Biology subjects

Saha, I.

Publications and source records attributed to Saha, I..

11 recordsLinked to original sources

bioRxiv2025

Mitochondrial proteostatic stress disrupts mitoribosome biogenesis and translation

Protein aggregation in various cellular compartments is a hallmark of proteostasis impairment linked to aging and numerous pathologies. Mitochondrial function depends on a balanced interplay of proteins imported from the cytosol as well as those synthesized on mitochondrial ribosomes (mitoribosomes). Here, we reveal an unexpected susceptibility of mitoribosome biogenesis to organellar proteostatic stress. Importing aggregation-prone proteins into yeast and human mitochondria triggered a chain of detrimental events involving extensive co-aggregation of newly-imported mitoribosome subunits and other RNA-binding proteins, as well as local disruption of mitochondrial cristae morphology. As a result, mitoribosome assembly and mitochondrial translation were severely impaired, leading to respiratory deficiency and, ultimately, loss of mitochondrial DNA. Surprisingly, dysfunction of mitochondrial HSP60 phenocopied the ribosome biogenesis defect and inhibition of translation, indicating a pronounced chaperone dependence of mitoribosome proteins. Declining mitochondrial translation likely contributes to aging and diseases associated with deficiencies in mitochondrial protein quality control machinery.

cell biology↗

bioRxiv2025

The targeting of non-fibrillar polyQ via distinct VCP-proteasome coupling

The aggregation of proteins containing expanded poly-glutamine (polyQ) repeat sequences is a cytopathological hallmark of several dominantly inherited neurodegenerative diseases (ND), including Huntingtons disease (HD). Previously, we observed that long polyQ repeats form a fibrillar core, surrounded by amorphous and soluble intermediates, which compared to the fibrils, are more readily taken up by autophagosomes 1. In this study, we investigated how the alternative major degradation pathway in cultured cells, ubiquitin proteasome system (UPS), interacts with the different pools of polyQ. We observed that the AAA+ ATPase, valosin-containing protein VCP/p97, in collaboration with proteasomes, plays a crucial role in degrading non-fibrillar polyQ. As both VCP and proteasomes were recruited to the polyQ intermediates peripheral to the fibrils, we imaged these regions by in situ cryo-ET for subtomogram averaging. VCP predominantly adopts an ATP-bound state, often in an active processing conformation with resolved NPLOC4-like cofactor density. The region was also enriched with the 26S proteasome (26S), 20S proteasome core (20S), and 19S regulatory particles (19S). Distance analysis of the macromolecules revealed a striking proximity between VCP and the 20S in situ. This is likely mediated by VCPs C-terminal hydrophobic-tyrosine-X (HbYX) motif, which binds the 20S via hydrophobic interactions, as confirmed by cryo-EM single-particle analysis (SPA) and in vitro assays. Our study highlights a distinct VCP-20S coupling mechanism, where VCP functionally overlaps with the 19S complex in substrate unfolding, facilitating the degradation of polyQ intermediates by the 20S catalytic core.

biochemistry↗

bioRxiv2024

Unveiling epigenetic regulatory elements associated with breast cancer development

Breast cancer is the most common cancer in women and the 2nd most common cancer worldwide, yearly impacting over 2 million females and causing 650 thousand deaths. It has been widely studied, but its epigenetic variation is not entirely unveiled. We aimed to identify epigenetic mechanisms impacting the expression of breast cancer related genes to detect new potential biomarkers and therapeutic targets. We considered The Cancer Genome Atlas database with over 800 samples and several omics datasets such as mRNA, miRNA, DNA methylation, which we used to select 2701 features that were statistically significant to differ between cancer and control samples using the Monte Carlo Feature Selection and Interdependency Discovery algorithm, from an initial total of 417,486. Their biological impact on cancerogenesis was confirmed using: statistical analysis, natural language processing, linear and machine learning models as well as: transcription factors identification, drugs and 3D chromatin structure analyses. Classification of cancer vs control samples on the selected features returned high classification weighted Accuracy from 0.91 to 0.98 depending on feature-type: mRNA, miRNA, DNA methylation, and classification algorithm. In general, cancer samples showed lower expression of differentially expressed genes and increased {beta}-values of differentially methylated sites. We identified mRNAs whose expression is well explained by miRNA expression and differentially methylated sites {beta}-values. We recognized differentially methylated sites possibly affecting NRF1 and MXI1 transcription factors binding, causing a disturbance in NKAPL and PITX1 expression, respectively. Our 3D models showed more loosely packed chromatin in cancer. This study successfully points out numerous possible regulatory dependencies.

cancer biology↗

bioRxiv2024

Competitive assembly resolves the stoichiometry of essential proteins in infectious HIV-1 virions.

During assembly on the plasma membrane, HIV-1 virions incorporate Gag-Pol as well as gp120/gp41 trimers. The Pol region consists of protease, reverse transcriptase and integrase precursors which are essential enzymes required for maturation, reverse transcription, and integration of the viral genome in the next host. gp120/gp41 trimers catalyze the fusion of the virion with its next host. Only a fraction of released virions are infectious. The stoichiometry of gp120/gp41 and Gag-Pol proteins in HIV virions was previously measured using cryotomography and ratiometric protein analysis, but what is the stoichiometry of these proteins in infectious virions remained to be determined. Here we developed a method based on competition between infectious HIV backbones with noninfectious mutants and measured 100 {+/-} 10 Gag-Pol and 15 {+/-} 3 gp120/gp41 proteins incorporated in infectious virions assembled in HEK293 cells from NL4.3 HIV-1 backbone. Our measurements are in broad agreement with cryotomography and ratiometric protein analysis and therefore stoichiometry of gp120/gp41 and Gag-Pol in infectious virions is the same as all released virions. With the development of appropriate mutants and infectivity assays, our method is applicable to other infectious viruses. Statement of significanceThere are 30 million people who have succumbed to the AIDS pandemic with 600,000 additional deaths per year. HIV has an accelerated rate of mutational accumulation with the virus mutating out of neutralizing antibodies within the same patient making development of vaccines challenging. Like most enveloped viruses, only a fraction of released virions are infectious and the question of what selects these virions has remained a mystery. The method developed in this article will allow stoichiometric measurements on infectious virions and therefore allows further studies of causes of infectivity.

biophysics↗

bioRxiv2023

Autophagy preferentially degrades non-fibrillar polyQ aggregates

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntingtons disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

cell biology↗

bioRxiv2022

Defects in the HIV immature lattice support essential lattice remodeling within budded virions

For HIV virions to become infectious, the immature lattice of Gag polyproteins attached to the virion membrane must be cleaved. Cleavage cannot initiate without the protease formed by the homo-dimerization of domains linked to Gag. However, only 5% of the Gag polyproteins, termed Gag-Pol, carry this protease domain, and they are embedded within the structured lattice. The mechanism of Gag-Pol dimerization is unknown. Here, we use reaction-diffusion simulations of the immature Gag lattice as derived from experimental structures, showing that dynamics of the lattice on the membrane is unavoidable due to the missing 1/3 of the spherical protein coat. These dynamics allow for Gag-Pol molecules carrying the protease domains to detach and reattach at new places within the lattice. Surprisingly, dimerization timescales of minutes or less are achievable for realistic binding energies and rates despite retaining most of the large-scale lattice structure. We derive a formula allowing extrapolation of timescales as a function of interaction free energy and binding rate, thus predicting how additional stabilization of the lattice would impact dimerization times. We further show that during assembly, dimerization of Gag-Pol occurs stochastically and therefore must be actively suppressed to prevent early activation. By direct comparison to recent biochemical measurements within budded virions, we find that only moderately stable hexamer contacts (-12kBT<{Delta}G<-8kBT) retain both the dynamics and lattice structures that are consistent with experiment. These dynamics are likely essential for proper maturation, and our models quantify and predict lattice dynamics and protease dimerization timescales that define a key step in understanding formation of infectious viruses. Statement of SignificanceFor retroviruses such as HIV-1, the Gag polyprotein assembles an immature lattice that ensures successful budding from the cell plasma membrane. The first step in the subsequent maturation requires a pair of protease domains embedded within the lattice to form a homodimer. We show here that this homo-dimerization can proceed within minutes despite involving a small subset of Gag monomers, due to the incompleteness of the immature lattice. Using reaction-diffusion simulations, we quantify timescales of first dimerization events between the protease domains and define a formula to extrapolate across a range of energies and rates. Our models illustrate how protein contacts can be weakened to disrupt lattice assembly or stabilized to slow the remodeling essential for viral infectivity.

biophysics↗

bioRxiv2022

Competitive HIV budding suggests that a self-packaging gRNA:Gag-Pol complex directs HIV assembly and enforces infectivity.

To resolve the assembly mechanism of infectious HIV virions, we tested the ability of HIV to assemble infectious virions in the presence of a titrated mix of infectious/ non-infectious proviral genomes. The analysis of our assembly competitions shows that during translation, 15 {+/-} 5 Gag-Pols bind back to their parental gRNA creating a gRNA1: Gag-Pol15 complex. This complex initiates the infectious virion assembly through interactions mediated by cis packaged Gag/Gag-pols and the plasma membrane. Our analysis also shows the number of Gag-Pol and Env proteins packaged in an infectious HIV virion and the minimum functional units of these proteins required for viral infectivity. We suggest that aside from orchestrating the infectious virion assembly the gRNA1: Gag-Pol15 complex plays a major role in HIV evolution and likely hampers effectiveness of antiviral therapies. One-Sentence SummaryCompetitive HIV budding reveals gRNA1: Gag-Pol15 complex orchestrating viral assembly.

microbiology↗

bioRxiv2022

The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds

Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimers disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the AAA+ chaperone VCP is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with Hsp70 and the ubiquitin-proteasome machinery. Inhibition of VCP activity stabilizes large Tau aggregates, and is accompanied by a reduction in the amount of Tau species competent of prion- like aggregate seeding in recipient cells. Thus, disaggregation by VCP generates seeding-active Tau as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.

cell biology↗

bioRxiv2021

Human eosinophil cationic protein manifests alarmin activity through its basicity and ribonuclease activity

Eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN), and human pancreatic ribonuclease (HPR) are members of the RNase A superfamily having similar catalytic residues and diverse functions. Alarmins are the endogenous mediators of innate immunity which activate or alarm the adaptive immune system by activating antigen presenting cells (APCs). EDN acts as an alarmin molecule and plays an important role in innate as well as adaptive immunity. EDN displays chemotactic activity for dendritic cells (DCs) and activates them, has antiviral and antiparasitic activities, and is rapidly released from immune cells. HPR only displays chemotactic activity while no such activity has been reported for ECP. In this study we show that ECP displays the chemotactic activity comparable to that of HPR and EDN. ECP also interacts with TLR-2 to activate NF-{kappa}B/AP-1 expression like EDN. The RNase activity of ECP, EDN and HPR, and basicity of ECP were found to be crucial determinants for their chemotactic activity for APCs, however for the DC maturation activity, RNase activity was not found to be essential. Bovine RNase A did not show any chemotactic activity despite having a very high RNase activity indicating that other determinants in addition to the RNase activity are involved in the chemotactic activity of ECP, EDN and HPR. The current study establishes that ECP also can act like an alarmin.

molecular biology↗

bioRxiv2019

Interferometric Fluorescent Cross Correlation Spectroscopy

We present a method utilizing single photon interference and fluorescence correlation spectroscopy (FCS) to simultaneously measure transport of fluorescent molecules within aqueous samples. Our method, within seconds, measures transport in thousands of homogenous voxels (100 nm)3 and eliminates photo-physical artifacts associated with blinking of fluorescent molecules. A comprehensive theoretical framework is presented and validated by measuring transport of quantum dots, associated with VSV-G receptor along cellular membranes as well as within viscous gels.

biophysics↗

bioRxiv2018

Haplotype-resolved and integrated genome analysis of ENCODE cell line HepG2

The HepG2 cancer cell line is one of the most widely-used biomedical research and one of the main cell lines of ENCODE. Vast numbers of functional genomics and epigenomics datasets have been produced to characterize its biology. However, the correct interpretation such data requires an understanding of the cell lines genome sequence and genome structure. Using a variety of sequencing and analysis methods, we identified a wide spectrum of HepG2 genome characteristics: copy numbers of chromosomal segments, SNVs and Indels (corrected for aneuploidy), phased haplotypes extending to entire chromosome arms, loss of heterozygosity, retrotransposon insertions, structural variants (SVs) including complex and somatic genomic rearrangements. We also identified allele-specific expression and DNA methylation genome-wide and assembled an allele-specific CRISPR/Cas9 targeting map.\n\nSIGNIFICANCEHaplotype-resolved and comprehensive whole-genome analysis of a widely-used cell line for cancer research and ENCODE, HepG2, serves as an essential resource for unlocking complex cancer gene regulation using a genome-integrated framework and also provides genomic context for the analysis of ~1,000 functional datasets to date on ENCODE for biological discovery. We also demonstrate how deeper insights into genomic regulatory complexity are gained by adopting a genome-integrated framework.

genomics↗