Biology subjects

Leung, A.

Publications and source records attributed to Leung, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Glucose-regulated O-GlcNAcylation of DNMT1 inhibits DNA methyltransferase activity and maintenance of genomic methylation

The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on DNMT1. Functional studies further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that is preferentially at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.

molecular biology↗

bioRxiv2022

Emergence of integrated information at macro timescales in real neural recordings

How a system generates conscious experience remains an elusive question. One approach towards answering this is to consider the information available in the system from the perspective of the system itself. Integrated information theory (IIT) proposes a measure to capture this, integrated information ({Phi}). While {Phi} can be computed at any spatiotemporal scale, IIT posits that it be applied at the scale at which the measure is maximised. Importantly, {Phi} should emerge to be maximal not at the smallest spatiotemporal scale, but at some macro scale where system elements or timesteps are grouped into larger elements or timesteps. Emergence in this sense has been demonstrated in artificial binary systems, but it remains unclear whether it occurs in real neural recordings which are generally continuous and noisy. Here we first utilise a computational model to confirm that {Phi} becomes maximal at the temporal scales underlying its generative mechanisms. Second, we search for emergence in local field potentials from the fly brain recorded during wakefulness and anaesthesia, finding that normalised {Phi} (wake/anaesthesia), but not raw {Phi} values, peaks at 5 ms. Lastly, we extend our model to investigate why raw {Phi} values themselves did not peak. This work extends the application of {Phi} to simple artificial systems consisting of logic gates towards searching for emergence of a macro spatiotemporal scale in real neural systems.

neuroscience↗

bioRxiv2022

Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening

The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen [~]38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100{micro}M, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors.

biochemistry↗

bioRxiv2022

Design, Synthesis and Evaluation of Inhibitors of the SARS-CoV-2 nsp3 Macrodomain

A series of amino acid based 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low M Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/482176v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@167aceorg.highwire.dtl.DTLVardef@1d88c47org.highwire.dtl.DTLVardef@1e1b34borg.highwire.dtl.DTLVardef@c2321a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model

Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

cell biology↗

bioRxiv2021

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a new mode of engaging poly(ADP-ribose)

PARP13/ZAP acts against multiple viruses through recognizing and promoting degradation of cytoplasmic viral mRNA. PARP13 has four N-terminal Zn-finger motifs that bind CG-rich nucleotide sequences, and a C-terminal ADP ribosyltransferase fold similar to other PARPs. A central region predicted to contain a fifth Zn-finger and two tandem WWE domains is implicated in binding poly(ADP-ribose); however, there are limited insights into the structure and function of this PARP13 region (ZnF5-WWE1-WWE2). Here, we present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and with ATP. ZnF5-WWE1-WWE2 crystallized as a dimer with major contacts formed between WWE1 and WWE2 originating from different monomers, indicative of a more compact monomeric arrangement of the tandem WWE domains. Solution scattering experiments and biophysical analysis indicated a monomer in solution, suggesting that the crystal dimer represents domain swapping that could potentially represent a PARP13 conformation assumed when signaling viral RNA detection. The crystal structure and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. The shape of the WWE2 binding pocket disfavors interaction with the ribose-ribose linkage of poly(ADP-ribose). Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose), and the composite structure of ZnF5-WWE1-WWE2 forms an extended surface to engage polymer chains of ADP-ribose. This model represents a novel mode of poly(ADP-ribose) recognition and provides a structural framework for investigating poly(ADP-ribose) impact on PARP13 function.

biochemistry↗

bioRxiv2021

Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter

AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.

molecular biology↗

bioRxiv2021

High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodomain inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

biochemistry↗

bioRxiv2021

The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

microbiology↗

bioRxiv2021

Exposure route, sex, and age influence disease outcome in a golden Syrian hamster model of SARS-CoV-2 infection

The emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resultant pandemic of coronavirus disease 2019 (COVID-19) has led to over one hundred million confirmed infections, greater than three million deaths, and severe economic and social disruption. Animal models of SARS-CoV-2 are critical tools for the pre-clinical evaluation of antivirals, vaccines, and candidate therapeutics currently under urgent development to curb COVID-19-associated morbidity and mortality. The golden (Syrian) hamster model of SARS-CoV-2 infection recapitulates key characteristics of severe COVID-19, including high-titer viral replication in the upper and lower respiratory tract and the development of pathogenic lesions in the lungs. In this work we examined the influence of the route of exposure, sex, and age on SARS-CoV-2 pathogenesis in golden hamsters. We report that delivery of SARS-CoV-2 primarily to the nasal passages (low-volume intranasal), the upper and lower respiratory tract (high-volume intranasal), or the digestive tract (intragastric) results in comparable viral titers in the lung tissue and similar levels of viral shedding during acute infection. However, low-volume intranasal exposure results in milder weight loss during acute infection while intragastric exposure leads to a diminished capacity to regain body weight following the period of acute illness. Further, we examined both sex and age differences in response to SARS-CoV-2 infection. Male hamsters, and to a greater extent older male hamsters, display an impaired capacity to recover from illness and a delay in viral clearance compared to females. Lastly, route of exposure, sex, and age were found to influence the nature of the host inflammatory cytokine response, but they had a minimal effect on both the quality and durability of the humoral immune response as well as the susceptibility of hamsters to SARS-CoV-2 re-infection. Together, these data indicate that the route of exposure, sex, and age have a meaningful impact SARS-CoV-2 pathogenesis in hamsters and that these variables should be considered when designing pre-clinical challenge studies.

microbiology↗

bioRxiv2021

Mechanisms regulating interindividual epigenetic variability at transposable elements

Transposable elements (TEs) are mobile genetic elements that make up a large fraction of mammalian genomes. While select TEs have been co-opted in host genomes to have function, the majority of these elements are epigenetically silenced by DNA methylation in somatic cells. However, some TEs in mice, including the Intracisternal A-particle (IAP) subfamily of retrotransposons, have been shown to display interindividual variation in DNA methylation. Recent work has revealed that IAP sequence differences and strain-specific KRAB zinc finger proteins (KZFPs) may influence the methylation state of these IAPs. However, the mechanisms underlying the establishment and maintenance of interindividual variability in DNA methylation still remain unclear. Here we report that sequence content and genomic context influence the likelihood that IAPs become variably methylated. IAPs that differ from consensus IAP sequences have altered KZFP recruitment that can lead to decreased KAP1 recruitment when in proximity of constitutively expressed genes. These variably methylated loci have a high CpG density, similar to CpG islands, and can be bound by ZF-CxxC proteins, providing a potential mechanism to maintain this permissive chromatin environment and protect from DNA methylation. These observations indicate that variably methylated IAPs escape silencing through both attenuation of KZFP binding and recognition by ZF-CxxC proteins to maintain a hypomethylated state.

genomics↗

bioRxiv2021

Substantial Differences in SARS-CoV-2 Antibody Responses Elicited by Natural Infection and mRNA Vaccination

We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8,476 finger stick blood specimens were collected before and after an aggressive mRNA vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing 10 SARS-CoV-2 antigens, 4 SARS, 3 MERS, 12 Common CoV, and 8 Influenza antigens. Twenty-six percent of 3,347 specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive. The Ab response was predominantly against nucleocapsid (NP), full length spike and the spike S2 domain. Anti-receptor binding domain (RBD) reactivity was low and there was no cross-reactivity against SARS S1 or SARS RBD. An aggressive mRNA vaccination campaign at the UCI Medical Center started on December 16, 2020 and 6,724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% in December to 79% in January, 93% in February and 99% in March. mRNA vaccination induced much higher Ab levels especially against the RBD domain and significant cross-reactivity against SARS RBD and S1 was also observed. Nucleocapsid protein Abs can be used to distinguish individuals in a population of vaccinees to classify those who have been previously infected and those who have not, because nucleocapsid is not in the vaccine. Previously infected individuals developed higher Ab titers to the vaccine than those who have not been previously exposed. These results indicate that mRNA vaccination rapidly induces a much stronger and broader Ab response than SARS-CoV-2 infection.

immunology↗

bioRxiv2020

PARprolink: a photoaffinity probe for identifying poly(ADP-ribose)-binding proteins

Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite its clinical significance, a molecular understanding of PAR function, including its binding partners, remains incomplete. In this work, we synthesize a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel binders involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer over 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteome-wide view of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.

molecular biology↗

bioRxiv2020

nearBynding: A flexible pipeline characterizing protein binding to local RNA structure

The etiology of diseases driven by dysregulated mRNA metabolism can be elucidated by characterizing the responsible RNA-binding proteins (RBPs). Although characterizations of RBPs have been mainly focused on their binding sequences, not much has been investigated about their preferences for RNA structures. We present nearBynding, an R/Bioconductor pipeline that incorporates RBP binding sites and RNA structure information to discern structural binding preferences for an RBP. nearBynding visualizes RNA structure at and proximal to sites of RBP binding transcriptome-wide, analyzes CLIP-seq data without peak-calling, and provides a flexible scaffold to study RBP binding preferences relative to diverse RNA structure data types.

bioinformatics↗

bioRxiv2020

Discovery and biosynthesis of clostyrylpyrones from the obligate an-aerobe Clostridium roseum

Anaerobic bacteria are a promising new source for natural product discovery. Examination of extracts from the obligate anaerobe Clostridium roseum led to discovery of a new family of natural products, the clostyrylpyrones. The polyketide synthase-based biosynthetic mechanism of clostyrylpyrones is further proposed based on bioinformatic, gene knockout, biochemical analysis and heterologous expression studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/245514v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1f24265org.highwire.dtl.DTLVardef@1e51bd7org.highwire.dtl.DTLVardef@86c761org.highwire.dtl.DTLVardef@8bd052_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

North American deer mice are susceptible to SARS-CoV-2

The zoonotic spillover of the pandemic SARS-coronavirus 2 (SARS-CoV-2) from an animal reservoir, currently presumed to be the Chinese horseshoe bat, into a naive human population has rapidly resulted in a significant global public health emergency. Worldwide circulation of SARS-CoV-2 in humans raises the theoretical risk of reverse zoonosis events with wildlife, reintroductions of SARS-CoV-2 into permissive non-domesticated animals, potentially seeding new host reservoir species and geographic regions in which bat SARS-like coronaviruses have not historically been endemic. Here we report that North American deer mice (Peromyscus maniculatus) and some closely related members of the Cricetidae family of rodents possess key amino acid residues within the angiotensin-converting enzyme 2 (ACE2) receptor known to confer SARS-CoV-2 spike protein binding. Peromyscus rodent species are widely distributed across North America and are the primary host reservoirs of several emerging pathogens that repeatedly spill over into humans including Borrelia burgdorferi, the causative agent of Lyme disease, deer tick virus, and Sin Nombre orthohantavirus, the causative agent of hantavirus pulmonary syndrome (HPS). We demonstrate that adult deer mice are susceptible to SARS-CoV-2 infection following intranasal exposure to a human isolate, resulting in viral replication in the upper and lower respiratory tract with little or no signs of disease. Further, shed infectious virus is detectable in nasal washes, oropharyngeal and rectal swabs, and viral RNA is detectable in feces and occasionally urine. We further show that deer mice are capable of transmitting SARS-CoV-2 to naive deer mice through direct contact. The extent to which these observations may translate to wild deer mouse populations remains unclear, and the risk of reverse zoonosis and/or the potential for the establishment of Peromyscus rodents as a North American reservoir for SARS-CoV-2 is unknown. Nevertheless, efforts to monitor wild, peri-domestic Peromyscus rodent populations are likely warranted as the SARS-CoV-2 pandemic progresses.

microbiology↗

bioRxiv2020

Aging leads to stochastic loss of silencing at transposons in mammary luminal epithelial cells

Luminal epithelial cells (LEps) are a key cell lineage implicated in age-related luminal breast cancers. Alterations to the epigenome are a hallmark of aging cells. However, the extent of age-associated DNA methylation alterations in LEps, and the corresponding functional consequences of these alterations, have remained unclear. We report here that aging leads to distinct methylation changes in LEps. Luminal lineage-specific genes gain promoter methylation, whereas myoepithelial-specific genes lose promoter methylation. Regulatory elements display methylation changes at lineage-specific TF binding sites consistent with the loss of lineage fidelity. CpG islands (CGIs) and transposable elements (TEs) have stochastic methylation gain and loss, respectively. PRC2 target genes that are hypermethylated in luminal breast cancer exhibit stochastic methylation increase with age. TEs with stochastic methylation loss are activated in breast cancer and potentially function as regulatory elements contributing to the loss of lineage fidelity with age. Each of these classes of methylation changes impact the regulation of genes associated with luminal breast cancer. Altogether, our results indicate that aging leads to DNA methylation changes that could determine breast cancer susceptibility. SignificanceMammary luminal epithelial cells lose lineage-specific expression with age and accumulate stochastic methylation changes that lead to loss of silencing at transposable elements. These age-dependent events potentially promote breast cancer susceptibility.

genomics↗

bioRxiv2020

Team flow is a unique brain state associated with enhanced information integration and neural synchrony

Team flow occurs when a group of people reaches high task engagement while sharing a common goal as in sports teams and music bands. While team flow is a superior enjoyable experience to individuals experiencing flow or regular socialization, the neural basis for such superiority is still unclear. Here, we addressed this question utilizing a music rhythm task and electroencephalogram hyper-scanning. Experimental manipulations held the motor task constant while disrupted the hedonic musical correspondence to blocking flow or occluded the partners body and task feedback to block social interaction. The manipulations effectiveness was confirmed using psychometric ratings and an objective measure for the depth of flow experience through the inhibition of the auditory-evoked potential to a task-irrelevant stimulus. Spectral power analysis revealed higher beta/gamma power specific to team flow at the left temporal cortex. Causal interaction analysis revealed that the left temporal cortex receives information from areas encoding individual flow or socialization. The left temporal cortex was also significantly involved in integrated information at both the intra- and inter-brains levels. Moreover, team flow resulted in enhanced global inter-brain integrated information and neural synchrony. Thus, our report presents neural evidence that team flow results in a distinct brain state and suggests a neurocognitive mechanism by which the brain creates this unique experience. Data AvailabilityAll data and analysis codes used in the preparation of this article are available at https://osf.io/3b4hp.

neuroscience↗