Biology subjects

Meng, B.

Publications and source records attributed to Meng, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural basis of underwound DNA topology bearing PAM-mutant recognition by AtCas9

The CRISPR-Cas9 system locates genomic targets through gRNA pairing and recognition a protospacer-adjacent motif (PAM). While PAM specificity is typically sequence-determined, we previously found that DNA topology can regulate PAM specificity, relaxing PAMs of various Cas9 effectors and allowing near-PAMless cleavage activity of a type II-C Alicyclobacillus tengchongensis Cas9 (AtCas9). However, the structural mechanism underlying this regulation remains unknown. Here, we report cryo-EM structures of AtCas9 bound to B-form DNA or a 340-bp underwound minicircle DNA bearing WT or mutant PAMs. Despite differences in PAM sequences, all three underwound complexes adopt an almost identical architecture that is distinct from the B-form DNA-bound state. On B-form DNA, AtCas9 recognizes the PAM via base-specific hydrogen bonds and steric exclusion, conferring preference for N4CNNN and N4RNNA (R = A/G). In contrast, underwound minicircle DNA widens the PAM major groove, reduces steric clashes, and enables a compensatory mode involving sequence-independent backbone contacts, explaining near-PAMless cleavage under underwound conformation. These findings uncover a topology-dependent mechanism of PAM recognition and establish a cryo-EM platform using underwound minicircle DNA for structural studies under native-like topological states.

biochemistry↗

bioRxiv2025

Methane Cycling Microbes are Important Predictors of Methylmercury Accumulation in Rice Paddies

Microbial production of methylmercury from inorganic mercury in rice paddies poses health risks to consumers of this essential dietary staple. Although mercury-methylating communities are well characterized, the microbial guilds contributing to methylmercury accumulation in rice paddies remain unclear. Here, we collected paddy soils across a mercury concentration gradient throughout the rice growing season to identify microbial and environmental factors influencing methylmercury dynamics. We show that hgcA gene abundance, the key gene required for methylation, was not a significant predictor of methylmercury concentration in paddy soils. We also show that merB gene abundance correlated with methylmercury in mercury-polluted rhizosphere samples. Methane cycling genes were actively expressed, and their beta-diversity was significantly associated with methylmercury levels. Methanogen abundance correlated with higher methylmercury under elevated total mercury concentrations. Analysis of the methanotroph-associated mbnT gene, implicated in demethylation, revealed an unexpected positive correlation with methylmercury. Multiple regression and machine learning models converged on mercury bioavailability and methanogen/methanotroph abundances as key predictors of methylmercury, with methanogen-associated hgcA gene abundance and methanogen-methanotroph interactions highlighted under flooded, low-redox conditions. These findings suggest that methane-cycling microbes play key roles in methylmercury cycling dynamics and point to management strategies that could simultaneously mitigate mercury pollution and greenhouse gas emissions. ImportanceMethylmercury is a microbially-derived neurotoxin that accumulates in rice, which is a global food staple. Predicting mercurys fate in rice paddies is challenging because of the interplay between microbes responsible for methylmercury cycling and variables that control mercury availability. Our study coupled genomic and geochemical measurements with machine learning to identify the key predictors of methylmercury accumulation in paddy soils. We demonstrate that methanogen and methanotroph abundance, and mercury bioavailability, are major predictors of methylmercury variability in paddies. We show that considering interactions between methane cycling guilds improves our capacity to predict methylmercury accumulation in soils compared to approaches that rely solely on mercury cycling genes. This work can inform remediation strategies for mercury in rice paddies but also wetlands and permafrost where methane and mercury cycling are tightly coupled. Such strategies could provide a solution to simultaneously mitigate methylmercury exposure and reduce greenhouse gas emissions amid global environmental change.

microbiology↗

bioRxiv2025

The 'Iceberg region of the Grapevine leafroll-associated virus 3 replicase polyprotein contains signals for mitochondrial targeting and outer membrane association

Positive-sense single-stranded (+ss) RNA viruses such as Grapevine leafroll-associated virus 3 (GLRaV3) replicate their genomes within membrane-bound viral replication complexes (VRCs). The biogenesis of such VRCs is driven by viral "replicase" polyproteins containing several replication-related domains. Past electron microscopy evidence suggests that GLRaV3 forms VRCs from the outer mitochondrial membrane of host grapevine plants. Here, we report the subcellular localization of the replicase polyprotein encoded by GLRaV3 ORF1a (PP1a) towards understanding its putative role in VRC formation. Through confocal laser scanning microscopy analysis of distinct EGFP-tagged PP1a domains, interdomain regions and truncations expressed in model plants, we report the molecular signals responsible for the targeting and association of PP1a to the mitochondria. This signal, located in the "Iceberg" region downstream from the methyltransferase-guanylyltransferase (M/GTase) domain, comprises an amphipathic -helix and a downstream transmembrane domain (TMD). Mutagenesis studies suggest that the polar face of the amphipathic -helix functions as the targeting signal, whereas the non-polar face, together with the TMD, act in membrane-anchoring. Microscopy observations are confirmed through mitochondrial isolation via gradient centrifugation and Western blotting. Structure prediction of the GLRaV3 M/GTase domain and its downstream TMD suggests a putative dodecameric oligomeric state. This dodecamer may gate the neck of GLRaV3 VRCs and contribute to their biogenesis, a hypothesis to be tested in follow-up studies. SIGNIFICANCE STATEMENTPositive-sense RNA viruses such as GLRaV3 form specialized membrane-bound viral replication complexes (VRCs) within host cells to sequester viral genome replication. Past evidence suggests that GLRaV3 targets the outer mitochondrial membrane (OMM) of grapevine host cells for VRC formation, however the viral protein responsible remained unknown. Here, we report a newly identified amphipathic -helix and a downstream transmembrane domain in the replicase polyprotein encoded by GLRaV3 ORF1a that are crucial for OMM targeting and membrane association. Since OMM targeting mechanisms in plants are poorly understood, our findings not only shed light on how GLRaV3 assembles VRCs, but also provides insight into OMM targeting more broadly. This work lays the foundation towards elucidating the molecular mechanisms of GLRaV3 replication and host-pathogen interactions.

plant biology↗

bioRxiv2024

Metabolic Divergence Between Healthy and Conditional Presenilin-1/2 knockout Dementia Model Mice under Chronic Nicotine exposure

Nicotine, a primary bioactive compound in tobacco, influences a wide range of metabolic pathways, with potential implications for neurodegenerative diseases such as Alzheimers disease (AD). This study investigates the metabolic effects of chronic nicotine administration in presenilin-1/2 double knockout (DKO) mice and wild-type (WT) control mice. Eight-month-old DKO and WT mice underwent a three-month oral nicotine administration which did not significantly affect body weight or spontaneous locomotor activity in either DKO or WT mice, as measured by weekly body weight measurement and open field test. The metabolomic analyses were performed using untargeted LC-MS and GC-MS techniques. The results revealed distinct metabolic profiles between DKO and WT mice under nicotine exposure. In DKO mice, nicotine exacerbated deficits in purine and nicotinamide metabolism, further highlighting dysregulation in energy homeostasis. Key findings include reduced adenosine and elevated dopamine levels in DKO serum, alongside increased cotinine concentrations, suggesting altered nicotine metabolism. WT mice showed significant changes in energy and lipid metabolism pathways, though with less pronounced alterations compared to DKO mice. Enrichment analyses identified significant pathway disruptions, particularly in choline metabolism, amino acid metabolism, and central carbon metabolism, with genotype-dependent responses to nicotine. This study highlights the intrinsic metabolic alterations in DKO mice, such as disruptions in purine and nicotinamide metabolism, which contribute to their neurodegenerative pathology and unique responses to nicotine. By revealing the differential metabolic impacts of nicotine on healthy and neurodegenerative states, the findings provide valuable insights into its dual therapeutic and pathological roles. These results may guide future research into the metabolic underpinnings of neurodegeneration and the potential for nicotine as a targeted therapeutic intervention.

neuroscience↗

bioRxiv2024

Lack of Ameliorative Effect of Chronic Oral Nicotine on Olfactory Dysfunction in Young Adult Presenilin-1/2 Double Knockout Mice of a Dementia Model

Olfactory dysfunction is a prevalent and early feature of neurodegenerative diseases including Alzheimers disease while the features and underlying mechanisms remain inadequately understood. This investigation aimed to elucidate the olfactory function characteristics of presenilin-1/2 double knockout (DKO) mice, an established mouse model of dementia. Through a series of behavioral tests on DKO and wildtype (WT) mice at 2 and 6 months of age, we assessed their abilities in odor detection, discrimination, and olfactory memory. The findings revealed a substantial deficit in odor detection in 2-month-old DKO mice compared to WT mice, with a noteworthy age-related deterioration observed from 2 to 6 months. Although DKO mice exhibited normal odor discrimination and olfactory memory capabilities at 2 months, these functions declined with age, falling significantly lower levels than observed in WT mice by the age of 6 months. Subsequently, 2-month-old DKO and WT mice underwent a 4-month chronic nicotine treatment through drinking water. This intervention failed to ameliorate olfactory dysfunctions in DKO mice and exhibited no significant impact on WT mice, despite a discernible trend of a potential negative effect. In summary, this behavioral study elucidates that Presenilin-1/2 double knockout impairs odor detection in young adult mice and accelerates declines of olfactory discrimination and memory. The chronic administration of nicotine did not effectively mitigate olfactory function deficiencies in young adult DKO mice, and the results also highlight age as an important contributing factor.

animal behavior and cognition↗

bioRxiv2024

Nasopharyngeal specific selection and emergence of SARS-CoV-2 spike fusion peptide mutation P812S during chronic infection

Omicron emergence represented a seismic event in the COVID-19 pandemic, demonstrating what was essentially antigenic shift in a virus that cannot reassort its genome as influenza can. Understanding the success of Omicron is essential, and yet we have little understanding of the biological underpinnings of its ability to accommodate diverse mutations and bring together deleterious mutations to generate a highly successful new serotype. Persistent SARS-CoV-2 infections are a likely source of new variants and may provide valuable insight into past and future evolutionary trajectories, in particular those involving allosteric interactions that defy genotype to phenotype prediction. Here we observe upper airway specific evolution of SARS-CoV-2 demonstrating fusion peptide (FP) domain mutation S:P812S adjacent to the S2 cleavage site that emerged during a chronic infection in an immunocompromised individual. Indeed, this mutation had previously emerged in an ancestral B lineage as well as the delta variant lineage and transmitted successfully in populations globally, though remains uncharacterised. P812S in spike pseudotyped virus particles did not impact entry efficiency across cell lines expressing endogenous ACE2 and TMPRSS2. However, efficiency of spike cleavage at S1/S2 was reduced and molecular dynamics simulation demonstrated altered S1/S2 loop conformations that possibly impacted furin mediated cleavage. Consistent with impaired S1/S2 cleavage, and reminiscent of Omicron BA.1, cell-cell fusogenicity was severely impaired by introduction of P812S. The mutation also introduced significant perturbations to the FP region and also affected protomer-protomer packing. P812S conferred evasion of a neutralising monoclonal antibody targeting the fusion peptide, consistent with significant structural rearrangements in the FP region. Finally, P812S bearing viruses showed evasion of polyclonal neutralising antibodies in sera from vaccinated individuals at 32{degrees}C (simulating upper respiratory tract) and to a lesser extent at 37{degrees}C. Thus we report a novel mutational adaptation to the upper airway allowing enhanced immune evasion to fusion peptide targeting neutralising antibodies that also incurs a defect in ability to induce syncytia. These data shed light on the balance between upper airway adaptation/immune evasion by SARS-CoV-2, ability to induce syncytia formation, and disease severity given the established link between syncytia and severe COVID-19.

immunology↗

bioRxiv2024

Spike N354 glycosylation augments SARS-CoV-2 fitness for human adaptation through multiple mechanisms

Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity could be recovered in the presence of heparin sulfate, which targets the "N354 pocket" to ease restrictions of conformational transition resulting in a "RBD-up" state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms. HIGHLIGHTSN354 glycosylation acts as a conformational control element to modulate infectivity Reduced infectivity could be recovered by altered binding mode of heparin sulfate N354 glycosylation improved fusogenicity and conferred escape from ADCC antibodies N354 glycosylation reduced immunogenicity and conferred immune evasion

microbiology↗

bioRxiv2023

Hypoxia drives HIF2-dependent macrophage cell cycle entry and susceptibility to lentivira transduction

Macrophages play critical roles across health and disease. Low oxygen conditions (hypoxia) have been associated primarily with cell cycle arrest in cultured dividing cells. Macrophages are typically quiescent in G0, though yolk sac and bone marrow derived macrophages frequently proliferate and monocyte-derived tissue macrophages are able to proliferate in response to tissue signals. Here we show that hypoxia (1% oxygen tension) results in reversible entry into the cell cycle in monocyte derived macrophages (MDM) and mouse peritoneal macrophages. Cell cycle progression is largely limited to G1/S phase with very little progression to G2/M. Mechanistically, this cell cycle transitioning is triggered by a HIF2-directed transcriptional program. The response is accompanied by increased expression of cell cycle-associated proteins, including CDK1, and reversible activation of the canonical mitogen-activated MEK-ERK proliferation pathway. CDK1 associated SAMHD1 phosphorylation at T592 in hypoxic macrophages renders them hyper-susceptible to lentiviral transduction. Furthermore, PHD inhibitors, which activate HIFs, are able to recapitulate HIF2-dependent cell cycle entry in macrophages, as well as susceptibility to lentiviral transduction. Finally, we demonstrate that tumour associated macrophages (TAM) in lung cancers exhibit transcriptomic profiles representing responses to low oxygen and cell cycle progression at single cell level. This work uncovers HIF2 driven macrophage cell cycle progression in low oxygen conditions that culminates in SAMHD1 phosphorylation and high susceptibility to lentiviral transduction. These findings have implications for inflammation, neoplasia and pathogen defence.

cell biology↗

bioRxiv2023

Pharmacokinetic profile of the synthetic mu-opioid receptor agonist Dermorphin-IRDye(R)800CW and its feasibility as a biomarker for opioid use disorder

BackgroundOpioid use disorder (OUD) affects more than 14 million Americans and poses a high risk of relapse, overdose, and death. Current treatments are not tailored to individual needs and do not monitor the effectiveness of the medication. We propose a novel method to measure the occupancy of mu opioid receptors (MOR), which are key targets for opioid pharmacotherapy, in peripheral tissues with high MOR density. We developed a fluorescent peptide agonist that binds to MOR and can be detected by non-invasive point-of-care techniques. We present in vitro and in vivo results that demonstrate the feasibility and potential of this method to assess MOR availability and treatment efficacy in OUD patients. MethodsA new fluorescent-labeled synthetic peptide agonist [Lys7]Dermorphin-IRDye800CW, called DRM-800, was synthesized and characterized in vitro to evaluate binding and internalization. Wildtype and MOR knock-out mice were used to quantify plasma kinetics and, using a cyromacrotome, fluorescence images were acquired post-mortem on whole-body sections 150 um apart. These volumes were used to compare in vivo enhancement of MOR-rich structures. ResultsIn vitro assays and microscope visualization of DRM-800 showed high MOR-affinity and rapid, robust internalization. Plasma half-life following intravenous injection in mice was 8-12 minutes. Specific binding by tissue structures of interest, measured by the ratio of relative fluorescent units in wild-type vs. MOR knockout mice showed high binding in dorsal root ganglia, spiral ganglia and trigeminal ganglion, as well as in the small and large intestine. ConclusionsThe pharmacokinetics and distribution, binding kinetics and rapid internalization suggests that MOR-specific fluorescence enhancement corresponding to opioid rich structures could serve as a potential biomarker in opioid use disorder.

bioengineering↗

bioRxiv2022

Structural basis of a high-affinity antibody binding to glycoprotein region with consecutive glycosylation sites

Consecutive glycosylation sites occur in both self and viral proteins. Glycan-shielding of underneath peptide region is a double-edged sword, that avoids immune attack to self-proteins, but helps viruses including HIV-1 and SARS-CoV2 to escape antibody binding. Here we report a high-affinity antibody, 16A, binding to linear peptide containing consecutive glycosylation sites. Co-crystallization of 16A Fab and glycopeptides with GalNAc modifications at different sites showed that STAPPAHG is the sequence recognized by 16A antibody. GalNAc modification at Threonine site on STAPPAHG sequence significantly increased the affinity of Fab binding by 30.6 fold (KD=6.7nM). The increased affinity is conferred by hydrophilic and pi-stacking interactions between the GalNAc residue on Threonine site and a Trp residue from the CDR1 region of the heavy chain. Furthermore, molecular modeling suggested that GalNAc on T site causes more favorable conformation for antibody binding. These results showed that glycan modification most proximal to linear peptide core epitope significantly increases antigenicity of a glycopeptide epitope. The antibody recognition mode by peptide-binding CDR groove with a glycan-binding edge, may shed light on designing of linear glycopeptide-based vaccines for cancer and viral diseases. TeaserA high-affinity antibody was found to bind densely glycosylated glycoprotein region by a peptide binding groove of the antibodys variant region, with a glycan-binding edge specific to glycosylation site most proximal to core peptide epitope.

immunology↗

bioRxiv2022

SARS-CoV-2 Spike N-Terminal Domain modulates TMPRSS2-dependent viral entry and fusogenicity

Over 20 mutations have been identified in the N-Terminal Domain (NTD) of SARS-CoV-2 spike and yet few of them are fully characterised. Here we first examined the contribution of the NTD to infection and cell-cell fusion by constructing different VOC-based chimeric spikes bearing B.1617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus (PV). We found the Delta NTD on a Kappa or WT background increased spike S1/S2 cleavage efficiency and virus entry, specifically in Calu-3 lung cells and airway organoids, through use of TMPRSS2. We have previously shown Delta spike confers rapid cell-cell fusion kinetics; here we show that increased fusogenicity can be conferred to WT and Kappa variant spikes by transfer of the Delta NTD. Moving to contemporary variants, we found that BA.2 had higher entry efficiency in a range of cell types as compared to BA.1. BA.2 showed higher fusogenic activity than BA.1, but the BA.2 NTD could not confer higher fusion to BA.1 spike. There was low efficiency of TMPRSS2 usage by both BA.1 and BA.2, and chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD did not result in more efficient use of TMRPSS2 or cell-cell fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions such as entry and cell-cell fusion in a spike context dependent manner, and allosteric interactions may be lost when combining regions from more distantly related spike proteins. These data may explain the lack of successful SARS-CoV-2 inter-variant recombinants bearing breakpoints within spike.

microbiology↗

bioRxiv2021

SARS-CoV-2 Omicron spike mediated immune escape, infectivity and cell-cell fusion

The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.

microbiology↗

bioRxiv2021

SARS-CoV-2 B.1.617 emergence and sensitivity to vaccine-elicited antibodies

The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD), in particular to the clinically approved bamlavinimab and imdevimab monoclonal antibodies. B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems as compared to B.1.1.7, associated with B.1.617.2 spike being in a predominantly cleaved state compared to B.1.1.7. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed substantially reduced ChAdOx-1 vaccine efficacy against B.1.617.2 relative to non-B.1.617.2. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

microbiology↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2020

Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

The scission of lipid membranes is a common biological process, often mediated by ESCRT complexes in concert with VPS4 assembling around the separation point. The functions of the ESCRT-I and ESCRT-III complexes are well established in certain of these cellular processes; however, the role of ESCRT-II remains contentious. Here, we devised a SNAP-tag fluorescent labelling strategy to understand the domain requirements of EAP45, the main component of ESCRT-II, in HIV egress, late endosome recruitment, and cytokinesis. We used TIRF microscopy to measure the spatial co-occurrence of the HIV structural polyprotein Gag with full length EAP45 in both fixed and live cells. Gag colocalises with the full length EAP45 comparably to ALIX, but this is lost on deletion of the EAP45 N terminus. Our findings reveal the H0 domain of the EAP45 protein is essential for linking to ESCRT-I during HIV budding and in anchoring at the late endosomal membrane, however in cytokinesis it is the Glue domain that is critical.

biophysics↗

bioRxiv2019

Precision Genome Editing Using Bacteriophage-derived Peptides

BackgroundCRISPR-Cas9 has been developed as a therapeutic agent for various infectious and genetic diseases. In many clinically relevant applications, constitutively active CRISPR-Cas9 is delivered into human cells without a temporal control system. Excessive and prolonged expression CRISPR-Cas9 can lead to elevated off-target cleavage. The need for modulating CRISPR-Cas9 activity over the dimensions of time and dose has created the demand of developing CRISPR-Cas off-switches. Protein and small molecule-based CRISPR-Cas inhibitors have been reported in previous studies. ResultsWe report the discovery of Cas9-inhibiting peptides from inoviridae bacteriophages. These peptides, derived from the periplasmic domain of phage major coat protein G8P (G8PPD), can inhibit the in vitro activity of Streptococcus pyogenes Cas9 (SpCas9) proteins in an allosteric manner. Importantly, the inhibitory activity of G8PPD on SpCas9 is dependent on the order of guide RNA addition. Ectopic expression of full-length G8P (G8PFL) or G8PPD in human cells can inactivate the genome-editing activity of SpCas9 with minimum alterations of the mutation patterns. Furthermore, unlike the anti-CRISPR protein AcrII4A that completely abolishes the cellular activity of CRISPR-Cas9, G8P co-transfection can reduce the off-target activity of co-transfected SpCas9 while retaining its on-target activity. ConclusionG8Ps discovered in the current study represent the first anti-CRISPR peptides that can allosterically inactivate CRISPR-Cas9. This finding may provide insights into developing next-generation CRISPR-Cas inhibitors for precision genome engineering.

genomics↗

bioRxiv2019

The Telomerase Reverse Transcriptase (TERT) and p53 Regulate Mammalian PNS and CNS Axon Regeneration downstream of c-Myc

Although several genes have been identified to promote axon regeneration in the central nervous system, our understanding of the molecular mechanisms by which mammalian axon regeneration is regulated is still limited and fragmented. Here by using sensory axon and optic nerve regeneration as model systems, we revealed an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. We also provided strong evidence that TERT and p53 acted downstream of c-Myc to control sensory axon regeneration. More importantly, overexpression of p53 in sensory neurons and retinal ganglion cells (RGCs) was sufficient to promote sensory axon and optic never regeneration, respectively. The study revealed a novel c-Myc-TERT-p53 signaling pathway, expanding horizons for novel approaches promoting CNS axon regeneration.

neuroscience↗

bioRxiv2019

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca2+) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase (CaMKII), a target protein for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon growth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.

neuroscience↗