11 results on '"Wooi F. Lim"'
Search Results
2. GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain
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Ghulam Hassan Dar, Cláudia C. Mendes, Wei-Li Kuan, Alfina A. Speciale, Mariana Conceição, André Görgens, Inna Uliyakina, Miguel J. Lobo, Wooi F. Lim, Samir EL Andaloussi, Imre Mäger, Thomas C. Roberts, Roger A. Barker, Deborah C. I. Goberdhan, Clive Wilson, and Matthew J. A. Wood
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Science - Abstract
GAPDH is generally considered a housekeeping gene and functions in glycolysis. Here, the authors show that GAPDH has a role in promoting vesicle clustering in endosomes and can load siRNA onto the surface of extracellular vesicles, which can be exploited therapeutically.
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- 2021
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3. Author Correction: GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain
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Ghulam Hassan Dar, Cláudia C. Mendes, Wei-Li Kuan, Alfina A. Speciale, Mariana Conceição, André Görgens, Inna Uliyakina, Miguel J. Lobo, Wooi F. Lim, Samir EL Andaloussi, Imre Mäger, Thomas C. Roberts, Roger A. Barker, Deborah C. I. Goberdhan, Clive Wilson, and Matthew J. A. Wood
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Science - Published
- 2021
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4. GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain
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Roger A. Barker, Clive Wilson, Wooi F. Lim, Alfina A Speciale, Thomas C. Roberts, André Görgens, Samir El Andaloussi, Imre Mäger, Matthew J.A. Wood, Cláudia C. Mendes, Ghulam Hassan Dar, Mariana Conceição, I. Uliyakina, Wei-Li Kuan, Deborah C.I. Goberdhan, Miguel J Lobo, Speciale, Alfina A. [0000-0001-9728-254X], Görgens, André [0000-0001-9198-0857], Lim, Wooi F. [0000-0002-1238-3040], EL Andaloussi, Samir [0000-0003-4468-9113], Mäger, Imre [0000-0003-2242-7227], Roberts, Thomas C. [0000-0002-3313-7631], Goberdhan, Deborah C. I. [0000-0003-0645-6714], Wood, Matthew J. A. [0000-0002-5436-6011], Apollo - University of Cambridge Repository, Speciale, Alfina A [0000-0001-9728-254X], Lim, Wooi F [0000-0002-1238-3040], El Andaloussi, Samir [0000-0003-4468-9113], Roberts, Thomas C [0000-0002-3313-7631], Goberdhan, Deborah CI [0000-0003-0645-6714], Wood, Matthew JA [0000-0002-5436-6011], and Barker, Roger [0000-0001-8843-7730]
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Small interfering RNA ,General Physics and Astronomy ,13 ,14 ,59/5 ,59 ,Drug Delivery Systems ,631/61/391/3932 ,42/89 ,RNA, Small Interfering ,13/89 ,Glyceraldehyde 3-phosphate dehydrogenase ,64 ,Huntingtin Protein ,Multidisciplinary ,biology ,Chemistry ,Vesicle ,article ,food and beverages ,Brain ,Glyceraldehyde-3-Phosphate Dehydrogenases ,Extracellular vesicle ,Cell biology ,Huntington Disease ,64/60 ,Protein Binding ,RNAi therapy ,Science ,Phosphatidylserines ,General Biochemistry, Genetics and Molecular Biology ,38 ,Extracellular Vesicles ,stomatognathic system ,Cell Line, Tumor ,Gene silencing ,Animals ,Humans ,Secretion ,Phosphatidylserine binding ,42 ,fungi ,Mesenchymal Stem Cells ,General Chemistry ,Mice, Inbred C57BL ,Disease Models, Animal ,HEK293 Cells ,biology.protein ,Extracellular signalling molecules ,631/80/86/820 ,Biogenesis ,HeLa Cells - Abstract
Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington’s disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain., GAPDH is generally considered a housekeeping gene and functions in glycolysis. Here, the authors show that GAPDH has a role in promoting vesicle clustering in endosomes and can load siRNA onto the surface of extracellular vesicles, which can be exploited therapeutically.
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- 2022
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- View/download PDF
5. Dystrophin involvement in peripheral circadian SRF signalling
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Wooi F. Lim, Melissa Bowerman, Corinne A. Betts, Lara Cravo, Aarti Jagannath, Tirsa L.E. van Westering, Jinhong Meng, Katarzyna Chwalenia, Russell G. Foster, Jennifer E. Morgan, Carlo Rinaldi, Maria Sofia Falzarano, Elizabeth O’Donovan, Alessandra Ferlini, Graham McClorey, Katharina E. Meijboom, John R. Counsell, Amarjit Bhomra, Matthew J.A. Wood, Michael J. Gait, Amer F. Saleh, and Subhashis Banerjee
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Serum Response Factor ,genetic structures ,Utrophin ,Myoblasts, Skeletal ,Health, Toxicology and Mutagenesis ,Plant Science ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Cell Line ,Dystrophin ,Mice ,Mediator ,Transcription (biology) ,medicine ,Animals ,Circadian rhythm ,Muscular dystrophy ,Research Articles ,Ecology ,biology ,Myogenesis ,Suprachiasmatic nucleus ,musculoskeletal system ,medicine.disease ,eye diseases ,Actins ,Cell biology ,embryonic structures ,cardiovascular system ,biology.protein ,medicine.symptom ,rhoA GTP-Binding Protein ,RC ,Signal Transduction ,Research Article ,Muscle contraction - Abstract
Absence of integral sarcolemmal protein, dystrophin, disrupts the RhoA-actin-SRF cascade in skeletal muscle, with subsequent dysregulation of downstream-SRF circadian targets and circadian rhythm., Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.
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- 2021
6. Biallelic and de novo variants in ATP6V0A1 cause progressive myoclonus epilepsy and developmental and epileptic encephalopathy
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Wooi F. Lim, Richard I. Morimoto, Kenneth H. Fischbeck, Carolina Courage, Forouhan M, Ingo Helbig, Janel O. Johnson, Angelucci F, Laura C. Bott, Ruth Ellerington, Federico Zara, Maria Lieto, Francesco Brancati, Nemeth Ah, Mikko Muona, Matthew J.A. Wood, Pasquale Striano, Alfina A Speciale, Chiara Criscuolo, David Chitayat, Ambre Sala, Samuel F. Berkovic, A. Filla, Carlo Rinaldi, and A E Lehesjoki
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Genetics ,0303 health sciences ,Mutation ,Ataxia ,Endosome ,Autophagy ,Progressive myoclonus epilepsy ,Biology ,medicine.disease_cause ,medicine.disease ,Phenotype ,03 medical and health sciences ,Epilepsy ,0302 clinical medicine ,medicine ,Missense mutation ,medicine.symptom ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
The vacuolar H+-ATPase is a large multi-subunit proton pump, composed of an integral membrane V0 domain, involved in proton translocation, and a peripheral V1 domain, catalysing ATP hydrolysis. This complex is widely distributed on the membrane of various subcellular organelles, such as endosomes and lysosomes, and plays a critical role in cellular processes ranging from autophagy to protein trafficking and endocytosis. Variants in ATP6V0A1, the brain-enriched isoform in the V0 domain, have been recently associated with developmental delay and epilepsy in four individuals. Here we identified 17 individuals from 14 unrelated families with both with new and previously characterised variants in this gene, representing the largest cohort to date. Five affected subjects with biallelic variants in this gene presented with a phenotype of early-onset progressive myoclonus epilepsy with ataxia, while 12 individuals carried de novo missense variants and showed severe developmental and epileptic encephalopathy. The R740Q mutation, which alone accounts for almost 50% of the mutations identified among our cases, leads to failure of lysosomal hydrolysis by directly impairing acidification of the endolysosomal compartment, causing autophagic dysfunction and severe developmental defect in C. elegans. Altogether, our findings further expand the neurological phenotype associated with variants in this gene and provide a direct link with endolysosomal acidification in the pathophysiology of ATP6V0A1-related conditions.
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- 2021
7. Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain
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Merlin Crossley, Wooi F. Lim, Richard C. M. Pearson, Alister P. W. Funnell, Jon Burdach, and Kate G. R. Quinlan
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Vascular Endothelial Growth Factor A ,0301 basic medicine ,Recombinant Fusion Proteins ,Kruppel-Like Transcription Factors ,Biology ,DNA-binding protein ,03 medical and health sciences ,Protein structure ,Recognition sequence ,Genetics ,Humans ,Promoter Regions, Genetic ,Transcription factor ,Zinc finger ,Binding Sites ,Genome, Human ,Gene regulation, Chromatin and Epigenetics ,Zinc Fingers ,Promoter ,DNA ,DNA-binding domain ,Fusion protein ,Chromatin ,Protein Structure, Tertiary ,Cell biology ,DNA-Binding Proteins ,HEK293 Cells ,030104 developmental biology ,Protein Binding ,Transcription Factors - Abstract
Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA binding in vitro, it appears that in vivo FDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Growth Factor-A (VEGF-A) gene promoter. We compared genome-wide occupancy of the KLF3FD-AZF fusion to that observed with AZF. AZF bound to the VEGF-A promoter as predicted, but was also found to occupy approximately 25 000 other sites, a large number of which contained the expected AZF recognition sequence, GCTGGGGGC. Interestingly, addition of the KLF3 FD re-distributes the fusion protein to new sites, with total DNA occupancy detected at around 50 000 sites. A portion of these sites correspond to known KLF3-bound regions, while others contained sequences similar but not identical to the expected AZF recognition sequence. These results show that FDs can influence and may be useful in directing AZF DNA-binding proteins to specific targets and provide insights into how natural transcription factors operate.
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- 2015
8. Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor
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Wooi F. Lim, Richard C. M. Pearson, Alister P. W. Funnell, Merlin Crossley, Ka Sin Mak, Crisbel M. Artuz, Jon Burdach, Lit Yeen Tan, and Beeke Wienert
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Kruppel-Like Transcription Factors ,Biology ,Gene Regulation, Chromatin and Epigenetics ,Cell Line ,Mice ,Consensus Sequence ,Genetics ,Animals ,Promoter Regions, Genetic ,Transcription factor ,Zinc finger ,Zinc finger transcription factor ,Binding Sites ,Base Sequence ,DNA-binding domain ,DNA ,Chromatin ,Cell biology ,Protein Structure, Tertiary ,Gene Expression Regulation ,Mutation ,Chromatin immunoprecipitation ,Corepressor ,Binding domain ,Protein Binding - Abstract
Transcription factors (TFs) are often regarded as being composed of a DNA-binding domain (DBD) and a functional domain. The two domains are considered separable and autonomous, with the DBD directing the factor to its target genes and the functional domain imparting transcriptional regulation. We examined an archetypal zinc finger (ZF) TF, Kruppel-like factor 3 with an N-terminal domain that binds the corepressor CtBP and a DBD composed of three ZFs at its C-terminus. We established a system to compare the genomic occupancy profile of wild-type Kruppel-like factor 3 with two mutants affecting the N-terminal functional domain: a mutant unable to contact the cofactor CtBP and a mutant lacking the entire N-terminal domain, but retaining the ZFs intact. Chromatin immunoprecipitation followed by sequencing was used to assess binding across the genome in murine embryonic fibroblasts. Unexpectedly, we observe that mutations in the N-terminal domain generally reduced binding, but there were also instances where binding was retained or even increased. These results provide a clear demonstration that the correct localization of TFs to their target genes is not solely dependent on their DNA-contact domains. This informs our understanding of how TFs operate and is of relevance to the design of artificial ZF proteins.
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- 2013
9. Differential regulation of the α-globin locus by Krüppel-like factor 3 in erythroid and non-erythroid cells
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Alister P. W. Funnell, Douglas R. Higgs, Wooi F. Lim, Gabriella E. Martyn, Richard C. M. Pearson, Kate G. R. Quinlan, Crisbel M. Artuz, Jon Burdach, Ka Sin Mak, Emma Whitelaw, Douglas Vernimmen, Beeke Wienert, and Merlin Crossley
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Transcription, Genetic ,Kruppel-Like Transcription Factors ,Repressor ,KLF1 ,Biology ,Alpha globin ,Mice ,Erythroid Cells ,alpha-Globins ,Cell Line, Tumor ,hemic and lymphatic diseases ,Animals ,Humans ,Gene silencing ,Promoter Regions, Genetic ,Molecular Biology ,Cells, Cultured ,Regulation of gene expression ,Reporter gene ,KLF3 ,Binding Sites ,Promoter ,Fibroblasts ,Molecular biology ,Globin gene regulation ,Gene Expression Regulation ,Regulatory sequence ,COS Cells ,Transcription factor ,K562 Cells ,Research Article - Abstract
Background: Krüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription. Results: We found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3−/− mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3−/− erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions. Conclusions: These findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression.
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- 2016
10. Loss of Kruppel-like factor 3 (KLF3/BKLF) leads to upregulation of the insulin-sensitizing factor adipolin (FAM132A/CTRP12/C1qdc2)
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Kim S. Bell-Anderson, Hannah R. Nicholas, Hanapi Mat Jusoh, Alexander J. Knights, Nigel Turner, Alister P. W. Funnell, Tiffany Scully, Amanda Sainsbury, Helen Williams, Merlin Crossley, Jon Burdach, Andrew J. Hoy, Ka Sin Mak, Wooi F. Lim, and Richard C. M. Pearson
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medicine.medical_specialty ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Kruppel-Like Transcription Factors ,Adipokine ,Biology ,Mice ,Insulin resistance ,Adipokines ,Downregulation and upregulation ,Internal medicine ,Internal Medicine ,medicine ,Animals ,Promoter Regions, Genetic ,Original Research ,Mice, Knockout ,Adiponectin ,Insulin ,Leptin ,medicine.disease ,Up-Regulation ,Metabolism ,Endocrinology ,Gene Expression Regulation ,Adipogenesis ,KLF3 ,Energy Metabolism - Abstract
Krüppel-like factor 3 (KLF3) is a transcriptional regulator that we have shown to be involved in the regulation of adipogenesis in vitro. Here, we report that KLF3-null mice are lean and protected from diet-induced obesity and glucose intolerance. On a chow diet, plasma levels of leptin are decreased, and adiponectin is increased. Despite significant reductions in body weight and adiposity, wild-type and knockout animals show equivalent energy intake, expenditure, and excretion. To investigate the molecular events underlying these observations, we used microarray analysis to compare gene expression in Klf3+/+ and Klf3−/− tissues. We found that mRNA expression of Fam132a, which encodes a newly identified insulin-sensitizing adipokine, adipolin, is significantly upregulated in the absence of KLF3. We confirmed that KLF3 binds the Fam132a promoter in vitro and in vivo and that this leads to repression of promoter activity. Further, plasma adipolin levels were significantly increased in Klf3−/− mice compared with wild-type littermates. Boosting levels of adipolin via targeting of KLF3 offers a novel potential therapeutic strategy for the treatment of insulin resistance.
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- 2013
11. Loss of Krüppel-Like Factor 3 (KLF3/BKLF) Leads to Upregulation of the Insulin-Sensitizing Factor Adipolin (FAM132A/CTRP12/C1qdc2).
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Bell-Anderson, Kim S., Funnell, Alister P., Williams, Helen, Hanapi Mat Jusoh, Scully, Tiffany, Wooi F. Lim, Burdach, Jon G., Ka Sin Mak, Knights, Alexander J., Hoy, Andrew J., Nicholas, Hannah R., Sainsbury, Amanda, Turner, Nigel, Pearson, Richard C., and Crossley, Merlin
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KRUPPEL-like factors ,OBESITY in animals ,GLUCOSE intolerance ,LABORATORY mice ,LEPTIN ,BODY weight ,GENE expression in mammals ,MESSENGER RNA - Abstract
Krüppel-like factor 3 (KLF3) is a transcriptional regulator that we have shown to be involved in the regulation of adipogenesis in vitro. Here, we report that KLF3-null mice are lean and protected from diet-induced obesity and glucose intolerance. On a chow diet, plasma levels of leptin are decreased, and adiponectin is increased. Despite significant reductions in body weight and adiposity, wild-type and knockout animals show equivalent energy intake, expenditure, and excretion. To investigate the molecular events underlying these observations, we used microarray analysis to compare gene expression in Klf3
+/+ and Klf3-/- tissues. We found that mRNA expression of Fam132a, which encodes a newly identified insulin-sensitizing adipokine, adipolin, is significantly upregulated in the absence of KLF3. We confirmed that KLF3 binds the Fam132a promoter in vitro and in vivo and that this leads to repression of promoter activity. Further, plasma adipolin levels were significantly increased in Klf3-/- mice compared with wild-type littermates. Boosting levels of adipolin via targeting of KLF3 offers a novel potential therapeutic strategy for the treatment of insulin resistance. [ABSTRACT FROM AUTHOR]- Published
- 2013
- Full Text
- View/download PDF
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