Biology subjects

Paudel, B.

Publications and source records attributed to Paudel, B..

10 recordsLinked to original sources

bioRxiv2024

Mitophagosomes induced during EV-D68 infection promote viral nonlytic release

Enterovirus-D68 (EV-D68) is a plus-strand RNA virus that primarily causes infant respiratory infections. In rare pediatric cases, infection with EV-D68 has been associated with acute flaccid myelitis, a polio-like paralytic disease. We have previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that EV-D68 induces mitophagy, the specific autophagic degradation of mitochondria. EV-D68 infection induces mitophagosome formation and several hallmarks of mitophagy, including mitochondrial fragmentation, mitochondrial membrane potential loss, and Parkin translocation to the mitochondria were observed in EV-D68 infected cells. The 3C protease of EV-D68 cleaves the mitochondrial fusion protein, mitofusin-2, near the C-terminal HR2 domain to induce mitochondrial fragmentation, and these fragmented mitochondria colocalized with double-stranded RNA (dsRNA), which labels viral RNA replication sites after peak viral RNA replication. Depleting components of mitophagy signaling specifically reduced EV-D68 release without impacting viral intracellular titers. Our results suggest that whereas the machinery of macroautophagy supports various stages of enterovirus replication, including viral genomic RNA replication and capsid maturation, mitophagy is the specific form of autophagy that regulates the nonlytic release of enteroviruses from cells. SignificanceEV-D68 is a respiratory pathogen and a significant cause of acute respiratory illness in infants, against which no prophylactic vaccines or antivirals exist. We have previously shown that EV-D68, like other enteroviruses, triggers and benefits from nonselective autophagy. However, it was unclear whether the virus induces selective autophagy. Here, we demonstrate that EV-D68 infection induces mitophagy and uses mitophagosomes to traffic virions to the surface of cells for nonlytic release. Our findings suggest that mitophagy may constitute the specific form of autophagy that facilitates the autophagosome-mediated nonlytic release of enteroviruses from infected cells and that targeting mitophagy could be an effective strategy to impede EV-D68 nonlytic release.

microbiology↗

bioRxiv2022

Death induced by survival gene elimination (DISE) contributes to neurotoxicity in Alzheimer's disease

Alzheimers disease (AD) is characterized by progressive neurodegeneration, but the specific events that cause cell death remain poorly understood. Death Induced by Survival gene Elimination (DISE) is a cell death mechanism mediated by short (s) RNAs acting through the RNA induced silencing complex (RISC). DISE is thus a form of RNA interference, in which G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich 6mer seed matches in genes essential for cell survival, resulting in the activation of cell death pathways. Here, using Argonaute precipitation and RNAseq (Ago-RP-Seq), we analyze RISC-bound sRNAs to quantify 6mer seed toxicity in several model systems. In mouse AD models and aging brain, in induced pluripotent stem cell-derived neurons from AD patients, and in cells exposed to A{beta}42 oligomers, RISC-bound sRNAs show a shift to more toxic 6mer seeds compared to controls. In contrast, in brains of "SuperAgers", humans over age 80 who have superior memory performance, RISC-bound sRNAs are shifted to more nontoxic 6mer seeds. Cells depleted of nontoxic sRNAs are sensitized to A{beta}42-induced cell death, and reintroducing nontoxic RNAs is protective. Altogether, the correlation between DISE and A{beta}42 toxicity suggests that increasing the levels of nontoxic miRNAs in the brain or blocking the activity of toxic RISC-bound sRNAs could ameliorate neurodegeneration.

neuroscience↗

bioRxiv2022

The role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families

CHD4 is an essential, widely conserved ATP-dependent translocase that is also a broad tumour dependency. In common with other SF2-family chromatin remodelling enzymes, it alters chromatin accessibility by repositioning histone octamers. Besides the helicase and adjacent tandem chromodomains and PHD domains, CHD4 features 1000 residues of N- and C-terminal sequence with unknown structure and function. We demonstrate that these regions regulate CHD4 activity through different mechanisms. An N-terminal intrinsically disordered region (IDR) promotes remodelling integrity in a manner that depends on the composition but not sequence of the IDR. The C-terminal region harbours an auto-inhibitory region that contacts the helicase domain. Auto-inhibition is relieved by a previously unrecognized C-terminal SANT-SLIDE domain split by [~]150 residues of disordered sequence, most likely by binding of this domain to substrate DNA. Our data shed light on CHD4 regulation and reveal strong mechanistic commonality between CHD family members, as well as with ISWI-family remodellers.

biochemistry↗

bioRxiv2022

Real-time single-molecule observation of chaperone-assisted protein folding

The ability of Hsp70 molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes towards efficient protein folding. Here, we used Total Internal Reflection Fluorescence (TIRF) microscopy and single-molecule Fluorescence Resonance Energy Transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase (Fluc) proteins as they are folded by the bacterial Hsp70 system. For the first time, we observed multiple cycles of chaperone binding-and-release to an individual client during refolding and that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.

biochemistry↗

bioRxiv2021

SPOROS: A pipeline to analyze DISE/6mer seed toxicity

micro(mi)RNAs are (18-22nt long) noncoding short (s)RNAs that suppress gene expression by targeting the 3 untranslated region of target mRNAs. This occurs through the seed sequence located in position 2-7/8 of the miRNA guide strand, once it is loaded into the RNA induced silencing complex (RISC). G-rich 6mer seed sequences can kill cells by targeting C-rich 6mer seed matches located in genes that are critical for cell survival. This results in induction of Death Induced by Survival gene Elimination (DISE), also referred to as 6mer seed toxicity. miRNAs are often quantified in cells by aligning the reads from small (sm)RNA sequencing to the genome. However, the analysis of any smRNA Seq data set for 6mer seed toxicity requires an advanced workflow, solely based on the exact position 2-7 of any sRNA that can enter the RISC. Therefore, we developed SPOROS, an automated pipeline that produces multiple useful outputs to compare 6mer seed toxicity of all cellular sRNAs, regardless of their nature, between different samples. We provide two examples to illustrate the capabilities of SPOROS: Example one involves the analysis of RISC-bound sRNAs in a cancer cell line (either wild-type or two mutant lines unable to produce most miRNAs). Example two is based on a publicly available smRNA Seq data set from postmortem brains (either from normal or Alzheimers patients). Our methods are designed to be used to analyze a variety of smRNA Seq data in various normal and disease settings.

bioinformatics↗

bioRxiv2020

Identification of the toxic 6mer seed consensus in human cancer cells

6mer seed toxicity is a novel anti-cancer mechanism that kills cancer cells by triggering death induced by survival gene elimination (DISE). It is based on si- or shRNAs with a specific G-rich nucleotide composition in position 2-7 of their guide strand. An arrayed screen of 4096 6mer seeds on two human and two mouse cell lines identified a consensus GGGGGC as the most toxic seed. After testing two more cell lines, one human and one mouse, we found that the GGGGGC seed while also toxic to murine cells, is more toxic to human cells, suggesting that the evolution to use of Gs as part of the toxic seeds is still slowly evolving, with Gs more common in the human toxic seeds. While new RNA Seq and bioinformatics analyses suggest that the GGGGGC seed is toxic to cancer cells by targeting GCCCCC seed matches in the 3 UTR of a set of genes critical for cell survival, we now directly confirm this by identifying a number of genes targeted by this seed. Furthermore, by using a luciferase reporter fused to the 3 UTR of these genes we confirm direct and specific on-targeting of GCCCCC seed matches. Targeting is strongly attenuated after mutating the GCCCCC seed matches in these 3 UTRs. Our data confirm that an siRNA containing the GGGGGC seed kills cancer cells through its miRNA like activity and points at artificial miRNAs, si- or shRNAs containing this seed as a potential new cancer therapeutics.

cancer biology↗

bioRxiv2020

Single-molecule fluorescence-based approach reveals novel mechanistic insights into small heat shock protein chaperone function

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones that are up-regulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation, however fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. Traditionally, it has been difficult to study sHsp function due to the dynamic and heterogenous nature of the species formed between sHsps and aggregation-prone proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic protein complexes and we have therefore developed a novel single-molecule fluorescence-based approach to observe the chaperone action of human B-crystallin (Bc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between Bc and a model client protein, chloride intracellular channel 1 (CLIC1). By examining the polydispersity and stoichiometries of these complexes over time, and in response to different concentrations of Bc, we have uncovered unique and important insights into a two-step mechanism by which Bc interacts with misfolded client proteins to prevent their aggregation. Understanding this fundamental mechanism of sHsp action is crucial to understanding how these molecular chaperone function to protect the cell from protein misfolding and their overall role in the cellular proteostasis network.

biochemistry↗

bioRxiv2019

Disruption of Redox Balance Enhances the Effects of BRAF-inhibition in Melanoma Cells

SummaryMelanomas harboring BRAF mutations can be treated with BRAF inhibitors (BRAFi), but responses are varied and tumor recurrence is inevitable. Here, using an integrative approach of experimentation and mathematical flux balance analyses in BRAF-mutated melanoma cells, we report that elevated antioxidant capacity is linked to BRAFi sensitivity in melanoma cells. High levels of antioxidant metabolites in cells with reduced BRAFi sensitivity confirm this conclusion. By extending our analyses to other melanoma subtypes in TCGA, we predict that elevated redox capacity is a general feature of melanomas, not previously observed. We propose that redox vulnerabilities could be exploited for therapeutic benefits and identify unsuspected combination targets to enhance the effects of BRAFi in any melanoma, regardless of mutational status.

systems biology↗

bioRxiv2019

Drug-tolerant idling melanoma cells exhibit theory-predicted metabolic low-low phenotype

Cancer cells adjust their metabolic profiles to evade treatment. Metabolic adaptation is complex and hence better understood by an integrated theoretical-experimental approach. Using a minimal kinetic model, we predicted a previously undescribed Low/Low (L/L) phenotype, characterized by low oxidative phosphorylation (OXPHOS) and low glycolysis. Here, we report that L/L metabolism is observed in BRAF-mutated melanoma cells that enter a drug-tolerant "idling state" upon long-term MAPK inhibition (MAPKi). Consistently, using publicly available RNA-sequencing data of both cell lines and patient samples, we show that melanoma cells decrease their glycolysis and/or OXPHOS activity upon MAPKi and converge toward the L/L phenotype. L/L metabolism is unfavorable for tumor growth, yet supports successful cell division at ~50% rate. Thus, L/L drug-tolerant idling cells are a reservoir for accumulating mutations responsible for relapse, and it should be considered as a target subpopulation for improving MAPKi outcomes in melanoma treatment.

systems biology↗

bioRxiv2018

Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response

The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the {lambda} repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.

biophysics↗