Biology subjects

Chauhan, R. D.

Publications and source records attributed to Chauhan, R. D..

6 recordsLinked to original sources

bioRxiv2026

The scaffold nucleoporin Nup188 undergoes liquid-liquid phase separation

Components of nuclear pore complexes are broadly classified into two categories: intrinsically disordered FG-repeat nucleoporins such as Nup98, which can undergo liquid-liquid phase separation, and scaffold nucleoporins, which form the NPC's structural framework. The basis for crosstalk of these nucleoporins within NPC and beyond is poorly understood. Here we show that human Nup188, a helical inner-ring scaffold protein, forms condensates both in purified form as well as at endogenous level. This condensate exhibits fusion, dissolved in 1,6-hexanediol and recovered after photobleaching, with dynamics and material properties comparable to those of hNup98 condensates. Moreover, these condensates are not colocalized to annulate lamellae. Our analysis also revealed that hNup188CTD is sufficient to form condensates and MD simulations revealed the key roles of IDR1-3 for condensation. Notably, hNup93 expression along with hNup188 suppressed the condensate formation. Together, these findings indicate that a structured scaffold nucleoporin can adopt a condensate-forming state via its CTD and Nup93 binding can suppress this phenomenon.

biochemistry↗

bioRxiv2023

The actin-associated protein Kaptin modulates F-actin barbed-end dynamics

Living cells require a dynamic and precisely regulated actin cytoskeleton to carry out normal cellular functions. In addition to well-established actin cytoskeleton regulators, such as nucleators, capping proteins, and bundlers, cells likely have uncharacterized modulators that regulate cytoskeleton dynamics, the detailed functions of which are not yet fully understood. In this study, we conducted biochemical exploration to identify the actin-regulatory activity of Kaptin (KPTN), a protein known to co-localize with actin-rich structures at the cells periphery. Using single-molecule assays, we demonstrated that KPTN inhibits actin nucleation. Our results revealed that KPTN possesses a novel barbed-end capping activity, which stabilizes and bundles actin filaments. Structural modeling, based on AlphaFold, suggests that KPTN is a member of the WD-repeat-containing protein family. Furthermore, we identified a crucial cationic residue in the putative N-terminal beta-propeller region of KPTN that plays a critical role in modulating actin dynamics. In summary, our data unveil the mechanistic underpinning functions of KPTN and establish its novel role as a regulator of the actin cytoskeleton.

cell biology↗

bioRxiv2021

CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production

Cassava (Manihot esculenta Crantz) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces toxic cyanogenic compounds and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. Reducing the cyanogen content by conventional breeding is problematic due to the heterozygous nature of the crop; recombination will generally disrupt a clonally propagated cultivars suite of desirable traits. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444 and the West African, farmer-preferred cultivar TME 419. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. Our work demonstrates cassava genome editing for food safety, reduced processing requirements, and environmental benefits that could be readily extended to other farmer-preferred cultivars.

plant biology↗

bioRxiv2021

Cryo-EM structure of human Nup155 reveals the biochemical basis for atrial fibrillation linked genetic mutation R391H

Human nuclear pore complexes are composed of [~]32 distinct nucleoporins to facilitate bidirectional nucleo-cytoplasmic transport. Many of them have been associated with various human diseases such as an inherited mutation (R391H) in Nup155 is shown as the clinical cause of atrial fibrillation and sudden cardiac arrest. Due to the lack of structural knowledge and mechanistic insights, the roles of Nups in NPC assembly and relevance in human diseases are very restricted. Here, we show the cryo-EM structure of human Nup155 at 5.2-5.7. [A] resolution deciphered from 3 distinct particle classes: N-terminus (19-863), C-terminus (864-1337), and longer N-terminus (19-1069). It revealed intrinsic plasticity at the middle domain of Nup155 and the role of species-specific loop regions in an atypical 7-bladed {beta}-propeller domain to provide a distinct interface for Nup93 and Nup35. Due to the proximity of these Nups interacting sites near the Arginine-391 position, atrial fibrillation linked genetic mutation (R391H) causes dissociation from NPC in absence of N-terminal 112 residues. HighlightsO_LICryo-EM structure of human Nup155 at 5.2 [A] resolution C_LIO_LISeven bladed {beta}-propeller domain at N-terminus of Nup155 exhibited distinct features for interaction with Nup35 and Nup93 C_LIO_LIThe middle domain of Nup155 is highly dynamic in nature C_LIO_LIStructural mapping allows mechanistic interpretation of AF linked R391H mutation C_LI

cell biology↗

bioRxiv2020

CASSPER: A Semantic Segmentation based Particle Picking Algorithm for Single Particle Cryo-Electron Microscopy

Single-particle cryo-electron microscopy has emerged as the method of choice for structure determination of proteins and protein complexes. However, particle identification and selection which is a prerequisite for achieving high-resolution still poses a major bottleneck for automating the steps of structure determination. Here, we present a generalised deep learning tool, CASSPER, for the automated detection and isolation of protein particles in transmission microscope images. This deep learning tool uses Semantic Segmentation and a collection of visually prepared training samples to capture the differences in the transmission intensities of protein, ice, carbon and other impurities found in the micrograph. CASSPER is the first method to do pixel level classification and completely eliminates the need of manual particle picking. Integration of Contrast Limited Adaptive Histogram Equalization (CLAHE) in CASSPER enables high-fidelity particle detection even in micrographs with variable ice thickness and contrast. In addition, our generalized model for cross molecule picking works with high efficiency on unseen datasets and can potentially pick particles on-the-fly, thereby, enabling automation of data processing.

biophysics↗

bioRxiv2017

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 confers elevated resistance to cassava brown streak disease

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome-linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E-1, eIF(iso)4E-2, novel cap-binding protein-1 (nCBP-1), and nCBP-2. Protein-protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9-mediated genome editing was employed to generate ncbp-1, ncbp-2, and ncbp-1/ncbp-2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp-1/ncbp-2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titer in storage roots relative to wild-type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

plant biology↗