Biology subjects

Das, H.

Publications and source records attributed to Das, H..

12 recordsLinked to original sources

bioRxiv2026

Palmitoylation of p75 neurotrophin receptor is required for retrograde regressive signaling

Target-derived neurotrophins regulate neuronal survival and pruning via retrograde signaling. Unlike Trk receptors, which are transported as transmembrane proteins, the p75 neurotrophin receptor (p75NTR) undergoes regulated intramembrane proteolysis to release a soluble intracellular domain (ICD) that must somehow associate with axonal endosomes for retrograde transport. Here, we identify palmitoylation of a conserved juxta membrane cysteine (C281) as a critical requirement for this process. In sympathetic neurons, we demonstrate that p75NTR palmitoylation is dispensable for surface trafficking and proteolytic cleavage but is strictly required for ligand-induced internalization and the biogenesis of p75ICD-positive signaling endosomes. Consequently, blocking palmitoylation, either pharmacologically or via a palmitoylation-deficient C281A mutation, prevents the accumulation of p75ICD in axonal endosomes and blocks axon-initiated apoptosis. In vivo, C281A knock-in mice exhibit reduced developmental apoptosis of sympathetic neurons, similar to p75NTR deletion. These findings define palmitoylation as an essential checkpoint that couples p75NTR cleavage to endosomal segregation, thereby enabling the long-distance transmission of regressive signals.

neuroscience↗

bioRxiv2025

Evolutionary Remodelling of a Remnant GET-Pathway Factor into PEX38, a Novel and Essential Peroxin in Euglenozoa

PEX19 is a cytosolic receptor that directs membrane proteins post-translationally to peroxisomes, as well as to mitochondria, lipid droplets, and the endoplasmic reticulum. A comprehensive Trypanosoma PEX19 interactome analysis uncovered PEX38 as a novel and essential Euglenozoa-specific peroxin. PEX38 contains distinct domains that bind the co- chaperone Hip and the PEX3-binding motif of PEX19, suggesting a role in stabilizing membrane protein and preventing premature membrane docking. PEX38 illustrates functional repurposing in organelle biogenesis. It originated from a remnant of the GET/TRC pathway, typically responsible for the targeting of tail-anchored proteins to the endoplasmic reticulum. While most components of this machinery are absent in Euglenozoa, PEX38 has been retained and adapted to mediate peroxisomal membrane protein targeting. This evolutionary adaptation is unique to Euglenozoa. Because the PEX19-PEX38 interaction is essential for parasite viability and PEX38 has no human homologs, this complex is a promising therapeutic target against trypanosomatid parasites.

cell biology↗

bioRxiv2025

The yeast peroxisomal proteome at absolute quantitative scale

Peroxisomes are dynamic organelles vital for lipid metabolism and redox homeostasis. In Saccharomyces cerevisiae, the expression of peroxisomal proteins is tightly regulated in response to metabolic conditions. Here, we provide the first absolute quantification of the yeast peroxisomal proteome under peroxisome-inducing (oleate) and fermentative (glucose) conditions using a label-free mass spectrometry approach. We determined protein copy numbers for [~]4,500 proteins, including 99 peroxisomal and peroxisome-associated proteins. Our data reveal that the peroxisomal proteome is [~]3-fold higher abundant in oleate-grown cells, constituting 2.8% of the total proteome (corresponding to 2.01 x 106 protein copies) compared to 0.8% (6.67 x 105 protein copies) in glucose, reflecting the necessity for peroxisomal functions such as fatty acid beta-oxidation. Enzymes of the beta-oxidation and glyoxylate cycle showed up to >500-fold higher abundance in oleate. In contrast, core components of the peroxisomal protein import machinery (e.g., Pex5, Pex14) exhibited only moderate changes ([~]2- to 8-fold). In addition to metabolic enzymes and components of the peroxisomal protein import pathways, we provide copy number data for proteins involved in cellular stress response, peroxisome proliferation, division and organization, peroxisome-associated membrane contact sites, and metabolite transporter. Taken together, our dataset offers a quantitative framework of peroxisomal remodeling under different metabolic conditions and highlights the organelles adaptive flexibility, providing a valuable resource for future studies on peroxisome biology.

cell biology↗

bioRxiv2025

Ca2+-dependent nanodomain enrichment is crucial for the perception of bacterial outer membrane vesicles in plants

O_LIXanthomonas oryzae pv. oryzae (Xoo) releases OMVs; however, the role of Xoo-OMVs in Xoo-rice interaction has not yet been studied. We investigated the early signalling events underlying Xoo-OMV perception by rice to gain insight into early plant-pathogen interactions. C_LIO_LIWe observed that Xoo-OMVs are perceived by rice through a rapid and unique Ca2+ signal that is essential to evoke an immune response. By confocal imaging and proteomic analysis of the rice PM, we found that the Ca2+ signal is required for Xoo-OMV-induced nanodomain enrichment and for the aggregation of defence response-associated proteins in rice PM. C_LIO_LIThe differential assembly of proteins in the PM helps the plant defend itself against Xoo. In the absence of the early Ca2+ signal, Xoo-OMVs are not perceived by the plant, resulting in a compromised immune response. C_LIO_LICollectively, our study shows that Xoo-OMVs are recognised by rice via Ca2+ signal-induced nanodomain assembly leading to protein reorganisation in the PM to prepare the plant for an imminent pathogen invasion. C_LI

plant biology↗

bioRxiv2024

PEX39 facilitates the peroxisomal import of PTS2 proteins

Peroxisomes are metabolic organelles essential for human health. Defects in peroxisomal biogenesis proteins (peroxins/PEXs) cause devastating disease. PEX7 binds newly synthesized proteins containing a type 2 peroxisomal targeting signal (PTS2) to enable their import from the cytosol into peroxisomes, although many aspects of this import pathway remain enigmatic. Utilizing in vitro assays, yeast, and human cells, we show that PEX39, a previously uncharacterized protein, is a cytosolic peroxin that facilitates PTS2-protein import by binding PEX7 and stabilizing its interaction with PTS2 cargo. PEX39 and PEX13, a peroxisomal membrane translocon protein, both possess a KPWE motif necessary for PEX7 binding. Sequential binding of PEX7 to this motif in PEX39 and PEX13 provides a novel paradigm for how PTS2 cargo engage the translocation machinery. Collectively, our work uncovers an ancient and functionally important relationship among PEX39, PEX7, and PEX13, offering insights that will advance our understanding of peroxisomal biogenesis and disease.

cell biology↗

bioRxiv2024

Integrative Omics reveals changes in the cellular landscape of yeast without peroxisomes

Peroxisomes are organelles that are crucial for cellular metabolism. However, these organelles play also important roles in non-metabolic processes, such as signalling. To uncover the consequences of peroxisome deficiency, we compared two extremes, namely Saccharomyces cerevisiae wild-type and pex3 cells, which lack functional peroxisomes, employing transcriptomics and quantitative proteomics technology. Cells were grown on acetate, a carbon source that involves peroxisomal enzymes of the glyoxylate cycle and does not repress peroxisomal proteins. Transcripts of peroxisomal {beta}-oxidation genes and the corresponding proteins were enhanced in pex3 cells. Peroxisome-deficiency also caused reduced levels of membrane bound peroxins, while the soluble receptors Pex5 and Pex7 were enhanced at the protein level. In addition, we observed alterations in non-peroxisomal transcripts and proteins, especially mitochondrial proteins involved in respiration or import processes. Our results not only reveal the impact of the absence of peroxisomes in yeast, but also represent a rich resource of candidate genes/proteins that are relevant in peroxisome biology. SummaryOmics comparison of wild-type and peroxisome-deficient (pex3) yeast cells uncovered processes that are affected by loss of peroxisomes. {beta}-oxidation enzymes were upregulated, whereas most peroxins were decreased. Also, several non-peroxisomal transcripts/proteins were significantly altered. Our data represent a rich source of candidate genes connected to peroxisome biology.

cell biology↗

bioRxiv2023

High confidence glycosomal membrane protein inventory unveils trypanosomal Peroxin PEX15

Infections by trypanosomatid parasites cause Chagas disease, Human African Trypanosomiasis, and Leishmaniasis, affecting over 12 million people worldwide. Glycosomes, the unique peroxisome-related organelles of trypanosomes are essential for their survival, and hence their metabolic functions and biogenesis mediated by peroxins (PEX) are suitable as drug targets. Here we report on a comprehensive protein inventory of glycosomal membranes through advanced subcellular membrane protein profiling employing quantitative mass spectrometry. Our quantitative analysis resulted in the identification of 28 novel high confidence glycosomal membrane proteins. Our in-depth protein inventory of glycosomal membranes serves as an important resource for characterizing glycosome biology and drug development. We validated four so far unknown glycosomal membrane proteins, including two tail-anchored (TA) proteins, a homolog of human peroxisomal PXMP4, and a Macrodomain-containing protein. Using a structure-based approach, we identified one of the TA proteins as the long-sought Trypanosoma PEX15. Despite its low sequence similarity, Trypanosoma PEX15 exhibits structural and topological similarities with its yeast (Pex15) and human counterparts (PEX26). We show that PEX15 is an essential integral glycosomal membrane protein that interacts with PEX6. Accordingly, RNAi knockdown of PEX15 in bloodstream form trypanosomes demonstrates that it is essential for glycosome biogenesis and parasite survival. Considering the low degree of conservation with its human counterpart, PEX15 is a promising molecular target for drug development.

cell biology↗

bioRxiv2022

Structural basis of Omicron neutralization by affinity-matured public antibodies

The SARS-CoV-2 Omicron1 Variant of Concern (B.1.1.529) has spread rapidly in many countries. With a spike that is highly diverged from that of the pandemic founder, it escapes most available monoclonal antibody therapeutics2,3 and erodes vaccine protection4. A public class of IGHV3-53-using SARS-CoV-2 neutralizing antibodies5,6 typically fails to neutralize variants carrying mutations in the receptor-binding motif7-11, including Omicron. As antibodies from this class are likely elicited in most people following SARS-CoV-2 infection or vaccination, their subsequent affinity maturation is of particular interest. Here, we isolated IGHV3-53-using antibodies from an individual seven months after infection and identified several antibodies capable of broad and potent SARS-CoV-2 neutralization, extending to Omicron without loss of potency. By introducing select somatic hypermutations into a germline-reverted form of one such antibody, CAB-A17, we demonstrate the potential for commonly elicited antibodies to develop broad cross-neutralization through affinity maturation. Further, we resolved the structure of CAB-A17 Fab in complex with Omicron spike at an overall resolution of 2.6 [A] by cryo-electron microscopy and defined the structural basis for this breadth. Thus, public SARS-CoV-2 neutralizing antibodies can, without modified spike vaccines, mature to cross-neutralize exceptionally antigenically diverged SARS-CoV-2 variants.

immunology↗

bioRxiv2021

A bispecific monomeric nanobody induces SARS-COV-2 spike trimer dimers

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we isolated a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the B.1.351 variant, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the B.1.351 variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes a novel strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.

immunology↗

bioRxiv2020

Modified split application of nitrogen with biochar improved grain yield and nitrogen use efficiency in rainfed maize grown in Vertisols of India

Conventionally, non-judicious and blanket fertilizer nitrogen (N) used in rainfed maize lead to higher N losses, low N use efficiency (NUEs) and poor yields due to substandard agronomic management practices. To avoid such N losses, fertilizer additions are synchronized with plant uptake requirements. In this context, agronomic based management focused on optimizing N rates and biochar application is essential for improved NUEs and crop productivity. Keeping this in view, a field experiment was conducted during 2014, 2015 and 2016 in rainfed maize (Zea mays L.) grown in Vertisols of India. In this study, twelve treatments that comprised of N omission plot (N0), skipping of basal rate, multi-split topdressing at varying time as broadcast and band placement, soil test crop response (STCR) based NPK with target yield 6.0 t ha-1 in maize and biochar application (10 t ha-1) were investigated. The experiment was conducted following a Randomized Complete Block Design (RCBD) set up with three replications. Pooled analysis of three years data revealed that the application of N rates (120 kg Nha-1) in 2 equal splits (60 kg Nha-1) at knee high (V8) and tasseling (VT) stages with skipped basal N rate, achieved higher maize grain yield (5.29 t ha-1) ascribed to the greater growth parameters, yield components and N uptake compared to the recommended practices. Biochar application (10 t ha-1) as soil amendments along with multi top dressed N (120 kg N ha-1) into 3 splits also increased the grain yield. Delayed N application at V8 and VT growth stages, resulted in higher N uptake, agronomy efficiency (AE), partial factor productivity (PFP), physiology efficiency (PE) and recovery efficiency (RE). Biochar along with N fertilizer also improved the soil organic carbon (5.47g kg-1), ammonium-N (2.40 mg kg-1) and nitrate-N (0.52 mg kg-1) concentration in soil (P<0.05) as compared to non-biochar treatments. Application of biochar along with chemical fertilizer (120 kg Nha-1) significantly increased the concentration of ammonium (2.40 mg kg-1) and nitrate (0.52 mg kg-1) in soil (P<0.05) as compared to non-biochar treatments. The perfect positive linear relationship illustrated that the grain yield of rainfed maize was highly dependent (R2=0.99 at p<0.0001) on N availability, as indicated by the fitted regression line of maize grain yield on N uptake. On the other hand, factor analysis revealed, the one to one positive function relationship of biomass with N uptake at V8 and VT growth stages. Principal Component Regression (PCR) analysis showed that PC1 acted as a major predictor variable for total dry matter yield (TDMY) and dominated by LAI and N uptake. Consequently, these results expressed that the agronomic management based multi-top dressed N application and biochar application to achieve higher yield and greater NUEs in rainfed maize is strongly linked with N application into splits.

plant biology↗

bioRxiv2020

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 {micro}g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the up ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

biophysics↗

bioRxiv2020

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

SARS-CoV-2 is the etiologic agent of COVID-19, currently causing a devastating pandemic for which pharmacological interventions are urgently needed. The virus enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. The nanobody binds with high affinity in the low nM range to the RBD, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations and that Ty1 sterically hinders RBD-ACE2 binding. This 12.8 kDa nanobody does not need an Fc domain to neutralize SARS-CoV-2, and can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗