Biology subjects

Flynn, J.

Publications and source records attributed to Flynn, J..

12 recordsLinked to original sources

bioRxiv2025

Mutational scan of self-cleavage by HIV-1 protease provides new views of a conformationally dynamic mechanism

The mechanism of conformationally dynamic proteins remains understudied because they are difficult to analyze structurally. For HIV-1 protease the mechanism of cleavage by mature protease is well understood in large part because it forms a stable structure that is amenable to x-ray crystallography. However, self-cleavage or autoproteolysis of protease from the viral polyprotein involves transiently populated structures and is poorly understood. We probed autoproteolysis in HIV-1 using a yeast reporter and mutational scanning. We compared our results with mutational scanning of protease on viral fitness, which integrates both autocleavage and cutting by mature enzyme. We identified 220 mutations that were well tolerated for self-cleavage but not fitness. We analyzed three of these mutations (D30E, W42M and P44L) using independent approaches. All three were capable of efficient self-cleavage in a bacterial assay of autoproteolysis, but had strong defects in mature form for cleavage of a peptide substrate. These separation of function mutations from the mutational scan clustered at hot-spot locations that do not impact autoproteolysis likely because they are conformationally dynamic during self-cleavage. We used the mutational scanning results and molecular simulations to provide models of autoproteolysis conformations. These models provide new views of a structurally dynamic mechanism.

biochemistry↗

bioRxiv2025

Systemic neonatal AAV9 gene therapy delivery improves behavioral and phenotypic outcomes comparable to intracerebroventricular delivery in a mouse model of Niemann-Pick disease, type C1

Niemann-Pick disease, type C (NPC), is an inherited fatal lysosomal storage disorder caused by a mutation in the NPC1 or NPC2 genes and characterized by impaired lysosomal cholesterol export. Previous studies have demonstrated that delivery of the NPC1 gene to the central nervous system (CNS) via an adeno-associated virus (AAV) can substantially improve lifespan and mitigate signs of disease in Npc1-deficient mouse models of NPC. To determine the optimal parameters for an efficacious AAV-based gene therapy for NPC, we measured the survival and disease phenotypes of mice treated systemically as neonates or at weaning age, along with neonatal mice treated via intracerebroventricular (ICV) delivery, with a construct containing either a ubiquitous truncated EF1 promoter or a truncated Mecp2 promoter. While all constructs and delivery methods resulted in improvement compared with baseline, mice treated as neonates survived significantly longer and experienced slower disease progression compared with those treated systemically at weaning age. Systemic delivery to neonates was capable of increasing survival and phenotypic improvement comparable to that of ICV delivery, and neonatal systemic and ICV delivery were both similarly capable of near-total Purkinje cell rescue. We also found no difference between a ubiquitous EF1-derived promoter and an Mecp2-derived promoter. Ultimately, early treatment with maximal access to the CNS, whether via systemic or direct CNS delivery, is key to the efficacy of gene therapy in treating NPC.

neuroscience↗

bioRxiv2025

Defective splicing of Y-chromosome-linked gigantic genes underlies hybrid male sterility in Drosophila

The Y chromosome evolves rapidly, often differing dramatically even between closely related species. While such divergence has long been suspected to contribute to hybrid male sterility, leading to reproductive isolation and thus speciation, the underlying mechanisms remain elusive. Here, we identify a molecular basis linking Y chromosome divergence to reproductive isolation in Drosophila. We show that male hybrids between D. simulans and D. mauritiana fail to properly express key Y-linked fertility genes. These genes contain unusually large introns, exceeding megabases and show substantial sequence divergence between species. In the hybrids, these gigantic introns are misprocessed, resulting in widespread splicing defects, including aberrant "back-splicing" events that join later exons to earlier ones. Our findings suggest that sequence divergence within introns can disrupt essential gene expression through defective splicing, providing a mechanistic link between rapid Y chromosome evolution and hybrid sterility. This work highlights the underappreciated role of intronic divergence in speciation.

evolutionary biology↗

bioRxiv2025

Molecular Mechanisms of Drug Resistance and Compensation in SARS-CoV-2 Main Protease: The Interplay Between E166 and L50

The SARS-CoV-2 main protease (Mpro) is essential for viral replication, and a primary target for COVID-19 antivirals. Direct-acting antivirals such as nirmatrelvir, the active component of Paxlovid, target the Mpro active site to block viral polyprotein cleavage and thus replication. However, drug resistance mutations at the active site residue Glu166 (E166) have emerged in in vitro selection studies, raising concerns about the durability of current antiviral strategies. Here, we investigate the molecular basis of drug resistance conferred by E166A and E166V mutations against nirmatrelvir and the related PF-00835231, individually and in combination with the distal mutation L50F. We found that E166 mutations reduce nirmatrelvir potency by up to 3000-fold while preserving substrate cleavage, with catalytic efficiency reduced by only up to 2- fold. This loss of catalytic efficiency was compensated for by the addition of L50F in the double- mutant variants. We have determined three cocrystal structures of the E166 variants (E166A, E166V, and E166V/L50F) bound to PF-00835231. Comparison of these structures with wild- type demonstrated that E166 is crucial for dimerization and for shaping the substrate-binding S1 pocket. Our findings highlight the mutability of E166, a prime site for resistance for inhibitors that leverage direct interactions with this position, and the potential emergence of highly resistant and active variants in combination with the compensatory mutation L50F. These insights support the design of inhibitors that target conserved protease features and avoid E166 side- chain interactions to minimize susceptibility to resistance.

biochemistry↗

bioRxiv2022

Crowdsourcing Temporal Transcriptomic Coronavirus Host Infection Data: resources, guide, and novel insights.

The emergence of SARS-CoV-2 reawakened the need to rapidly understand the molecular etiologies, pandemic potential, and prospective treatments of infectious agents. The lack of existing data on SARS-CoV-2 hampered early attempts to treat severe forms of COVID-19 during the pandemic. This study coupled existing transcriptomic data from SARS-CoV-1 lung infection animal studies with crowdsourcing statistical approaches to derive temporal meta-signatures of host responses during early viral accumulation and subsequent clearance stages. Unsupervised and supervised machine learning approaches identified top dysregulated genes and potential biomarkers (e.g., CXCL10, BEX2, and ADM). Temporal meta-signatures revealed distinct gene expression programs with biological implications to a series of host responses underlying sustained Cxcl10 expression and Stat signaling. Cell cycle switched from G1/G0 phase genes, early in infection, to a G2/M gene signature during late infection that correlated with the enrichment of DNA Damage Response and Repair genes. The SARS-CoV-1 meta-signatures were shown to closely emulate human SARS-CoV-2 host responses from emerging RNAseq, single cell and proteomics data with early monocyte-macrophage activation followed by lymphocyte proliferation. The circulatory hormone adrenomedullin was observed as maximally elevated in elderly patients that died from COVID-19. Stage-specific correlations to compounds with potential to treat COVID-19 and future coronavirus infections were in part validated by a subset of twenty-four that are in clinical trials to treat COVID-19. This study represents a roadmap to leverage existing data in the public domain to derive novel molecular and biological insights and potential treatments to emerging human pathogens. The data from this study is available in an interactive portal (http://18.222.95.219:8047).

bioinformatics↗

bioRxiv2022

Identification and characterization of the T cell receptor (TCR) repertoire of the Cynomolgus macaque (Macaca Fascicularis)

BackgroundNon-human primates (NHP) are desirable as animal models of human disease because they share behavioral, physiological, and genomic traits with people. Hence, NHP recapitulate manifestations of disease not observed in other animal species. The Macaca fascicularis (i.e., Cynomolgus macaque) is an NHP species extensively used for biomedical research, but the TCR repertoire hasnt been characterized yet. ResultWe used the genomic sequences to design primers to identify the expressed TCR repertoire by single cell RNAseq. The data analysis from 22 unique samples were used to assign a functional status to each TCR genes. We identified and analyzed the TRA/D, TRB and TRG loci of the Cynomolgus macaque. ConclusionThe genomic organization of the Cynomolgus macaque has great similarity with Macaca mulatta (i.e., Rhesus macaque) and they shared >90% sequence similarity with the human TCR repertoire. These data will facilitate the analysis of T cell immunity in Cynomolgus macaques.

immunology↗

bioRxiv2022

Defining the Substrate Envelope of SARS-CoV-2 Main Protease to Predict and Avoid Drug Resistance

Coronaviruses, as exemplified by SARS-CoV-2, can evolve and spread rapidly to cause severe disease morbidity and mortality. Direct acting antivirals (DAAs) are highly effective in decreasing disease burden especially when they target essential viral enzymes, such as proteases and polymerases, as demonstrated in HIV-1 and HCV and most recently SARS-CoV-2. Optimization of these DAAs through iterative structure-based drug design has been shown to be critical. Particularly, the evolutionarily conserved molecular mechanisms underlying viral replication can be leveraged to develop robust antivirals against rapidly evolving viral targets. The main protease (Mpro) of SARS-CoV-2, which is evolutionarily constrained to recognize and cleave 11 specific sites to promote viral maturation, exemplifies one such target. In this study we define the substrate envelope of Mpro by determining the molecular basis of substrate recognition, through nine high-resolution cocrystal structures of SARS-CoV-2 Mpro with the viral cleavage sites. These structures enable identification of evolutionarily vulnerable sites beyond the substrate envelope that may be susceptible to drug resistance and compromise binding of the newly developed Mpro inhibitors.

biochemistry↗

bioRxiv2020

Exploring the coronavirus epidemic using the new WashU Virus Genome Browser

Since its debut in mid-December, 2019, the novel coronavirus (2019-nCoV) has rapidly spread from its origin in Wuhan, China, to several countries across the globe, leading to a global health crisis. As of February 7, 2020, 44 strains of the virus have been sequenced and uploaded to NCBIs GenBank [1], providing insight into the viruss evolutionary history and pathogenesis. Here, we present the WashU Virus Genome Browser, a web-based portal for viewing virus genomic data. The browser is home to 16 complete 2019-nCoV genome sequences, together with hundreds of related viral sequences including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and Ebola virus. In addition, the browser features unique customizability, supporting user-provided upload of novel viral sequences in various formats. Sequences can be viewed in both a track-based representation as well as a phylogenetic tree-based view, allowing the user to easily compare sequence features across multiple strains. The WashU Virus Genome Browser inherited many features and track types from the WashU Epigenome Browser, and additionally incorporated a new type of SNV track to address the specific needs of viral research. Our Virus Browser portal can be accessed at https://virusgateway.wustl.edu, and documentation is available at https://virusgateway.readthedocs.io/.

genomics↗

bioRxiv2019

The adaptive potential of the M-domain of yeast Hsp90

The distribution of fitness effects (DFE) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in others. So far, results regarding the cost of adaptation across environments have been mixed, and most studies have sampled random mutations across different genes. Here, we quantify systematically how costs of adaptation vary along a large stretch of protein sequence by studying the DFEs of the same {approx}2300 amino-acid changing mutations obtained from deep mutational scanning of 119 amino acids in the middle domain of the heat-shock protein Hsp90 in five environments. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-terminal-Middle and Middle-C-terminal interdomains, and regulation of ATPase-chaperone activity. Interestingly, we find that fitness correlates well across diverse stressful environments, with the exception of one environment, diamide. Consistent with this result, we find little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client binding interfaces, or residues that are involved in ATPase chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information.

evolutionary biology↗

bioRxiv2019

Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including zebrafish Danio rerio, we identified two populations of white pigment cells--leucophores--one of which arises by transdifferentiation of adult melanophores and another that develops from a yellow/orange xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical and ultrastructural analyses of zebrafish leucophores revealed cell-type specific chemical compositions, organelle configurations and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching and we show that leucophore complement influences behavior. Together, our studies revealed new, independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish, and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.

developmental biology↗

bioRxiv2018

Highly accelerated rates of heritable large-scale mutations under prolonged exposure to a metal mixture of copper and nickel

Mutation rate variation has been under intense investigation for decades. Despite these efforts, little is known about the extent to which environmental stressors accelerate mutation rates and influence the genetic load of populations. Moreover, most studies have focused on point mutations rather than large-scale deletions and duplications (copy number variations or \"CNVs\"). We estimated mutation rates in Daphnia pulex exposed to low levels of environmental stressors as well as the effect of selection on de novo mutations. We conducted a mutation accumulation (MA) experiment in which selection was minimized, coupled with an experiment in which a population was propagated under competitive conditions in a benign environment. After an average of 103 generations of MA propagation, we sequenced 60 genomes and found significantly accelerated rates of deletions and duplications in MA lines exposed to ecologically relevant concentrations of metals. Whereas control lines had gene deletion and duplication rates comparable to other multicellular eukaryotes (1.8 x 10-6 per gene per generation), a mixture of nickel and copper increased rates fourfold. The realized mutation rate under selection was reduced to 0.4x that of control MA lines, providing evidence that CNVs contribute to mutational load. Our CNV breakpoint analysis revealed that nonhomologous recombination associated with regions of DNA fragility is the primary source of CNVs, plausibly linking metal-induced DNA strand breaks with higher CNV rates. Our findings suggest that environmental stress, in particular multiple stressors, can have profound effects on large-scale mutation rates and mutational load of populations.

genomics↗

bioRxiv2018

Cross-feeding modulates antibiotic tolerance in bacterial communities

Microbes frequently rely on metabolites excreted by other bacterial species, but little is known about how this cross-feeding influences the effect of antibiotics. We hypothesized that when species rely on each other for essential metabolites, the minimum inhibitory concentration (MIC) for all species will drop to that of the \"weakest link\" - the species least resistant in monoculture. We tested this hypothesis in an obligate cross-feeding system that was engineered between Escherichia coli, Salmonella enterica, and Methylobacterium extorquens. The effect of tetracycline and ampicillin were tested on both liquid and solid media. In all cases, resistant species were inhibited at significantly lower antibiotic concentrations in the cross-feeding community than in monoculture or a competitive community. However, deviation from the \"weakest link\" hypothesis was also observed in cross-feeding communities apparently as result of changes in the timing of growth and cross-protection. Comparable results were also observed in a clinically relevant system involving facultative cross-feeding between Pseudomonas aeruginosa and an anaerobic consortium found in the lungs of cystic fibrosis patients. P. aeruginosa was inhibited by lower concentrations of ampicillin when cross-feeding than when grown in isolation. These results suggest that cross-feeding significantly alters tolerance to antibiotics in a variety of systems.

microbiology↗