Biology subjects

Gupta, S.

Publications and source records attributed to Gupta, S..

At least 325 records · Page 18Linked to original sources

bioRxiv2020

Control of Systemic Iron Homeostasis by the 3' Iron-Responsive Element of Divalent Metal Transporter 1 in Mice.

Divalent metal transporter 1 (DMT1) is essential for dietary iron assimilation and erythroid iron acquisition. The 3 untranslated region of the murine DMT1 mRNA contains an iron responsive element (IRE) that is conserved in humans but whose functional role remains unclear. We generated and analyzed mice with targeted disruption of the DMT1 3IRE. These animals display hypoferremia during the suckling period, associated with a reduction of DMT1 mRNA and protein in the intestine. In contrast, adult mice exhibit hyperferremia, accompanied by enlargement of hepatic and splenic iron stores. Intriguingly, disruption of the DMT1 3IRE in adult animals augments intestinal DMT1 expression, in part due to increased mRNA translation. Hence, during postnatal growth, the DMT1 3IRE promotes intestinal DMT1 expression and secures iron sufficiency; in adulthood, it suppresses DMT1 and prevents systemic iron loading. This work demonstrates that the 3IRE of DMT1 plays a role in the control of DMT1 expression and systemic iron homeostasis, and reveals an age-dependent switch in its activity. Key pointsO_LITargeted mutagenesis of the 3IRE of DMT1 in mice reveals its importance for maintenance of systemic iron homeostasis. C_LIO_LIThe 3IRE stimulates intestinal DMT1 expression and prevents hypoferremia during early life, but exerts opposite effects in adulthood C_LI

molecular biology↗

bioRxiv2020

Investigating the dynamics of microbial consortia in spatially structured environments

The spatial organization of microbial communities arises from a complex interplay of biotic and abiotic interactions and is a major determinant of ecosystem functions. We design a microfluidic platform to investigate how the spatial arrangement of microbes impacts gene expression and growth. We elucidate key biochemical parameters that dictate the mapping between spatial positioning and gene expression patterns. We show that distance can establish a low-pass filter to periodic inputs, and can enhance the fidelity of information processing. Positive and negative feedback can play disparate roles in the synchronization and robustness of a genetic oscillator distributed between two strains to spatial separation. Quantification of growth and metabolite release in an amino-acid auxotroph community demonstrates that the interaction network and stability of the community are highly sensitive to temporal perturbations and spatial arrangements. In sum, our microfluidic platform can quantify spatiotemporal parameters influencing diffusion-mediated interactions in microbial consortia.

systems biology↗

bioRxiv2020

The Atlas of Inflammation-Resolution (AIR)

Acute inflammation is a protective reaction by the immune system in response to invading pathogens or tissue damage. Ideally, the response should be localized, self-limited, and returning to homeostasis. If not resolved, acute inflammation can result in organ pathologies leading to chronic inflammatory phenotypes. Acute inflammation and inflammation resolution are complex coordinated processes, involving a number of cell types, interacting in space and time. The biomolecular complexity and the fact that several biomedical fields are involved, make a multi and interdisciplinary approach necessary. This Atlas of Inflammation Resolution (AIR) is a web-based resource capturing the state-of-the-art in acute inflammation and inflammation resolution research. The AIR provides an interface for users to search thousands of interactions, arranged in inter-connected multi-layers of process diagrams, covering a wide range of clinically relevant phenotypes. The AIR serves as an open access knowledgebase, including a gateway to numerous public databases. It is furthermore possible for the user to map experimental data onto the molecular interaction maps of the AIR, providing the basis for bioinformatics analyses and systems biology approaches. By mapping experimental data onto the Atlas, it can be used to elucidate drug action as well as molecular mechanisms underlying different disease phenotypes. For the visualization and exploration of information, the AIR uses the Minerva platform, which is a well-established tool for the presentation of disease maps. The molecular details of the AIR are encoded using international standards. The Atlas of Inflammation Resolution was created as a freely accessible resource, supporting research and education in the fields of acute inflammation and inflammation resolution. The AIR connects research communities, facilitates clinical decision making, and supports research scientists in the formulation and validation of hypotheses.

immunology↗

bioRxiv2020

Automated Workflow For Peptide-level Quantitation From DIA/ SWATH-MS Data

Data Independent Acquisition (DIA) is a powerful method to acquire spectra from all ionized precursors of a sample. Considering the complexity of the highly multiplexed spectral data, sophisticated workflows have been developed to obtain peptides quantification. Here we describe an open-source and easy-to-use workflow to obtain a quantitative matrix from multiple DIA runs. This workflow requires as prior information an "assay library", which contains the MS coordinates of peptides. It consists of OpenSWATH, pyProphet and DIAlignR software. For the ease of installation and to isolate operating system related dependency, docker-based containerization is utilized in this workflow.

bioinformatics↗

bioRxiv2020

DrawAlignR: An interactive tool for across run chromatogram alignment visualization

Multi-run alignment is widely used in proteomics to establish analyte correspondence across runs. Generally alignment algorithms return a cumulative score, which may not be easily interpretable for each peptide. Here we present a novel tool, DrawAlignR, to visualize each chromatographic alignment for DIA/SWATH data. Furthermore, we have developed a novel C++ based implementation of raw chromatogram alignment which is 35 times faster than the previously published algorithm. This not only enables users to plot alignment interactively by DrawAlignR, but also allows other software platforms to use the algorithm. DrawAlignR is an open-source web application using R Shiny that can be hosted using the source-code available at https://github.com/Roestlab/DrawAlignR.

bioinformatics↗

bioRxiv2020

Dynamin-like proteins are essential for vesicle biogenesis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) secretes pathogenicity factors and immunologically active molecules via membrane vesicles. However, nothing is known about the mechanisms involved in mycobacterial vesicle biogenesis. This study investigates molecular determinants of membrane vesicle production in Mtb by analyzing Mtb cells under conditions of high vesicle production: iron limitation and VirR restriction. Ultrastructural analysis showed extensive cell envelope restructuring in association with vesicle release that correlated with downregulation of cell surface lipid biosynthesis and peptidoglycan alterations. Comparative transcriptomics showed common upregulation of the iniBAC operon in association with high vesicle production in Mtb cells. Vesicle production analysis demonstrated that the dynamin-like proteins (DLPs) encoded by this operon, IniA and IniC, are necessary for release of EV by Mtb in culture and in infected macrophages. Isoniazid, a first-line antibiotic, used in tuberculosis treatment, was found to stimulate vesicle release in a DLP-dependent manner. Our results provide a new understanding of the function of mycobacterial DLPs and mechanistic insights into vesicle biogenesis. The findings will enable further understanding of the relevance of Mtb-derived extracellular vesicles in the pathogenesis of tuberculosis and may open new avenues for therapeutic research. IMPORTANCEIron is an essential nutrient that promotes survival and growth of M. tuberculosis, the bacterium that causes human tuberculosis (TB). Limited availability of iron, often encountered in the host environment, stimulates M. tuberculosis to secrete membrane-bound extracellular vesicles containing molecules that may help it evade the immune system. Characterizing the bacterial factors and mechanisms involved in the production of mycobacterial vesicles is important for envisioning ways to interfere with this process. Here, we report the discovery of proteins required by M. tuberculosis for vesicle biogenesis in culture and during host cell infection. We also demonstrate a connection between antibiotic response and extracellular vesicle production. The work provides insights into the mechanisms underlying vesicle biogenesis in M. tuberculosis and permits better understanding of the significance of vesicle production to M. tuberculosis-host interactions and antibiotic stress response.

microbiology↗

bioRxiv2020

Development of novel anti-malarial from structurally diverse library of molecules, targeting plant-like Calcium Dependent Protein Kinase 1, a multistage growth regulator of P. falciparum

Upon Plasmodium falciparum merozoites exposure to low [K+] environment in blood plasma, there is escalation of cytosolic [Ca2+] which activates Ca2+-Dependent Protein Kinase 1 (CDPK1), a signaling hub of intra-erythrocytic proliferative stages of parasite. Given its high abundance and multidimensional attributes in parasite life-cycle, this is a lucrative target for desiging antimalarials. Towards this, we have virtually screened MyriaScreenII diversity collection of 10,000 drug-like molecules, which resulted in 18 compounds complementing ATP-binding pocket of CDPK1. In vitro screening for toxicity in mammalian cells revealed that these compounds are non-toxic in nature. Further, SPR analysis demonstrated differential binding affinity of these compounds towards recombinantly purified CDPK1 protein. Selection of lead compound 1 was performed by evaluating their inhibitory effects on phosphorylation and ATP binding activities of CDPK1. Further, in vitro biophysical evaluations by ITC and FS revealed that binding of compound 1 is driven by formation of energetically favorable non-covalent interactions, with different binding constants in presence and absence of Ca2+, and TSA authenticated stability of compound 1 bound CDPK1 complex. Finally, compound 1 strongly inhibited intra-erythrocytic growth of P. falciparum in vitro. Concievably, we propose a novel CDPK1-selective inhibitor, step towards developing pan-CDPK kinase inhibitors, prerequisite for cross-stage anti-malarial protection.

biochemistry↗

bioRxiv2020

Interaction of 14-3-3I and CDPK1 mediates the growth of human malaria parasite

Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs of target proteins and confer conformational changes leading to modulation of their functional parameters. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have characterized Plasmodium falciparum 14-3-3 and its interaction with key kinase of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Towards this, we annotated PF3D7_0818200 as 14-3-3 isoform I through extensive phylogenetic and comparative sequence analysis. Molecular dynamics simulation studies indicated that phosphoSer64 present in CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The protein-protein interaction was validated in vitro with ELISA and SPR, which confirmed that CDPK1 interacts with 14-3-3I in a phosphorylation dependent manner, with binding affinity constant of 670 {+/-} 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: ARSHpSYPA and RLYHpSLPA as CDPK1 mimetics, by simulation studies and ITC. Further, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.

cell biology↗

bioRxiv2019

The effects of a 6-week core exercises on swimming performance of national level swimmers

The aim of this study was to assess the impact of a 6-week specialized training program aimed at strengthening core muscles to improve the effectiveness of selected elements of a swimming race in group of elite polish swimmers. Sixteen male national and international level swimmers (21.6 {+/-} 2.2 years) participated in the research. The competitors were randomly assigned to 1 of 2 groups before the data collection process: an experimental (EG, n = 8) and control (CG, n = 8) group. Both groups of swimmers carried out the same training program in the water environment (volume and intensity), while swimmers from EG additionally carried out the specific core muscle training. The task of the swimmers was an individual swim of 50 meters freestyle, during which the kinematic parameters of start, turn and swimming techniques were recorded using the video camera system. In both groups a minor increase in the flight phase was observed during the start (EG=0.06 m, 1.8%; p=0.088; CG = 0.08 m, 2.7%; p=0.013). The time of the distance 5 m after the turn and the recorded average speed of swimming this distance in EG statistically significant improved accordingly 0.1 s (-28.6%; p<0.001) and 3.56 m{middle dot}s-1 (23.2%; p=0.001). In EG, a statistically significant improvement in 50-m freestyle performance was observed by 0.3 s (-1.2%, p = 0.001). The results of the research show that the implementation of isolated strength of the stabilizing muscles seems to be a valuable addition to the standard training of swimmers.

scientific communication and education↗

bioRxiv2019

LncRNA and predictive model to improve the diagnosis of clinically diagnosed pulmonary tuberculosis

BackgroundClinically diagnosed pulmonary tuberculosis (PTB) patients lack Mycobacterium tuberculosis (MTB) microbiologic evidence, and misdiagnosis or delayed diagnosis often occurs as a consequence. We investigated the potential of lncRNAs and corresponding predictive models to diagnose these patients. MethodsWe enrolled 1372 subjects, including clinically diagnosed PTB patients, non-TB disease controls and healthy controls, in three cohorts (Screening, Selection and Validation). Candidate lncRNAs differentially expressed in blood samples of the PTB and healthy control groups were identified by microarray and qRT-PCR in the Screening Cohort. Logistic regression models were developed using lncRNAs and/or electronic health records (EHRs) from clinically diagnosed PTB patients and non-TB disease controls in the Selection Cohort. These models were evaluated by AUC and decision curve analysis, and the optimal model was presented as a Web-based nomogram, which was evaluated in the Validation Cohort. The biological function of lncRNAs was interrogated using ELISA, lactate dehydrogenase release analysis and flow cytometry. ResultsThree differentially expressed lncRNAs (ENST00000497872, n333737, n335265) were identified. The optimal model (i.e., nomogram) incorporated these three lncRNAs and six EHR variables (age, hemoglobin, weight loss, low-grade fever, CT calcification and TB-IGRA). The nomogram showed an AUC of 0.89, sensitivity of 0.86 and specificity of 0.82 in the Validation Cohort, which demonstrated better discrimination and clinical net benefit than the EHR model. ENST00000497872 may regulate inflammatory cytokine production, cell death and apoptosis during MTB infection. ConclusionsLncRNAs and the user-friendly nomogram could facilitate the early identification of PTB cases among suspected patients with negative MTB microbiologic evidence.

molecular biology↗

bioRxiv2019

A random walk down personalized single-cell networks: predicting the response of any gene to any drug for any patient

BackgroundThe last decade has seen a major increase in the availability of genomic data. This includes expert-curated databases that describe the biological activity of genes, as well as high-throughput assays that measure gene expression in bulk tissue and single cells. Integrating these heterogeneous data sources can generate new hypotheses about biological systems. Our primary objective is to combine population-level drug-response data with patient-level single-cell expression data to predict how any gene will respond to any drug for any patient. MethodsWe take 2 approaches to benchmarking a "dual-channel" random walk with restart (RWR) for data integration. First, we evaluate how well RWR can predict known gene functions from single-cell gene co-expression networks. Second, we evaluate how well RWR can predict known drug responses from individual cell networks. We then present two exploratory applications. In the first application, we combine the Gene Ontology database with glioblastoma single cells from 5 individual patients to identify genes whose functions differ between cancers. In the second application, we combine the LINCS drug-response database with the same glioblastoma data to identify genes that may exhibit patient-specific drug responses. ConclusionsOur manuscript introduces two innovations to the integration of heterogeneous biological data. First, we use a "dual-channel" method to predict up-regulation and down-regulation separately. Second, we use individualized single-cell gene co-expression networks to make personalized predictions. These innovations let us predict gene function and drug response for individual patients. Taken together, our work shows promise that single-cell co-expression data could be combined in heterogeneous information networks to facilitate precision medicine.

systems biology↗

bioRxiv2019

A Simplest Bioinformatics Pipeline for Whole Transcriptome Sequencing: Overview of the Processing and Steps from Raw Data to Downstream Analysis.

Recent advances in next generation sequencing (NGS) technologies have heralded the genomic research. From the good-old inferring differentially expressed genes (DEG) using microarray to the current adage NGS-based whole transcriptome or RNA-Seq pipelines, there have been advances and improvements. With several bioinformatics pipelines for analysing RNA-Seq on rise, inferring the candidate DEGs prove to be a cumbersome approach as one may have to reach consensus among all the pipelines. To Check this, we have benchmarked the well known cufflinks-cuffdiff pipeline on a set of datasets and outline it in the form of a protocol where researchers interested in performing whole transcriptome shotgun sequencing and its downstream analysis can better disseminate the analysis using their datasets.

bioinformatics↗

bioRxiv2019

Paradoxical role of AT-rich interactive domain 1A in restraining pancreatic carcinogenesis

Background & AimsARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model, PDAC cell lines. MethodsPancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre;KrasG12D;Arid1af/f or "KAC") was generated by crossing Ptf1a-Cre;KrasG12D ("KC") mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cells lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. ResultsArid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in "KAC" as compared to "KC" mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of "KAC" mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous "KAC" mice revealed epigenetic changes underlying the various compensatory mechanisms to overcome the growth suppressive effects of Arid1a loss. ConclusionsArid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models.

cancer biology↗

bioRxiv2019

Replacement of microglia by monocyte-derived macrophages prevents long-term memory deficits after therapeutic irradiation

Brain resident microglia have a distinct origin compared to macrophages in other organs. Under physiological conditions, microglia are maintained by self-renewal from the local pool, independent of hematopoietic progenitors. Pharmacological depletion of microglia during therapeutic whole-brain irradiation prevents synaptic loss and long-term recognition memory deficits but the mechanisms behind these protective effects are unknown. Here we demonstrate that after a combination of therapeutic whole-brain irradiation and microglia depletion, macrophages originating from circulating monocytes engraft into the brain and replace the microglia pool. Comparisons of transcriptomes reveal that brain-engrafted macrophages have an intermediate phenotype that resembles both monocytes and embryonic microglia. Brain-engrafted macrophages display reduced phagocytic activity for synaptic compartments compared to microglia from normal brains in response to a secondary concussive brain injury. In addition to sparing mice from brain radiotherapy-induced long-term cognitive deficits, replacement of microglia by brain-engrafted macrophages can prevent concussive injury-induced memory loss. These results demonstrate the long-term functional role of brain-engrafted macrophages as a possible therapeutic tool against radiation-induced cognitive deficits.

neuroscience↗

bioRxiv2019

PDGFRA Defines the Mesenchymal Stem Cell Kaposi’s Sarcoma Progenitors by Enabling KSHV Oncogenesis in an Angiogenic Environment

Kaposis sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (P(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected P(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected P(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.\n\nAUTHOR SUMMARYIdentification of the KS progenitor cell creates the possibility of studying viral oncogenesis and its determinants from its initial steps as a continuum. It also increases our understanding of pathogenic mechanisms and disease preferential tropism. Hereby we identify P(+)S-MSCs as KS progenitors, in which KSHV infection has oncogenic consequences; only when these cells are in a pro-angiogenic environment in which PDGFRA activation enables an oncogenic de-repressed KSHV epigenome. These results identify a KS-progenitor population in the P(+)S-MSCs and point to pro-angiogenic environmental conditions as essential for oncogenic viral gene expression and transformation. We designed a novel model of KSHV oncogenesis, creating a very robust platform to identify KSHV oncogenic pathways and their relationship with cellular lineages and extracellular growth environments.

cancer biology↗

bioRxiv2019

LRR-extensins of vegetative tissues are a functionally conserved family of RALF1 receptors interacting with the receptor kinase FERONIA

Plant cell growth requires the coordinated expansion of the protoplast and the cell wall that confers mechanical stability to the cell. An elaborate system of cell wall integrity sensors monitors cell wall structures and conveys information on cell wall composition and growth factors to the cell. LRR-extensins (LRXs) are cell wall-attached extracellular regulators of cell wall formation and high-affinity binding sites for RALF (rapid alkalinization factor) peptide hormones that trigger diverse physiological processes related to cell growth. RALF peptides are also perceived by receptors at the plasma membrane and LRX4 of Arabidopsis thaliana has been shown to also interact with one of these receptors, FERONIA (FER). Here, we demonstrate that several LRXs, including the main LRX protein of root hairs, LRX1, interact with FER and RALF1 to coordinate growth processes. Membrane association of LRXs correlate with binding to FER, indicating that LRXs represent a physical link between intra- and extracellular compartments via interaction with membrane-localized proteins. Finally, despite evolutionary diversification of the LRR domains of various LRX proteins, many of them are functionally still overlapping, indicative of LRX proteins being central players in regulatory processes that are conserved in very different cell types.\n\nAuthor SummaryCell growth in plants requires the coordinated enlargement of the cell and the surrounding cell wall, which is ascertained by an elaborate system of cell wall integrity sensors, proteins involved in the exchange of information between the cell and the cell wall. In Arabidopsis thaliana, LRR-extensins (LRXs) are localized in the cell wall and are binding RALF peptides, hormones that regulate cell growth-related processes. LRX4 also binds the plasma membrane-localized receptor kinase FERONIA (FER), establishing a link between the cell and the cell wall. It is not clear, however, whether the different LRXs of Arabidopsis have similar functions and how they interact with their binding partners. Here, we demonstrate that interaction with FER and RALFs requires the LRR domain of LRXs and several but not all LRXs can bind these proteins. This explains the observation that mutations in several of the LRXs induce phenotypes comparable to a fer mutant, establishing that LRX-FER interaction is important for proper cell growth. Some LRXs, however, appear to influence cell growth processes in different ways, which remain to be identified.

plant biology↗

bioRxiv2019

Allosteric priming of E. coli CheY by the flagellar motor protein FliM

Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY.FliMN with and without D13K-Y106W have shown FliMN bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D13K at the D57 phosphorylation site and Y/W106 at the FliMN interface; namely the closure of the 4-{beta}4 hinge and inward rotation of Y/W106 with W58. We used hydroxy-radical foot-printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other sidechains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay sidechains reported by XFMS confirmed the intermediate conformation of the native CheY.FliMN complex, the inactive state of free D13K-Y106W CheY and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch with Y/W106 sidechain burial correlated with increased FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated community sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN increased sector size and interactions. These sectors fused between the substituted K13K-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far. Statement of SignificanceCheY affinity for FliMN, its binding target at the flagellar motor, is increased by phosphorylation to switch rotation sense. Atomistic simulations based on CheY and CheY.FliMN crystal structures with and without the phospho-mimetic double substitution (D13K-Y106W) showed CheY compaction is entrained to increased FliMN affinity. Burial of exposed aromatic sidechains drove compaction, as validated by tracking sidechain solvent accessibility with hydroxyl-radical foot-printing. The substitutions were localized at the phosphorylation pocket (D13K) and FliMN interface (Y106W). Mutual information measures revealed these locations were allosterically coupled by a specialized conduit when the conformational landscape of FliMN-tethered CheY was modified by the substitutions. Novel surfaces stabilized by the conduit may bind additional motor sites, essential for the high cooperativity of the flagellar switch.

biophysics↗

bioRxiv2019

The Dynamics of Cytoplasmic mRNA Metabolism

For all but a few mRNAs, the dynamics of metabolism are unknown. Here, we developed an experimental and analytical framework for examining these dynamics for mRNAs from thousands of genes. mRNAs of mouse fibroblasts exit the nucleus with diverse intragenic and intergenic poly(A)-tail lengths. Once in the cytoplasm, they have a broad (1000-fold) range of deadenylation rate constants, which correspond to cytoplasmic lifetimes. Indeed, with few exceptions, degradation appears to occur primarily through deadenylation-linked mechanisms, with little contribution from either endonucleolytic cleavage or deadenylation-independent decapping. Most mRNA molecules degrade only after their tail lengths fall below 25 nt. Decay rate constants of short-tailed mRNAs vary broadly (1000-fold) and are more rapid for short-tailed mRNAs that had previously undergone more rapid deadenylation. This coupling helps clear rapidly deadenylated mRNAs, enabling the large range in deadenylation rate constants to impart a similarly large range in stabilities.\n\nHighlightsO_LImRNAs enter the cytoplasm with diverse intra- and intergenic lengths\nC_LIO_LImRNA deadenylation rates span a 1000-fold range and correspond to mRNA half-lives\nC_LIO_LIAfter their tails become short, mRNAs decay at rates that span a 1000-fold range\nC_LIO_LIMore rapidly deadenylated mRNAs decay more rapidly upon reaching short tail lengths\nC_LI

genomics↗