Biology subjects

Sahu, D.

Publications and source records attributed to Sahu, D..

11 recordsLinked to original sources

bioRxiv2026

An extended N-terminus restrains the plant cell death-inducing ability of the catalytically competent ribonuclease domain in a pea powdery mildew RALPH effector

RALPH (RNase-like proteins associated with haustoria) effectors, which are preferentially expressed in haustoria and structurally resemble fungal T1/F1 RNases, constitute one of the largest effector families in powdery mildew (PM) fungi, yet their functions in dicot-adapted PM species remain poorly understood. Unlike cereal PM RALPHs, which lack the catalytic residues required for RNase activity, some dicot PM RALPHs retain these residues. Here, we performed a comprehensive structural and expression-based characterization of the pea PM Erysiphe pisi RALPH (EpRALPH) repertoire and functionally characterized EpRALPH11, a RALPH effector with partial conservation of the catalytic residues of T1/F1 fungal RNases. Comparative analyses identified multi-RNase-domain RALPHs as a conserved feature of the Erysiphe lineage, while expression profiling showed that many EpRALPHs are preferentially expressed in haustoria during early host colonization. AlphaFold 3-based structural analyses revealed a conserved T1/F1 RNase-like fold despite substantial sequence and surface charge divergence, indicating functional diversification among EpRALPHs. EpRALPH11 enhanced susceptibility to E. pisi in Medicago truncatula, localized to the nucleolus, and induced nucleolar fragmentation when heterologously expressed in Nicotiana benthamiana leaves. Its RNase domain exhibited T1 RNase activity in vitro, supporting the retention of a catalytically competent RNase domain and, together with its nucleolar localization, suggesting that EpRALPH11 targets plant rRNA and disrupts nucleolar functions. The RNase domain induced cell death in N. benthamiana, whereas the full-length protein and catalytic mutants did not. Cell death induction required exclusive nucleolar localization of the RNase domain, and an extended N-terminal intrinsically disordered region suppressed this activity in the full-length protein. Together, our findings reveal a previously unrecognized mechanis regulating RNase activity in a dicot PM RALPH effector and provide new insights into the functional diversification of RALPHs and their adaptation to obligate biotrophy.

plant biology↗

bioRxiv2026

tRF-3021a, a tRNA-Ala-TGC derived 3' fragment, promotes glioblastoma cell invasion, suppresses apoptosis, and is required for normal levels of protein synthesis

tRNA-derived fragments (tRFs) are a relatively recently discovered class of small RNAs implicated in gene-regulatory processes in diverse biological contexts but there have been very few reports of a clear phenotypic role of these small RNAs in cancer progression. To select tRFs that should receive priority for mechanistic experiments, we analyzed small RNA-seq data from The Cancer Genome Atlas (TCGA) and found that high expression of three 3' tRFs (tRF-3a), tRF-3009a, tRF-3021a or tRF-3030a, is significantly associated with poor overall survival in low-grade glioma (LGG). In glioblastoma cells, tRF-3009a, tRF-3021a and tRF-3030a enhance cell invasion and migration but tRF-3021a was uniquely required for cell proliferation and suppression of apoptosis. Interestingly, tRF-3021a knockdown decreases global protein synthesis prior to and independent of apoptosis in a number of cancer cell lines, both from and outside the glioblastoma lineage. RNA-seq reveals that the results cannot be explained by microRNA-like functions of tRF-3021a. These data indicate that tRF-3021a supports cancer cell survival and particularly protein synthesis while promoting cellular invasion and migration and suggests that strategies to downregulate this short RNA could be a potential therapeutic approach in cancers. ImplicationtRF-3021a promotes malignant cell phenotypes, sustains global protein synthesis and prevents spontaneous apoptosis, motivating efforts to evaluate it as a biomarker and therapeutic target.

cancer biology↗

bioRxiv2025

Unlocking meiotic crossovers near plant centromeres

Crossovers (COs) ensure proper chromosome segregation during meiosis and generate genetic diversity. COs are non-uniformly distributed along chromosomes and almost universally suppressed in centromere-proximal regions, notably creating a significant bottleneck for plant breeding. The mechanism of this CO suppression is elusive, but chromatin state is a contributing factor. Here, we identify three factors that actively limit proximal CO in Arabidopsis, the cohesion establishment factor CTF18, the centromeric cohesin protector SGO2, and the deSUMOylase SPF2. The mutation of these factors allows both the formation of CO in centromere-proximal region where they were completely absent in the wild-type, and enhance their frequency where they were rare. COs can be further increased by combining these mutations together or with mutation in the DNA methylase CMT3, suggesting that multiple mechanism prevent proximal CO in parallel. The identification of the very conserved CTF18, SGO2 and SPF2 as suppressors of centromere-proximal CO highlights the importance of cohesin turnover in this process and opens up new possibilities for plant breeding.

genetics↗

bioRxiv2025

AllerStack: Predicting Allergenic Proteins with a Stacked Ensemble Approach

Accurate prediction of protein allergenicity is essential for ensuring food and drug safety. While machine learning and deep learning models have been explored for this task, limitations remain in dataset scale, feature representation, and model architecture. Here, we introduce AllerStack, a two-stage stacked ensemble model integrating handcrafted and ESM2-based learned features for allergenicity classification. The model was developed using a balanced dataset comprising 11,930 allergenic and 11,930 non-allergenic proteins. We extracted amino acid composition, dipeptide composition, and physicochemical features using the Biopython library, alongside contextual embeddings from the pre-trained ESM2 protein language model. Diverse classifiers (QDA, SVM, KNN, and ANN) were trained separately on these features in the base layer. Their predictions were used as input to a meta-classifier based on XGBoost. AllerStack achieved high predictive performance with 96.87% accuracy, 96.86% F1-score, 93.75% Matthews correlation coefficient (MCC), and an AUC of 0.99. A publicly accessible web server (https://cosylab.iiitd.edu.in/allerstack/) enables real-time allergenicity prediction from protein sequences. AllerStack provides a robust, interpretable, and user-friendly platform for allergen detection in computational biology. HighlightsO_LIAn extensive dataset of 23,860 proteins. C_LIO_LICombines handcrafted and ESM2-derived features. C_LIO_LIStacked ensemble model with an accuracy of 96.87%. C_LIO_LISHAP-based model interpretability at the model and feature level. C_LIO_LIPublic web server, AllerStack (https://cosylab.iiitd.edu.in/allerstack/). C_LI

bioinformatics↗

bioRxiv2025

Resolving the molecular niche of pulmonary fibrosis using cryopreserved human precision cut lung slices

Background and methodsPulmonary fibrosis (PF) manifests locally and heterogeneously in the diseased lung. To uncover mechanistic underpinnings, we have utilized cryopreserved fibrotic human precision-cut lung slices (PCLS), and using single-cell spatial transcriptomics, mapped and interpreted the transcriptomic regulation of effector cell types/states. ResultsCryopreserved-thawed fibrotic human PCLS retained canonical molecular and cellular PF hallmarks including fibroblast and myofibroblast accumulation, epithelial depletion, and vascular remodeling. Notably, these cellular changes were spatially co-incident with 1) collagen-rich regions enriched for extracellular matrix (ECM)-related genes (e.g., COL1A1, SPARC), 2) expanded fibroblast-endothelial crosstalk with enhanced chemokine signaling (CXCL12- CXCR4), 3) integrin-driven mesenchymal signaling targets (ANGPTL-integrin), 4) stress-adapted and immune-interacting Alveolar Type 2 cells with impaired surfactant gene expression, and 5) endothelial reprogramming toward a contractile and profibrotic phenotype. At the transcriptomic level, the spatially-defined fibrotic niche was characterized by increased gene expression related to immune responses (IGKC and IGHG1), fibrosis (collagen, fibronectin, and DCN), mechanotransduction (MYH11, TPM2, ACTG2, ACTA2), immune cell recruitment (CCL2, IL6, ICAM1), ECM remodeling (e.g. thrombospondin 1, THBS1), and decreased gene expression related to surfactant production (SFTPB, SFTPC). At the functional level, the fibrotic niches were defined by the activation of three fibroblast mediating signaling pathways: (1) the matricellular glycoprotein, SPARC, (2) Relaxin signaling, and (3) multiple immunoglobulin transcripts, including IGHA1, IGHG1, IGHG3, and IGKC. ConclusionsWe verify model fidelity, identify both known and unanticipated pro-fibrotic genes, and advocate for the expanded use of spatial transcriptomic measurements using cryopreserved human PCLS for disease modeling and drug discovery.

genomics↗

bioRxiv2025

The N-terminal Segment of the Human ANKZF1 negatively regulates its internal mitochondrial targeting signal to prevent its mitochondrial localization

ANKZF1, the human orthologue of yeast Vms1, is a multi-domain cytosolic protein. Occasionally, the protein is also found in mitochondria, although the reason for its mitochondrial localization and the mechanism of its mitochondrial targeting remains unclear. Despite the absence of any predicted Mitochondrial Targeting Sequence or Domain (MTS/MTD) in the protein, ANKZF1 possesses multiple internal Matrix-targeting sequence-like sequences (iMTS-Ls). In this study, we show that ANKZF1s N-terminal 73 residues negatively regulate its mitochondrial targeting. Interestingly, 1-72-ANKZF1 peptide hinders the mitochondrial targeting of {Delta}73-ANKZF1 upon co-expression. Using a series of truncation mutants of ANKZF1, we further show that iMTS-Ls constituted by residues 231-240 of ANKZF1 is indispensable for its mitochondrial localization. The sequence of 231-240 residues is extremely conserved across different organisms, indicating the importance of this segment for conditional mitochondrial targeting of the protein. Importantly, 231-324 residues containing two consecutive predicted iMTS-Ls constitute an independent mitochondrial signal sequence and can target Green Fluorescent Protein (GFP) to mitochondria when fused to N-terminus. Further, by Molecular Dynamic simulation, we show that the deletion of N-terminal 74 amino acids of ANKZF1 leads to a massive structural rearrangement within the protein leading to the opening of its C-terminal part and solvent exposure of the 231-240 residues. We posit that these structural rearrangements and exposure of the internal mitochondrial targeting sequence in the absence of N-terminal segment of ANKZF1 leads to its mitochondrial translocation.

cell biology↗

bioRxiv2025

Chaperone-mediated autophagy substrates bind to lid domain of Hsc70: Exploration using integrated approach

Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway crucial for cellular homeostasis. Its dysfunction is linked to cancer and neurodegenerative disorders. Hsc70 mediates CMA selectivity by recognizing proteins with KFERQ-type sequences, but the structural basis for substrate recognition and the substrate-binding mechanism of Hsc70 remain unclear. This study integrates literature, bioinformatics, AlphaFold 3 predictions, and 44 s of molecular dynamics (MD) simulations to elucidate the lid domain interactions of Hsc70 with fifteen experimentally verified CMA substrate segments. Structural and thermodynamic residue-level analyses suggest stable binding of substrates in two distinct sites. The first site resides in a hydrophobic cavity formed by alpha helix 1 and 3 in the lid domain of Hsc70, with complex stabilization primarily driven by hydrophobic residues within KFERQ-type motif and its flanking regions. The second stable site was observed to be canonical hydrophobic cleft in Hsc70, with complex stabilization primarily driven by hydrophobic residues within KFERQ-type motif as well as the through water-mediated interactions by the lid domain. The robustness of our findings is confirmed by consistent convergence across force fields, substrates, and supporting literature, all identifying the same site. We propose these sites may facilitate upstream or downstream recognition and activation events. Author SummaryCells must constantly remove damaged or unnecessary proteins in order to stay healthy. One important pathway that performs this task is chaperone-mediated autophagy. When this pathway fails, it has been linked to conditions such as cancer and neurodegenerative diseases. A key player in this process is the protein Hsc70, which recognizes short sequence signatures in target proteins and directs them for degradation. However, how Hsc70 physically recognizes these signatures has remained poorly understood. In this study, we investigated how Hsc70 interacts with a range of confirmed target protein segments. We combined information from prior experimental studies, with computational structure prediction and extensive molecular simulations to explore these interactions in detail. Our results indicate that Hsc70 can bind target proteins at two distinct regions, each stabilized mainly by hydrophobic amino acids within and/or around the recognition signal, with additional contribution from water-mediated interactions in the latter site. Notably, these binding modes were consistent across different targets and computational conditions. Together, our findings provide a clearer structural picture of how selectivity is achieved in this degradation pathway. This improved understanding may help guide future studies on how chaperone-mediated autophagy is regulated and how it becomes disrupted in disease.

biophysics↗

bioRxiv2024

A 36-base hairpin within lncRNA DRAIC, which is modulated by alternative splicing, interacts with the IKKα coiled-coil domain and inhibits NF-kB and tumor cell phenotypes

A tumor-suppressive long noncoding RNA (lncRNA) DRAIC (down-regulated RNA in cancers) inhibits NF-{kappa}B activity and physically interacts with IKK, a kinase component of the IKK complex, in several cancer types. Here we explore the precise molecular mechanism involved in this interaction and suppression. Using SHAPE-MaP, we identified a 36-nucleotide hairpin (A+B) within DRAIC that is necessary and sufficient for its anti-oncogenic function. RNA immunoprecipitation (RIP) and Electrophoretic mobility shift assays (EMSA) confirmed this hairpin physically interacts with the coiled coil domain of IKK. A+B RNA has a high binding affinity (KD [~]1-7 nM) to the coiled-coil domain of IKK. The binding of A+B disrupts the dimerization of NEMO and IKK coiled-coil domains, a critical step for IKK action. Consistent with this, A+B inhibits the phosphorylation of the NF-{kappa}B inhibitor I{kappa}B and suppresses NF-{kappa}B activity. Publicly available tumor RNAseq data revealed that alternative splicing modulates the presence of this critical hairpin: the inclusion of exon 4a (encoding one side of the A+B hairpin) in lung tumors correlates with reduced NF-{kappa}B activity. By demonstrating that the A+B hairpin is both necessary and sufficient to inhibit IKK and oncogenic phenotypes, this study underscores the centrality of IKK interaction and NF-{kappa}B inhibition in DRAIC-mediated cancer suppression and indicates that the activity of this lncRNA is regulated by alternative splicing. This study also reveals the first example of a short RNA disrupting coiled-coil dimerization, offering a new approach to disrupt such dimerization in cancer biology.

molecular biology↗

bioRxiv2022

Novel Peptide Inhibitor of Human Tumor Necrosis Factor-α has Antiarthritic Activity

The inhibition of tumor necrosis factor- (TNF) trimer formation renders it inactive for binding to its receptors thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNF monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Modeller) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNF. Fluorescence-activated cell sorting (FACS) and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNF-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NF{kappa}B) was significantly suppressed in the peptide-treated A549 cells as observed in immunofluorescence and gel mobility-shift assays. Furthermore, peptide protected against joint damage in collagen-induced arthritis (CIA) mouse model as revealed in the microfocal-CT scans. In conclusion, this TNF antagonist would be useful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an anti-arthritic drug.

immunology↗

bioRxiv2021

The human disease gene CLEC16A encodes an intrinsically disordered protein region required for mitochondrial quality control

CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. While CLEC16A has ubiquitin ligase activity, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs pancreatic {beta}-cell mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases its self-ubiquitination and destabilizes CLEC16A, thus impairing formation of a critical CLEC16A-dependent mitophagy complex. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we clarify how an IDPR in CLEC16A prevents diabetes, thus implicating the disruption of IDPRs as novel pathological contributors to diabetes and other CLEC16A-associated diseases.

molecular biology↗

bioRxiv2020

Troxerutin acts on complement mediated inflammation to ameliorate arthritic symptoms in rats.

Troxerutin (TXR), is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit anti-arthritic properties of TXR using an adjuvant induced arthritic (AIA) rat model. AIA induced rats showed highest arthritis score at disease onset and by oral administration of TXR (50, 100, 200 mg/kg body weight), reduced to basal level in a dose dependent manner. Isobaric tag for relative and absolute quantitative (iTRAQ) proteomics tool was employed to identify deregulated joint homogenate proteins in AIA and TXR treated rats to decipher probable mechanism of the TXR action in arthritis. iTRAQ analysis identified a set of 434 joint homogenate proteins with 65 deregulated proteins (log2 case/control [≥]1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from healthy were validated using Western blot analysis. Western blot data corroborated proteomics findings. In silico protein-protein interaction study of joint homogenate proteome revealed that complement component 9, the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, gets perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an anti-arthritis agent in AIA model and after additional investigation its arthritis ameliorating properties could be exploited for clinical usability.

biochemistry↗