therefore, comprehensive identification of the RNA-protein interactions, especially their interfaces with site-specific resolution, is significant to elucidate the intricate biological activities governed by RNA. Nevertheless, it remains challenging for data-dependent acquisition (DDA)-based proteomics to identify the RNA-cross-linked peptides in depth due to the low abundance and negative charge of modified peptides. To address such limitations, we developed an innovative method named "isoRIC" for profiling RNA-binding proteomes with site-specific resolution, which combines the metabolic labeling of isotopic nucleotides for photo-cross-linking of RNA-binding proteins and the real-time targeted LC-MS/MS analysis of RNA-cross-linked peptides. This method shows a dramatic improvement of sensitivity in identifying RNA-cross-linked peptides with low abundance as compared to the DDA-based proteomic approaches, enabling the discovery of novel RNA-binding proteins and precise mapping of RNA-protein binding interfaces at single amino acid resolution. We applied isoRIC in the context of pathogenic mutations and post-translational modifications to highlight the critical role of RNA-binding sites in modulating the RNA-binding ability.

RNA-Binding Proteins↗

PubMed2025

Functional integration of the bacteriophage T4 DNA replication complex: The multiple roles of the ssDNA binding protein (gp32).

Single-stranded DNA binding protein (gp32) serves as the central regulatory component of the multi-subunit T4 bacteriophage DNA replication system by coordinating the system's three functional sub-assemblies, resulting in phage DNA synthesis in T4-infected E. coli cells at the high speeds (~1,000 nts s-1) and the high fidelity (< 1 error per 107 nts) required for genomic function within this cellular eco-system. Gp32 proteins continuously bind to, slide as cooperatively-linked clusters on, and un-bind from transiently exposed single-stranded (ss) DNA templates to carry out their coordinating functions, as well as to protect genomic sequences from nuclease activity and block the formation of interfering secondary structures. The N-terminal domains (NTDs) of gp32 mediate cooperative interactions within ssb clusters, but the roles of the disordered C-terminal domains (CTD) in the nucleation of gp32-ssDNA filaments at ss-dsDNA junctions are less well understood. We here present microsecond-resolved single-molecule Förster resonance energy transfer studies of the initial steps of gp32 assembly on short oligo-deoxythymidine lattices of varying lattice length and polarity near model ss-dsDNA junctions. These data are analyzed to define the molecular steps and related free energy surfaces involved in initiating gp32 cluster formation, which show that the nucleation mechanisms and regulatory interactions driven by gp32 proteins at ss-dsDNA junctions are significantly directed by lattice polarity. We propose a model for the role of the CTDs in orienting gp32 monomers at lattice positions close to ss-dsDNA junctions that suggests how intrinsically disordered CTD domains might facilitate and control non-base-sequence-specific binding in both the nucleation and the dissociation of the gp32-ssDNA filaments involved in phage DNA replication and related processes.

Journal Article↗

PubMed2025

CaXML: Chemistry-informed machine learning explains mutual changes between protein conformations and calcium ions in calcium-binding proteins using structural and topological features.

Proteins' flexibility is a feature in communicating changes in cell signaling instigated by binding with secondary messengers, such as calcium ions, associated with the coordination of muscle contraction, neurotransmitter release, and gene expression. When binding with the disordered parts of a protein, calcium ions must balance their charge states with the shape of calcium-binding proteins and their versatile pool of partners depending on the circumstances they transmit. Accurately determining the ionic charges of those ions is essential for understanding their role in such processes. However, it is unclear whether the limited experimental data available can be effectively used to train models to accurately predict the charges of calcium-binding protein variants. Here, we developed a chemistry-informed, machine-learning algorithm that implements a game theoretic approach to explain the output of a machine-learning model without the prerequisite of an excessively large database for high-performance prediction of atomic charges. We used the ab initio electronic structure data representing calcium ions and the structures of the disordered segments of calcium-binding peptides with surrounding water molecules to train several explainable models. Network theory was used to extract the topological features of atomic interactions in the structurally complex data dictated by the coordination chemistry of a calcium ion, a potent indicator of its charge state in protein. Our design created a computational tool of CaXML, which provided a framework of explainable machine learning model to annotate ionic charges of calcium ions in calcium-binding proteins in response to the chemical changes in an environment. Our framework will provide new insights into protein design for engineering functionality based on the limited size of scientific data in a genome space.

Machine Learning↗

PubMed2025

The CGG triplet repeat binding protein 1 counteracts R-loop induced transcription-replication stress.

The CGG triplet repeat binding protein 1 (CGGBP1) binds to CGG repeats and has several important cellular functions, but how this DNA sequence-specific binding factor affects transcription and replication processes is an open question. Here, we show that CGGBP1 binds human gene promoters containing short (< 5) CGG-repeat tracts prone to R-loop formation. Loss of CGGBP1 leads to deregulated transcription, transcription-replication-conflicts (TRCs) and accumulation of Serine-5 phosphorylated RNA polymerase II (RNAPII), indicative of promoter-proximal stalling and a defect in transcription elongation. Consistently, an episomal CGG-repeat-containing model locus as well as endogenous genes show deregulated transcription, R-loop accumulation and increased RNAPII chromatin occupancy in CGGBP1-depleted cells. We identify the DEAD-box RNA:DNA helicases DDX41 and DHX15 as interaction partners specifically recruited by CGGBP1. Co-depletion experiments show that DDX41 and CGGBP1 work in the same pathway to unwind R-loops and avoid TRCs. Together, our work shows that short trinucleotide repeats are a source of genome-destabilizing secondary structures, and cells rely on specific DNA-binding factors to maintain proper transcription and replication coordination at short CGG repeats.

Humans↗

PubMed2025

Development and Validation of a Novel LC-MS/MS Based Proteomics Method for Quantitation of Retinol Binding Protein 4 (RBP4) and Transthyretin (TTR).

Retinol binding protein 4 (RBP4), the circulating carrier of retinol, complexes with transthyretin (TTR) and is a potential biomarker of cardiometabolic disease. However, RBP4 quantitation relies on immunoassays and western blots without retinol and TTR measurement. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous absolute quantitation of circulating RBP4 and TTR is critical to establishing their biomarker potential. Surrogate peptides with reproducible, linear LC-MS/MS response were selected. Purified proteins were used as quantitation standards and heavy-labelled peptides as internal standards. Matrix effects were evaluated. The validated method was applied to measure inter- and intra-individual variability in RBP4 and TTR concentrations in healthy individuals and patients with diabetic kidney disease. Quantitation was linear for the clinically relevant concentration ranges of RBP4 (0.5-6 μM) and TTR (5.8-69 μM). Assay inter-day variability was <12% and precision within 5%. The inter-individual variability for RBP4 and TTR concentrations was 18-26%, while intra-individual variability was similar to assay variability. RBP4 and TTR quantitation correlated with commercially available ELISA assays. The developed LC-MS/MS method enables simultaneous absolute quantitation of RBP4 and TTR in serum and plasma that can be applied to clinical biomarker studies and stoichiometric measurements of circulating RBP4, TTR, and retinol.

Retinol binding protein 4 (RBP4)↗

PubMed2025

A chemical epigenetic tool to probe site-specific DNA-binding protein complexes.

Site-specific DNA binding by proteins is critical for regulating transcriptional activity and cell fate decision. However, identifying proteins bound to specific genomic regions (e.g., promoter or enhancer regions) remains challenging. To address this, we developed a chemical epigenetic tool, named Site-specific noncanonical amino acid-mediated capture of protein (SCOPE), incorporating a photo-crosslinking amino acid into a nuclease-deficient dCas9 mutant. Human pluripotent stem cells (hPSCs) carrying SCOPE enable the capture of proteins bound to, in theory, any genomic location, facilitating the study of the cell context-dependent DNA-protein interactions. Using SCOPE, we identified the OCT4/SOX2/CARHSP1 complex binding to the NANOG promoter to maintain pluripotency in hPSCs. During ectoderm differentiation, ZIC2 acts as a competitive inhibitor, binding the same promoter region to downregulate NANOG expression and promote differentiation. Additionally, SCOPE identified that ZNF8 binds to the distal regulatory region of OCT4 to maintain naïve pluripotency. In summary, SCOPE provides a robust system for uncovering cell context-dependent, site-specific genome regulators, offering valuable insights into gene regulation networks driving cell fate transitions.

Humans↗

PubMed2024

An Intrinsically Disordered RNA Binding Protein Modulates mRNA Translation and Storage.

Proteins with intrinsically disordered regions (IDR) play diverse functions in regulating gene expression in the cell. Many of these proteins interact with cytoplasmic ribosomes. However, the molecular functions related to the interactions are largely unclear. In this study, using an abundant RNA-binding protein, Sbp1, with a structurally well-defined RNA recognition motif and an intrinsically disordered RGG domain as a model system, we investigated how an RNA binding protein with IDR modulates mRNA storage and translation. Using genomic and molecular approaches, we show that Sbp1 slows ribosome movement on cellular mRNAs and promotes polysome stacking or aggregation. Sbp1-associated polysomes display a ring-shaped structure in addition to a beads-on-string morphology visualized under the electron microscope, likely to be an intermediate slow translation state between actively translating polysomes and the translation-sequestered RNA granule. Moreover, the binding of Sbp1 to the 5'UTRs of mRNAs represses both cap-dependent and cap-independent translation initiation of proteins, many are functionally important for general protein synthesis in the cell. Finally, post-translational modifications at the arginine in the RGG motif change the Sbp1 protein interactome and play important roles in directing cellular mRNAs to either translation or storage. Taken together, our study demonstrates that under physiological conditions, intrinsically disordered RNA binding proteins promote polysome aggregation and regulate mRNA translation and storage using multiple distinctive mechanisms. This research also establishes a framework with which functions of other IDR-containing proteins can be investigated and defined.

RNA-Binding Proteins↗

PubMed2024

Mudskipper detects combinatorial RNA binding protein interactions in multiplexed CLIP data.

The uncovering of protein-RNA interactions enables a deeper understanding of RNA processing. Recent multiplexed crosslinking and immunoprecipitation (CLIP) technologies such as antibody-barcoded eCLIP (ABC) dramatically increase the throughput of mapping RNA binding protein (RBP) binding sites. However, multiplex CLIP datasets are multivariate, and each RBP suffers non-uniform signal-to-noise ratio. To address this, we developed Mudskipper, a versatile computational suite comprising two components: a Dirichlet multinomial mixture model to account for the multivariate nature of ABC datasets and a softmasking approach that identifies and removes non-specific protein-RNA interactions in RBPs with low signal-to-noise ratio. Mudskipper demonstrates superior precision and recall over existing tools on multiplex datasets and supports analysis of repetitive elements and small non-coding RNAs. Our findings unravel splicing outcomes and variant-associated disruptions, enabling higher-throughput investigations into diseases and regulation mediated by RBPs.

RNA-Binding Proteins↗

PubMed2026

Integrative analysis of the roles and prognostic value of RNA-binding proteins in papillary renal cell carcinoma.

RNA-binding proteins (RBPs) serve essential roles in various cancer types, but their functions in papillary renal cell carcinoma (pRCC) have not been elucidated to date. In our work, differentially expressed RBPs in pRCC were identified after acquisition of RNA-sequencing and clinical data related to pRCC from The Cancer Genome Atlas database(TCGA). Functional enrichment analysis and protein interaction network analysis, along with univariate and multivariate Cox regression analyses, were performed to uncover potential biological effects of the identified RBPs and screen the hub RBPs for pRCC prognosis. We identified 251 up-regulated and 129 down-regulated RBPs, and filtered out seven hub RBPs, namely, SRSF8, CD3EAP, HBS1L, ELAC2, MRPL34, NOP2 and IGF2BP2, for their prognostic relevance. A prognostic risk score model for overall survival of pRCC patients was constructed based on the seven hub RBPs. Further analysis showed that the low-risk group had higher survival rate than the high-risk group in both training and validation cohorts. The predictive accuracy was verified in the Human Protein Atlas database.In addition, we introduced the GSE15641 dataset from the Gene Expression Omnibus (GEO) database for independent external validation, and confirmed the expression levels of HBS1L, MRPL34 and IGF2BP2 through real-time quantitative PCR (RT-qPCR) and Western blotting (WB) using human renal tubular epithelial cell line HK-2 and human papillary renal cell carcinoma cell line Caki-2. In pRCC, CD3EAP was significantly elevated, while ELAC2, IGF2BP2, MRPL34, SRSF8 and HBS1L were significantly reduced. There was no significant difference between tumor and normal tissues in NOP2 expression. Risk score and tumor grade were independent prognostic factors associated with overall survival. In addition, we established a nomogram based on the seven prognostic RBPs to help predict overall survival at 1-3 years. In conclusion, seven differentially expressed hub RBPs were identified as potential prognostic biomarkers for pRCC. Our prognostic model might serve as a support for better treatment decision-making. Our work could provide potential new ideas for diagnosis and research on targeted drugs for pRCC.

Bioinformatics↗

PubMed2026

DBP-CanPred: a machine learning model for predicting cancer-causing mutations in DNA-binding proteins.

INTRODUCTION: The fundamental cellular processes, including transcriptional regulation, chromatin organization, and genome maintenance, are regulated by DNA-binding proteins (DBPs). Mutations in DBPs can alter protein-DNA interactions, leading to tumor development. However, identifying such driver mutations remains a major challenge due to limitations of experimental approaches. METHODS: We have trained a machine learning model, DBP-CanPred, to identify driver mutations in DBPs. We used the sequence-derived evolutionary features, as well as structure-based features such as mutation-perturbed structural descriptors. RESULTS: We evaluated DBP-CanPred using a curated test set, achieving an AU-ROC of 0.86 and a balanced accuracy of 0.79. Further analysis based on substitution-type showed consistent performance across different categories, especially higher performance on charged residues. In addition, we applied the model on an independent dataset and identified potential driver mutations with high confidence scores. DISCUSSION: The study contributes to understanding mutation patterns in DNA-binding proteins and supports variant interpretation in cancer research.

DNA-binding proteins↗

PubMed2024

Correlation between rs7041 and rs4588 polymorphisms in vitamin D binding protein gene and COVID-19-related severity and mortality.

BACKGROUND: The vitamin D binding protein (DBP) plays a critical role in both innate and adaptive immune systems, participating in several clinical conditions, including coronavirus disease 2019 infection severity, and mortality rate. The study aimed to investigate the correlation between rs7041 and rs4588 polymorphisms in the DBP gene and Coronavirus Disease-2019 (COVID-19) severity and mortality, in patients of Suez Canal University Hospitals in Ismailia, Egypt. METHODS: A case-control study enrolled 220 individuals; 140 COVID-19 patients and 80 healthy controls. Serum 25(OH) vitamin D levels were determined by the enzyme-linked immunosorbent assay (ELISA), and rs7041 and rs4588 polymorphisms of the DBP gene were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The study found that both groups had vitamin D deficiency, which was considerably lower in the COVID-19 patients group compared to controls. Among COVID-19 patients, there was a significant difference in vitamin D levels according to the disease severity indicating that vitamin D levels can be used as predictors of COVID-19 severity. Negative significant correlations between genetic variants rs4588 CA genotype and genetic variants rs7041 TT genotype and COVID-19 prevalence (p = 0.006 and 0.009 respectively) were proved. No significant correlations between all the genetic variants of both rs4588 and rs7041 and COVID-19 severity (p > 0.05). Positive significant correlations between both genetic variants rs4588 CA genotype and genetic variants rs7041 TG genotype and COVID-19 mortality (p = 0.029 and 0.031 respectively). CONCLUSION: vitamin D deficiency increased the severity of COVID-19. The DBP polymorphism correlated with vitamin COVID-19 prevalence and mortality.

Humans↗

PubMed2026

RNA Binding Protein PCBP1 Functions in the Endothelial-to-Hematopoietic Transition During Hematopoietic Stem Cell Formation.

Two paralogous RNA-binding proteins, PCBP1 and PCBP2, are individually essential for mouse development. Pcbp2-null embryos lose viability at midgestation, whereas Pcbp1-null embryos have an earlier, peri-implantation lethality. Inactivation of each gene alone in the differentiating erythroid lineage failed to impact embryonic viability, whereas combined Pcbp1/Pcbp2 inactivation resulted in repression of erythroid/hematopoietic gene expression midgestation, decreased blood cell formation, and fetal demise. Here, we assess the impacts of individual and combined conditional inactivation of Pcbp1 and Pcbp2 in endothelial cells on embryonic hematopoietic stem and progenitor cell (HSPC) formation. Inactivation of Pcbp1 in endothelial cells is embryonic lethal and reduces the number of HSPCs, whereas loss of Pcbp2 has no impact. Combined deletion of Pcbp1 and Pcbp2 in endothelial cells results in a more severe phenotype than loss of Pcbp1 alone. These results demonstrate that although Pcbp1 is a major regulator of HSPC formation, the two genes have nonredundant roles in endothelial cells.

Animals↗

PubMed2026

De novo variants in the poly(rC)-binding protein gene PCBP1 cause a neurodevelopmental disorder.

Poly(rC)-binding protein 1 (PCBP1), a splicing factor and key member of the hnRNP E family, was initially characterized for its tumor suppressive properties. More recently, its role in gene regulation in the brain and nervous system has attracted growing interest. Through an international multicenter collaboration, we identified 16 de novo pathogenic variants in PCBP1 across 17 subjects from 16 unrelated families. All affected individuals exhibited intellectual disability (ID), with autism spectrum disorder (ASD) as a prominent feature. Functional analysis in primary hippocampal mouse neuron cultures indicated that PCBP1 variants impair dendritic arborization, underscoring their deleterious effects. Transcriptomic profiling by RNA sequencing of subject-derived T cells showed a distinctive signature characterized by significantly increased exon skipping. These results highlight the contribution of PCBP1 in neurogenesis and neuritogenesis, which is impacted by loss-of-function variants expressed in neuronal cells, thereby supporting the link between splicing defects and neurodevelopmental disorders. Collectively, our findings demonstrate the prominent role of PCBP1 in neurodevelopment, reaffirming the importance of splicing regulation in mammalian neurodevelopment.

Journal Article↗

PubMed2025

The RNA-binding protein TRIM71 is essential for hearing in humans and mice and times auditory sensory organ development.

The RNA-binding protein TRIM71 is essential for brain development, and recent genetic studies in humans have identified TRIM71 as a risk gene for congenital hydrocephal-us (CH). Here, we show that monoallelic missense mutations in TRIM71 are associated with hearing loss (HL) and inner ear aplasia in humans. Utilizing conditional Trim71 knockout mice carrying a CH and HL-associated mutation, we demonstrate that loss of TRIM71 function during early otic development (embryonic day 9 to 10) causes severe HL. While inner ear morphogenesis occurs normally in Trim71 knockout mice, we find that early otic loss of TRIM71 function disrupts the highly stereotyped timing of cell cycle exit and differentiation within the inner ear auditory sensory organ (cochlea), resulting in the premature formation and innervation of mechanosensory hair cells. Transcriptomic profiling of Trim71-deficient cochlear progenitor cells identifies Inhba and Tgfbr2 as targets of TRIM71 repression, and our analysis of Inhba-Tgfbr1 double knockout mice indicates that TRIM71 maintains hair cell progenitors in a proliferative and undifferentiated state by restricting TGFβ-type signaling. Characterization of hair cells and their associated neurons in adult Trim71 knockout mice revealed reduced presynaptic terminals and neuronal degeneration in the outer hair cell region, providing a basis for the observed hearing deficits in Trim71 knockout mice.

Animals↗

PubMed2025

iNOME-seq: in vivo simultaneous genome-wide mapping of chromatin accessibility, nucleosome positioning, DNA-binding protein sites, and DNA methylation in Arabidopsis.

We present iNOMe-seq, a novel method for in vivo simultaneous profiling of chromatin accessibility, nucleosome occupancy, DNA-binding protein sites, and DNA methylation in living tissues. iNOMe-seq utilizes an m5C methyltransferase to mark accessible cytosines in a GpC context, bypassing nucleosome-restricted regions. Using Arabidopsis thaliana, we demonstrate that iNOMe-seq improves chromatin accessibility quantification compared to existing methods. Furthermore, it allows for the spatial and temporal analysis of chromatin dynamics, transcription factor binding, and DNA methylation, offering insight into the role of epigenetic components in transcriptional regulation across tissues and genetic variations in natural populations.

Arabidopsis↗

PubMed2026

Lineage structure and penicillin-binding protein variability in clinical Streptococcus pneumoniae isolates from Southwest China exhibiting reduced susceptibility to penicillin.

BACKGROUND: Reduced susceptibility to penicillin in Streptococcus pneumoniae is mediated primarily by alterations in penicillin-binding proteins (PBPs) and often coexists with multidrug resistance within successful lineages. The region-specific genomic characterization of clinically relevant pneumococci with reduced penicillin susceptibility in Southwest China remains limited. METHODS: We performed whole-genome sequencing of 204 clinical S. pneumoniae isolates collected from five institutions in Southwest China (2018-2022) that met our operational screening definition of reduced susceptibility to penicillin (PEN MIC ≥0.12 μg/mL). Molecular serotypes, MLST types, and Global Pneumococcal Sequence Clusters (GPSCs) were assigned; virulence and antimicrobial resistance determinants were profiled; and a core genome phylogeny was reconstructed with international contextualization through the use of PubMLST genomes meeting the same MIC criterion. Amino acid variability in PBP1a/PBP2b/PBP2x was quantified using TIGR4 numbering, and highly variable noncatalytic residues located within 15 Å of catalytic motifs were prioritized via structure-guided screening. RESULTS: The isolates showed a high burden of resistance to non-β-lactam antibiotics (erythromycin, 98.5%; tetracycline, 82.8%; trimethoprim-sulfamethoxazole, 64.7%), while fluoroquinolone susceptibility was largely preserved (≥97%), and vancomycin/linezolid resistance was not detected. Twenty-seven serotypes were identified, among which 19F (23.5%) and 19A (14.2%) were dominant, and the estimated PCV13 coverage was 69.6%. GPSC1 was the dominant lineage (36.8%), and the lineage composition among our isolates differed markedly from those in the PubMLST-USA and PubMLST-Thailand subsets. Virulence and resistance gene carriage differed markedly between GPSC1 and non-GPSC1 isolates, with enrichment of pilus operons, mef(A)/msr(D), and folA/folP in GPSC1. PBP variations were clustered in transpeptidase domains and motif-adjacent regions while essential catalytic residues were conserved; with the structure-guided filter, 12, 11, and 11 motif-proximal noncatalytic candidate sites were prioritized in PBP1a, PBP2b, and PBP2x, respectively. CONCLUSION: Clinical S. pneumoniae isolates with reduced penicillin susceptibility collected in Southwest China demonstrated resistance and accessory gene profiles that were strongly structured by a GPSC-defined lineage background. Our site-resolved, structure-guided PBP analysis provides a regional PBP variability landscape and a compact set of recurrent motif-proximal candidate substitutions to support surveillance and downstream functional validation.

Streptococcus pneumoniae↗

PubMed2025

In silico, in vitro, and in vivo characterization of thiamin-binding proteins from plant seeds.

Thiamin, an essential micronutrient, is a cofactor for enzymes involved in the central carbon metabolism and amino acid pathways. Despite efforts to enhance thiamin content in rice by incorporating thiamin biosynthetic genes, increasing thiamin content in the endosperm remains challenging, possibly due to a lack of thiamin stability and/or a local sink. The introduction of storage proteins has been successful in several biofortification strategies, and similar efforts targeting thiamin have been performed, leading to a 3-4-fold increase in white rice. However, only one thiamin-binding protein (TBP) sequence has been described in plants, more specifically from sesame seeds. Therefore, we aimed to identify and characterize TBPs, as well as to evaluate the effect of their expression on thiamin concentration, using a comprehensive approach integrating in silico, in vitro, and in vivo methods. We identified the sequences of putative TBPs from Oryza sativa (Os, rice), Fagopyrum esculentum (Fe, buckwheat), and Zea mays (Zm, maize) and pinpointed the thiamin-binding pockets through molecular docking. FeTBP and OsTBP contained one pocket with binding affinities similar to the Escherichia coli TBP, a well-characterized TBP, supporting their function as TBPs. In vivo expression studies of TBPs in tobacco leaves and rice callus resulted in increased thiamin levels, with FeTBP and OsTBP showing the most pronounced effects. Additionally, thermal shift assays confirmed the thiamin-binding capabilities of FeTBP and OsTBP, as observed by the significant increases in melting temperatures upon thiamin binding, indicating protein stabilization. These findings offer new insights into the diversity and function of plant TBPs and highlight the potential of FeTBP and OsTBP to modulate thiamin levels in crop plants.

Thiamine↗