Biology subjects

Chan, A. H.

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

5 recordsLinked to original sources

bioRxiv2024

Identification and characterization of thiamine analogues with antiplasmodial activity

Thiamine is metabolized into thiamine pyrophosphate (TPP), an essential enzyme cofactor. Previous work has shown that oxythiamine, a thiamine analogue, is metabolized by thiamine pyrophosphokinase (TPK) into oxythiamine pyrophosphate (OxPP) within the malaria parasite Plasmodium falciparum, and then inhibits TPP-dependent enzymes, killing the parasite in vitro and in vivo. To identify a more potent antiplasmodial thiamine analogue, 11 commercially available compounds were tested against P. falciparum and P. knowlesi. Five active compounds were identified, but only N3-pyridyl thiamine (N3PT), a potent transketolase inhibitor and candidate anticancer lead compound, was found to suppress P. falciparum proliferation with an IC50 value 10-fold lower than that of oxythiamine. N3PT was active against P. knowlesi and was >17 times less toxic to human fibroblast, as compared to oxythiamine. Increasing the extracellular thiamine concentration reduced the antiplasmodial activity of N3PT, consistent with N3PT competing with thiamine/TPP. A transgenic P. falciparum line overexpressing TPK was found to be hypersensitized to N3PT. Docking studies showed an almost identical binding mode in TPK between thiamine and N3PT. Furthermore, we show that [3H]thiamine accumulation, resulting from a combination of transport and metabolism, in isolated parasites is reduced by N3PT. Treatment of P. berghei-infected mice with 200 mg/kg/day N3PT reduced their parasitemia, prolonged their time to malaria symptoms, and appeared to be non-toxic to mice. Collectively, our studies are consistent with N3PT competing with thiamine for TPK binding and inhibiting parasite proliferation by reducing TPP production, as well as being converted into a TPP antimetabolite that inhibits TPP-dependent enzymes.

pharmacology and toxicology↗

bioRxiv2023

Caspase-4 dimerisation and D289 auto-processing elicits an interleukin-1β converting enzyme

The non-canonical inflammasome is a signalling complex critical for cell defence against cytosolic Gram-negative bacteria. A key step in the human non-canonical inflammasome pathway involves unleashing the proteolytic activity of caspase-4 within this complex. Caspase-4 induces inflammatory responses by cleaving gasdermin-D (GSDMD) to initiate pyroptosis, although the molecular mechanisms that activate caspase-4 and govern its capacity to cleave substrates are poorly defined. Caspase-11, the murine counterpart of caspase-4, acquires protease activity within the non-canonical inflammasome by forming a dimer that self-cleaves at D285 to directly cleave GSDMD. These cleavage events trigger signalling via the NLRP3-ASC-caspase-1 axis, leading to downstream cleavage of the pro-interleukin-1{beta} (pro-IL-1{beta}) cytokine precursor. Here, we show that caspase-4 first dimerises then self-cleaves at two sites - D270 and D289 - in the interdomain linker to acquire full proteolytic activity, cleave GSDMD and induce cell death. Surprisingly, caspase-4 dimerisation and self-cleavage at D289 generates a caspase-4 p34/p9 protease species that directly cleaves pro-IL-1{beta}, resulting in its maturation and secretion independently of the NLRP3 inflammasome in primary human myeloid and epithelial cells. Our study thus elucidates the key molecular events that underpin signalling by the caspase-4 inflammasome, and identifies IL-1{beta} as a natural substrate of caspase-4.

immunology↗

bioRxiv2022

Orphan C/D box snoRNAs modulate fear-related memory processes in mice

C/D box small nucleolar RNAs (snoRNAs) comprise a class of small noncoding RNAs with important regulatory effects on cellular RNA function. Although it is well established that snoRNAs coordinate the post-transcriptional modification of pre-ribosomal and small nuclear RNAs by 2-O-methylation, which leads to enhanced RNA stability, whether they are necessary for memory-related processes remains relatively unexplored. Using targeted sequencing, we have identified more than 150 C/D box snoRNAs in the prefrontal cortex of male C57BL/6J mice, 31 of which are differentially expressed in response to fear extinction learning. We have also discovered a subset of snoRNAs, including many orphans, that are enriched in the synaptic compartment, including the orphan snoRNA snord64. An extinction learning-induced increase in synapse-enriched snord64 led to increased 2-O-methylation within the 3-UTR of the mRNA encoding the ubiquitin ligase RNF146. This effect was blocked by snord64 knockdown and was accompanied by attenuated forgetting of conditioned fear and the enhanced retrieval of fear extinction memory. Localized activity of orphan snoRNAs therefore represents a novel mechanism associated with fear-related learning and memory. Significance statementWe have discovered a population of experience-dependent small nucleolar RNAs (snoRNAs), and found that several of these are recruited to the synaptic compartment in response to fear extinction learning. In particular, the orphan snoRNA, snord64, drives the methylation of the mRNA encoding the ubiquitin ligase, RNF146, with a reduction in snord64 attenuating forgetting of conditioned fear and enhancing the retrieval of fear extinction memory. This study reveals a new mechanism of gene regulation associated with fear-related memory that involves the activity of synapse-enriched snoRNAs.

neuroscience↗

bioRxiv2021

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein aggregates within processing bodies

RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.

cell biology↗

bioRxiv2020

KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

A vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

biophysics↗