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Results for “allele-specific proteomics”

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PubMed2026

Polysomal Profiling Coupled to Allele-Specific Proteomics Reveals an EIF4H TranSNP Allele Possessing Higher mRNA Translation Potential.

To search for genetic sources of allele-specific mRNA translation, we leveraged heterozygous polymorphisms and variants present in the exome of HCT116 colorectal adenocarcinoma-derived cells, computing allelic fractions from both total and polysome-associated RNA from RNA-Seq data. Allelic imbalance in polysomal RNA led us to nominate 52 coding variants associated with allele-specific mRNA translation, of which 16 are nonsynonymous. To validate instances of allele-specific translation, a proteomics workflow was developed that combines label-free shotgun analysis, high-pH reversed-phase peptide fractionation, and targeted parallel reaction monitoring using isotope-labeled peptide standards. Using this approach, we provide proof-of-concept validation of the heterozygous G>A, R183H missense single-nucleotide variant rs1554710467 in the eukaryotic initiation factor 4H (EIF4H) gene. The variant is present in two EIF4H alternatively spliced variants, which showed equivalent translation efficiency in HCT116 cells but differ in abundance. The alternative peptide containing H183 was significantly more abundant than the corresponding reference peptide containing R183, consistent with the over-representation of the alternative allele in polysomal RNA in HCT116 cells. A dual-fluorescence ribosome-stalling assay confirmed the enhanced translation potential of the variant allele. The two EIF4H allelic proteins exhibited similar stability and subpolysomal localization. This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein levels by exploiting heterozygous coding variants. Overall, our approach extends the toolbox available to investigate allele-specific differences in mRNA translation potential, a relatively underexplored layer of gene expression regulation that could reveal interindividual differences in disease-relevant phenotypes.

Humans↗

PubMed2026

Engineering cold stress resilience in capsicum annuum through functional genomics and precision breeding.

This review synthesizes the molecular mechanisms of cold tolerance in pepper, integrating multi-omics data,genome editing, and precision breeding strategies to accelerate the development of cold-resilient cultivars. Cold stress is a significant environmental factor that affects the growth, productivity, and fruit quality of Capsicum annuum by impairing membrane integrity photosynthesis and cellular redox homeostasis. Although pepper has several endogenous cold-responsive regulators such as CaNAC035 and CabHLH035, along with antioxidant defense systems, its cold tolerance remains limited due to low transcriptional activation of key regulators, functional redundancy among cold-responsive genes, and the polygenicity of cold tolerance. These complexities, combined with low genetic diversity and linkage drag, have hindered the improvement of cold-resistant cultivars through conventional breeding. This review brings together the recent progress in understanding the molecular mechanisms of cold stress perception, signal transduction, transcriptional regulation, metabolic reprogramming, and phytohormone interactions in pepper. Precision Breeding 2.0 is a new innovation that combines the integration of multi-omics-based target identification with next-generation genome-editing techniques, allowing precise and multiplex engineering of complex and interconnected regulatory networks instead of single genes. We cover new approaches such as engineering the DREB/CBF pathway, allele-specific editing and targeted disruption of negative regulators to enhance the pathway(s) involved in cold response. Moreover, we propose a roadmap for integration of transcriptomics, proteomics, metabolomics, high-throughput phenomics, and speed breeding to accelerate the identification, validation, and deployment of superior alleles to boost cold tolerance. This review provides a foundation for developing climate-resilient pepper cultivars by connecting functional genomics with precision genome engineering approaches to maintain productivity under variable environmental conditions.

Capsicum↗

PubMed2026

Innovative CRISPR/Cas9-Based Strategy for Allele-Specific HLA Peptidome Analysis Using a Pan-HLA Antibody.

Human leukocyte antigen (HLA) immunopeptidomics is restricted by the limited availability of allele-specific antibodies and by potential artifacts introduced by HLA overexpression systems. To address these challenges, we developed a CRISPR/Cas9-based strategy that selectively deletes undesired classical class I alleles while preserving a single endogenous allele, thereby enabling allele-resolved peptidome profiling with a pan-HLA class I antibody. As a proof of concept, we edited JY cells to eliminate HLA-B∗07:02 and HLA-C∗07:02 while retaining HLA-A∗02:01 (ΔBC clones). Peptide-HLA complexes were immunoprecipitated from WT and ΔBC clones using either the pan-HLA class I antibody W6/32 or the A∗02:01-specific antibody PA2.1, followed by nanoLC-MS/MS and computational HLA assignment. Deletion of HLA-B and HLA-C alleles caused an expected ∼55% reduction in total class I surface expression. Despite this, W6/32 immunoprecipitation from ΔBC clones recovered a comparable peptide yield to PA2.1 in WT cells. Binding predictions showed that most peptides identified in ΔBC clones using W6/32 were assigned to HLA-A∗02:01, with near-complete loss of HLA-B∗07:02- and HLA-C∗07:02-derived peptides. Sequence logo analysis confirmed the canonical A∗02:01 motif across conditions. The ΔBC W6/32 immunopeptidome exhibited a high degree of overlap (∼88%) with the WT PA2.1 repertoire, supporting the specificity and fidelity of the approach. These findings establish CRISPR-based editing of HLA alleles as a viable strategy for allele-specific immunopeptidome analysis using pan-HLA antibodies, supporting its potential application beyond this proof-of-concept system, reducing reliance on allele-specific reagents and facilitating the study of underrepresented HLA alleles.

Humans↗

PubMed2026

Immunopeptidomics-driven MHC class II peptide-binding motif discovery for 2 common canine DR alleles.

Despite the central role of major histocompatibility complex (MHC) class II in adaptive immunity, peptide-binding motifs have yet to be characterized for any canine MHC class II allele. Here, we report the first immunopeptidomics-derived binding motifs for DLA-DRB1*015:01 (DLA-DR15) and DLA-DRB1*012:01 (DLA-DR12), 2 alleles overrepresented in breeds predisposed to immune-mediated diseases. Because dogs co-express DLA-DR and DLA-DQ, the MHC class II Ab clone YKIX334.2 was validated to be DLA-DR-specific, enabling allele-selective immunoaffinity purification of DLA-DR molecules from homozygous DLA-DR15 and DLA-DR12 donor spleens. Mass spectrometry and GibbsCluster motif deconvolution of 838 DLA-DR15-associated and 644 DLA-DR12-associated peptides eluted from their respective peptide-binding grooves revealed distinct allele-specific binding motifs, with characterization of anchor residue preferences, peptide-length distributions, cross-species comparisons with human and murine MHC class II motifs, and source protein composition of the eluted self-peptidome. To evaluate the translational utility of these motifs, recombinant DLA-DR15 and DLA-DR12 molecules were used to screen rabies virus glycoprotein and nucleoprotein peptide libraries via fluorescence-based peptide competition assays, identifying high-affinity candidate binders for both alleles. Spearman rank correlation between immunopeptidomics-derived position-specific scoring matrix scores and peptide competition assay rankings demonstrated modest associations, consistent with these approaches capturing complementary dimensions of peptide-MHC class II interaction. Ultimately, these findings establish what we believe is the first allele-specific peptide-binding motif framework for canine MHC class II, providing a foundation for DLA-allele-informed CD4+ T-cell epitope discovery studies and Ag-specific immune response characterization in the dog.

Animals↗