Biology subjects

Chan, S. H.

Publications and source records attributed to Chan, S. H..

13 recordsLinked to original sources

bioRxiv2026

Cap-specific N6-adenosine methyltransferases and applications in m6A Cap-1 mRNA synthesis

In addition to the 5' Cap-1 structure, the initiating adenosine of mammalian mRNA can be further modified by PCIF1 (Phosphorylated C-terminal domain-interacting factor 1) to form 2'-O,N6-dimethyladenosine (m6A Cap-1). While the biological role is still under investigation, the implications of m6A Cap-1 in mRNA metabolism and viral-host interactions have spurred interest in PCIF1 and m6A Cap-1 as biomarkers, drug targets, and research tools. Here, we identify and characterize two PCIF1-like cap-specific N6-adenosine methyltransferases, PC04 and PC05 and evaluate their applicability in m6A Cap-1 mRNA synthesis in vitro. We found that both enzymes are highly specific to the m7G cap structure and highly active on Cap-1 RNA oligonucleotides and trinucleotide Cap-1 analogs at nanomolar concentrations. We demonstrate that PC04 or PC05 methylation reactions can be incorporated into established post-transcriptional capping and cap analog-based co-transcriptional in vitro transcription (IVT) workflows to produce m6A Cap-1 RNAs. Finally, we show that PC05 can be combined with a previously reported FCE::T7 RNA polymerase fusion to synthesize m6A Cap-1 mRNA from DNA templates in a single step, co-transcriptional manner. The robustness of the PCIF1-like cap N6-adenosine methyltransferases can accelerate research into the biological roles of cap m6A modification and development of highly effective mRNA-based therapeutics.

biochemistry↗

bioRxiv2023

MFSD1 in complex with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes

Lysosomal degradation of macromolecules in lysosomes produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here, we deorphanized the Major Facilitator Superfamily Domain Containing 1 (MFSD1) protein, which forms a tight complex with the Glycosylated Lysosomal Membrane Protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer, and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1/GLMP acts as a uniporter for cationic and neutral dipeptides. Cryo-EM structure of the dipeptide-bound MFSD1/GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.

biochemistry↗

bioRxiv2023

Co-transcriptional capping using an RNA capping enzyme-T7 RNA polymerase fusion protein

mRNA vaccines and therapeutics are highly effective and can be developed and manufactured with a relatively short lead time. Here we report an all-enzyme platform to generate capped synthetic RNA in a one-step process based on an RNA capping enzyme-T7 RNA polymerase fusion protein. Under standard in vitro transcription reaction conditions, the fusion protein, in conjunction with an RNA cap 2'-O-methyltransferase, can generate synthetic mRNA with up to 95% of Cap-1 incorporation, greatly simplifying mRNA manufacturing workflows.

biochemistry↗

bioRxiv2023

Streamlined DNA template preparation and co-transcriptional 5' capped RNA synthesis enabled by solid-phase catalysis

The success of SARS-CoV-2 mRNA vaccines demonstrated that rapid, large-scale manufacturing of synthetic mRNA is necessary for an effective and timely response to a pandemic. Innovations in areas such as template design and manufacturing processes are being implemented to facilitate more simple, cost-effective and scalable mRNA synthesis. In this study, for the first time, we demonstrate that the enzymatic steps in mRNA production (including DNA template linearization, RNA synthesis, 5' capping and methylation) can be carried out using enzymes immobilized to a solid support. Specifically, we demonstrate efficient IVT template DNA linearization using immobilized BspQI, where the linearized template DNA can be directly used in IVT without the need of purification. We also showed that immobilized T7 RNA polymerase, Faustovirus RNA capping enzyme (FCE), vaccinia cap 2'-O-methyltransfease (2'OMTase) and a novel FCE::T7RNAP fusion enable efficient enzymatic synthesis of Cap-1 RNA in a one-pot format. This solid-phase enzymatic platform may enable highly efficient, seamless and continuous mRNA synthesis workflows that minimizes sample loss and units of operation in biopharmaceutical manufacturing.

biochemistry↗

bioRxiv2023

Biochemical characterization of mRNA capping enzyme from Faustovirus

The mammalian mRNA 5 cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5- mediated mRNA turnover. Hence, installation of the proper 5 cap is crucial in therapeutic applications of synthetic mRNA. The core 5 cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5 triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity towards RNA containing secondary structures, and broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

biochemistry↗

bioRxiv2023

Screening in serum-derived medium reveals differential response to compounds targeting metabolism

INTRODUCTION INTRODUCTION RESULTS DISCUSSION KEY RESOURCES TABLE CONTACT FOR REAGENT AND... EXPERIMENTAL MODEL AND SUBJECT... METHOD DETAILS QUANTIFICATION AND STATISTICAL... Data resources DATA AND SOFTWARE AVAILABILITY AUTHOR CONTRIBUTIONS DECLARATION OF INTERESTS REFERENCES Studies of cancer cells in standard culture conditions have long been used as a tractable tool for drug discovery. Cell culture provides unparalleled experimental flexibility, scalability, and low cost to identify and understand the response to cancer therapeutics; however, drug responses in culture are not always predictive of drug response in animal models or in patients1 ...

cancer biology↗

bioRxiv2022

Coupling of protein condensates to ordered lipid domains determines functional membrane organization

During T-cell activation, the transmembrane adaptor Linker of Activation of T-cells (LAT) forms biomolecular condensates with Grb2 and Sos1, facilitating signaling. LAT has also been associated with cholesterol-rich condensed lipid domains. However, the potential coupling between protein condensation and lipid phase separation and its role in organizing T-cell signaling were unknown. Here, we report that LAT/Grb2/Sos1 condensates reconstituted on model membranes can induce and template lipid domains, indicating strong coupling between lipid- and protein-based phase separation. Correspondingly, activation of T-cells induces protein condensates that associate with and stabilize raft-like membrane domains. Inversely, lipid domains nucleate and stabilize LAT protein condensates in both reconstituted and living systems. This coupling of lipid and protein assembly is functionally important, since uncoupling of lipid domains from cytoplasmic protein condensates abrogates T-cell activation. Thus, thermodynamic coupling between protein condensates and ordered lipid domains regulates the functional organization of living membranes. SUMMARYMembrane-associated protein condensates couple to ordered membrane domains to determine the functional organization of T-cell plasma membranes

cell biology↗

bioRxiv2020

Synergistic activity of Nanog, Pou5f3, and Sox19b establishes chromatin accessibility and developmental competence in a context-dependent manner

Genome-wide chromatin reprogramming is a fundamental requirement for establishing developmental competence in the newly-formed zygote. In zebrafish, Nanog, Pou5f3 and Sox19b play partially redundant roles in zygotic genome activation, however their interplay in establishing chromatin competency, the context in which they do so and their mechanism of action remain poorly defined. Here, we generated a triple maternal-zygotic nanog-/-;pou5f3-/-;sox19b-/- mutant and assessed the causal relationship between transcription factor (TF) occupancy, chromatin accessibility and genome activation. Analyses of this triple mutant and combinatorial rescues revealed highly synergistic and context-dependent activity of Nanog, Pou5f3, and Sox19b (NPS) in establishing chromatin competency at >50% of active enhancers. Motif analysis revealed a network of TFs that depend on NPS for establishing chromatin accessibility, including the endodermal determinant Eomesa, whose binding we show is regulated by NPS pioneer-like activity. Finally, we demonstrated that NPS play an essential role in establishing H3K27ac and H3K18ac at enhancers and promoters, and that their function in transcriptional activation can be bypassed by targeted recruitment of histone acetyltransferases to individual genes. Altogether, our findings reveal a large network of TFs that function to establish developmental competency, many of which depend on the synergistic and highly context-dependent role of NPS in establishing chromatin accessibility and regulating histone acetylation in order to activate the genome.

developmental biology↗

bioRxiv2020

MCART1 is required for mitochondrial NAD transport

The nicotinamide adenine dinucleotide (NAD+/NADH) pair is a cofactor in redox reactions and is particularly critical in mitochondria as it connects substrate oxidation by the tricarboxylic acid (TCA) cycle to ATP generation by the electron transport chain (ETC) and oxidative phosphorylation. While a mitochondrial NAD+ transporter has been identified in yeast, how NAD enters mitochondria in higher eukaryotes is unknown. Here, we mine gene essentiality data from human cell lines to identify MCART1 (SLC25A51) as co-essential with ETC components. MCART1-null cells have large decreases in TCA cycle flux, mitochondrial respiration, ETC complex I activity, and mitochondrial levels of NAD+ and NADH. Isolated mitochondria from cells lacking or overexpressing MCART1 have greatly decreased or increased NAD uptake in vitro, respectively. Moreover, MCART1 and NDT1, a yeast mitochondrial NAD+ transporter, can functionally complement for each other. Thus, we propose that MCART1 is the long sought mitochondrial transporter for NAD in human cells.

cell biology↗

bioRxiv2020

Dihydroxyacetone phosphate signals glucose availability to mTORC1

In response to nutrients, the mTOR complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose, which provide the building blocks for the biosynthesis of most macromolecules. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells to identify glucose-derived metabolites required to activate mTORC1. We find that mTORC1 senses a metabolite downstream of the aldolase and upstream of the glyceraldehyde 3-phosphate dehydrogenase steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from dihydroxyacetone is sufficient to activate mTORC1 even in the absence of glucose. Genetic perturbations in the GATOR-Rag signaling axis abrogate glucose sensing by mTORC1. DHAP is the glycolytic metabolite along with glyceraldehyde 3-phosphate (GAP) that has the greatest fold-change between cells in high and low glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.

cell biology↗

bioRxiv2020

Family History Assessment Significantly Enhances Delivery of Precision Medicine in the Genomics Era

BackgroundFamily history has traditionally been an essential part of clinical care to assess health risks. However, declining sequencing costs have precipitated a shift towards genomics-first approaches in population screening programs, with less emphasis on family history assessment. We evaluated the utility of family history for genomic sequencing selection. MethodsWe analysed whole genome sequences of 1750 healthy research participants, with and without preselection based on standardised family history collection, screening 95 cancer genes. ResultsThe frequency of likely pathogenic/ pathogenic (LP/P) variants in 884 participants with no family history available (FH not available group) (2%) versus 866 participants with family history available (FH available group) (3.1%) was not significant (p=0.158). However, within the FH available group, amongst 73 participants with an increased family history cancer risk (increased FH risk), 1 in 7 participants carried a LP/P variant inferring a six-fold increase compared with 1 in 47 participants assessed at average family history cancer risk (average FH risk) and a seven-fold increase compared to the FH not available group. The enrichment was further pronounced (up to 18-fold) when assessing the 25 cancer genes in the ACMG 59-gene panel. Furthermore, 63 participants had an increased family history cancer risk in absence of an apparent LP/P variant. ConclusionOur findings show that systematic family history collection remains critical for health risk assessment, providing important actionable data and augmenting the yield from genomic data. Family history also highlights the potential impact of additional hereditary, environmental and behavioural influences not reflected by genomic sequencing.

genomics↗

bioRxiv2018

Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we developed a quantitative metabolomics method to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors.\n\nImpact StatementNutrient availability is an important tumor microenvironmental factor that impacts cancer cell biology; we developed methods to measure nutrients available to tumor cells and characterized factors that influence tumor nutrient availability.

cancer biology↗

bioRxiv2018

Brd4 and P300 regulate zygotic genome activation through histone acetylation

The awakening of the zygote genome, signaling the transition from maternal transcriptional control to zygotic control, is a watershed in embryonic development, but the factors and mechanisms controlling this transition are still poorly understood. By combining CRISPR-Cas9-mediated live imaging of the first transcribed genes (miR-430), chromatin and transcription analysis during zebrafish embryogenesis, we observed that genome activation is gradual and stochastic, and the active state is inherited in daughter cells. We discovered that genome activation is regulated through both translation of maternal mRNAs and the effects of these factors on the chromatin. We show that chemical inhibition of H3K27Ac writer (P300) and reader (Brd4) block genome activation, while induction of a histone acetylation prematurely activates transcription, and restore genome activation in embryos where translation of maternal mRNAs is impaired, demonstrating that they are limiting factors for the activation of the genome. In contrast to current models, we do not observe triggering of genome activation by a reduction of the nuclear-cytoplasmic (N/C) ratio or slower cell division. We conclude that genome activation is controlled by a time-dependent mechanism involving the translation of maternal mRNAs and the regulation of histone acetylation through P300 and Brd4. This mechanism is critical to initiating zygotic development and developmental reprogramming.

developmental biology↗