24 results on '"Lau HT"'
Search Results
2. The laser-hybrid accelerator for radiobiological applications
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Aymar, G, Becker, T, Boogert, S, Borghesi, M, Bingham, R, Brenner, C, Burrows, PN, Dascalu, T, Ettlinger, OC, Gibson, S, Greenshaw, T, Gruber, S, Gujral, D, Hardiman, C, Hughes, J, Jones, WG, Kirkby, K, Kurup, A, Lagrange, J-B, Long, K, Luk, W, Matheson, J, McKenna, P, Mclauchlan, R, Najmudin, Z, Lau, HT, Parsons, JL, Pasternak, J, Pozimski, J, Prise, K, Puchalska, M, Ratoff, P, Schettino, G, Shields, W, Smith, S, Thomason, J, Towe, S, Weightman, P, Whyte, C, and Xiao, R
- Subjects
Quantitative Biology::Tissues and Organs ,Physics::Medical Physics ,physics.bio-ph ,Physics::Accelerator Physics ,physics.acc-ph - Abstract
The `Laser-hybrid Accelerator for Radiobiological Applications', LhARA, is conceived as a novel, uniquely-flexible facility dedicated to the study of radiobiology. The technologies demonstrated in LhARA, which have wide application, will be developed to allow particle-beam therapy to be delivered in a completely new regime, combining a variety of ion species in a single treatment fraction and exploiting ultra-high dose rates. LhARA will be a hybrid accelerator system in which laser interactions drive the creation of a large flux of protons or light ions that are captured using a plasma (Gabor) lens and formed into a beam. The laser-driven source allows protons and ions to be captured at energies significantly above those that pertain in conventional facilities, thus evading the current space-charge limit on the instantaneous dose rate that can be delivered. The laser-hybrid approach, therefore, will allow the vast ``terra incognita'' of the radiobiology that determines the response of tissue to ionising radiation to be studied with protons and light ions using a wide variety of time structures, spectral distributions, and spatial configurations at instantaneous dose rates up to and significantly beyond the ultra-high dose-rate `FLASH' regime. It is proposed that LhARA be developed in two stages. In the first stage, a programme of in vitro radiobiology will be served with proton beams with energies between 10MeV and 15MeV. In stage two, the beam will be accelerated using a fixed-field accelerator (FFA). This will allow experiments to be carried out in vitro and in vivo with proton beam energies of up to 127MeV. In addition, ion beams with energies up to 33.4MeV per nucleon will be available for in vitro and in vivo experiments. This paper presents the conceptual design for LhARA and the R&D programme by which the LhARA consortium seeks to establish the facility.
- Published
- 2020
3. Automatically early detection of skin cancer: Study based on nueral netwok classification
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Lau, HT and Al-Jumaily, A
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In this paper, an automatically skin cancer classification system is developed and the relationship of skin cancer image across different type of neural network are studied with different types of preprocessing.. The collected images are feed into the system, and across different image processing procedure to enhance the image properties. Then the normal skin is removed from the skin affected area and the cancer cell is left in the image. Useful information can be extracted from these images and pass to the classification system for training and testing. Recognition accuracy of the 3-layers back-propagation neural network classifier is 89.9% and auto-associative neural network is 80.8% in the image database that include dermoscopy photo and digital photo. © 2009 IEEE.
- Published
- 2009
4. A clinicopathological study of non-functioning pituitary neuroendocrine tumours using the World Health Organization 2022 classification.
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Woo CS, Ho RS, Ho G, Lau HT, Fong CH, Chang JY, Leung EK, Tang LC, Ma IK, Lee AC, Lui DT, Woo YC, Chow WS, Leung GK, Tan KC, Lam KS, and Lee CH
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- Humans, Female, Male, Middle Aged, Adult, Aged, Prognosis, Young Adult, Follow-Up Studies, T-Box Domain Proteins metabolism, World Health Organization, Pituitary Neoplasms classification, Pituitary Neoplasms pathology, Pituitary Neoplasms metabolism, Neuroendocrine Tumors pathology, Neuroendocrine Tumors classification, Neuroendocrine Tumors metabolism
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Background: The 2022 World Health Organization (WHO) classification of pituitary neuroendocrine tumour (PitNET) supersedes the previous one in 2017 and further consolidates the role of transcription factors (TF) in the diagnosis of PitNET. Here, we investigated the clinical utility of the 2022 WHO classification, as compared to that of 2017, in a cohort of patients with non-functioning PitNET (NF-PitNET)., Methods: A total of 113 NF-PitNET patients who underwent resection between 2010 and 2021, and had follow-up at Queen Mary Hospital, Hong Kong, were recruited. Surgical specimens were re-stained for the three TF: steroidogenic factor (SF-1), T-box family member TBX19 (TPIT) and POU class 1 homeobox 1 (Pit-1). The associations of different NF-PitNET subtypes with tumour-related outcomes were evaluated by logistic and Cox regression analyses., Results: Based on the 2022 WHO classification, the majority of NF-PitNET was SF-1-lineage tumours (58.4%), followed by TPIT-lineage tumours (18.6%), tumours with no distinct lineage (16.8%) and Pit-1-lineage tumours (6.2%). Despite fewer entities than the 2017 classification, significant differences in disease-free survival were present amongst these four subtypes (Log-rank test p=0.003), specifically between SF-1-lineage PitNET and PitNET without distinct lineage (Log-rank test p<0.001). In multivariable Cox regression analysis, the subtype of PitNET without distinct lineage (HR 3.02, 95% CI 1.28-7.16, p=0.012), together with tumour volume (HR 1.04, 95% CI 1.01-1.07, p=0.017), were independent predictors of a composite of residual or recurrent disease., Conclusion: The 2022 WHO classification of PitNET is a clinically useful TF and lineage-based system for subtyping NF-PitNET with different tumour behaviour and prognosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Woo, Ho, Ho, Lau, Fong, Chang, Leung, Tang, Ma, Lee, Lui, Woo, Chow, Leung, Tan, Lam and Lee.)
- Published
- 2024
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5. Proximity biotinylation to define the local environment of the protein kinase A catalytic subunit in adrenal cells.
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Omar MH, Lauer SM, Lau HT, Golkowski M, Ong SE, and Scott JD
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- Humans, Catalytic Domain, Biotinylation, Cyclic AMP-Dependent Protein Kinases metabolism, Biotin metabolism, Proteomics methods
- Abstract
Mutant protein kinase A catalytic subunit (PKAc) drives adrenal Cushing's syndrome, though its signaling interactions remain unclear. This protocol details steps to use live-cell proximity labeling to identify subcellular compartments and proteins closely associated with variants of PKAc in human adrenal cells. We include instructions for clonal cell line generation, live biotin labeling of proximal proteins, isolation of biotinylated proteins, and sample processing for proteomic analysis using the biotin ligase miniTurbo with wild-type and mutant PKAc.
1 , 2 For complete details on the use and execution of this protocol, please refer to Omar et al. (2022).3 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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6. Multiplexed kinase interactome profiling quantifies cellular network activity and plasticity.
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Golkowski M, Lius A, Sapre T, Lau HT, Moreno T, Maly DJ, and Ong SE
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- Humans, Signal Transduction, Protein Interaction Maps, Neoplasms
- Abstract
Dynamic changes in protein-protein interaction (PPI) networks underlie all physiological cellular functions and drive devastating human diseases. Profiling PPI networks can, therefore, provide critical insight into disease mechanisms and identify new drug targets. Kinases are regulatory nodes in many PPI networks; yet, facile methods to systematically study kinase interactome dynamics are lacking. We describe kinobead competition and correlation analysis (kiCCA), a quantitative mass spectrometry-based chemoproteomic method for rapid and highly multiplexed profiling of endogenous kinase interactomes. Using kiCCA, we identified 1,154 PPIs of 238 kinases across 18 diverse cancer lines, quantifying context-dependent kinase interactome changes linked to cancer type, plasticity, and signaling states, thereby assembling an extensive knowledgebase for cell signaling research. We discovered drug target candidates, including an endocytic adapter-associated kinase (AAK1) complex that promotes cancer cell epithelial-mesenchymal plasticity and drug resistance. Our data demonstrate the importance of kinase interactome dynamics for cellular signaling in health and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)
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- 2023
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7. Mislocalization of protein kinase A drives pathology in Cushing's syndrome.
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Omar MH, Byrne DP, Jones KN, Lakey TM, Collins KB, Lee KS, Daly LA, Forbush KA, Lau HT, Golkowski M, McKnight GS, Breault DT, Lefrançois-Martinez AM, Martinez A, Eyers CE, Baird GS, Ong SE, Smith FD, Eyers PA, and Scott JD
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- Animals, Catalytic Domain genetics, Cyclic AMP-Dependent Protein Kinases metabolism, Hydrocortisone metabolism, Mice, Proteomics, Cushing Syndrome genetics, Cushing Syndrome metabolism
- Abstract
Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing's syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing's mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing's syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing's mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing's variant promotes the transmission of distinct downstream pathogenic signals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2022
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8. Early routine (erCT) versus selective computed tomography (sCT) for acute abdominal pain: A systematic review and meta-analysis of randomised trials.
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Lau HT, Liu W, Lam V, and Pang T
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- Abdominal Pain diagnostic imaging, Abdominal Pain etiology, Humans, Length of Stay, Randomized Controlled Trials as Topic, Tomography, X-Ray Computed, Abdomen, Acute
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Background: There are ongoing controversies about the routine use of computed tomography (CT) in the evaluation of acute abdominal pain (AAP), our study was designed to evaluate the impacts of early routine use CT (erCT) and selective CT (sCT) on clinical outcomes., Methods: We conducted a meta-analysis of randomized trials. We included non-quadrant and non-region-specific studies only. The primary outcomes were the number of correct diagnoses at 24 h, mortality, and length of stay (LOS). The secondary outcomes were the number of corrected diagnoses from an initial misdiagnosis, major changes in management, and non-specific abdominal pain (NSAP)., Results: 6 Studies from 3 RCTs were included, enrolling 570 patients. erCT showed a higher number of correct diagnoses and corrected diagnoses at 24 h, [risk ratio (RR) 1.13, 95% confidence interval (CI) 1.01-1.26, P = 0.03] and [RR 1.36, 95% CI 1.01-1.85, P = 0.04] respectively, and a lower mortality at 6 months [RR 0.36, 95% CI 0.15-0.87, P = 0.02]. However, no differences were shown in LOS [mean difference (MD) -0.65, 95% CI -2.88 - 1.58, P = 0.57], major changes in management [RR 1.45, 95% CI 0.94-2.22, P = 0.09] and NSAP [RR 0.92, 95% CI 0.57-1.50, P = 0.74]., Conclusion: erCT has demonstrated both diagnostic and survival benefits by having more correct diagnoses at 24 h and lower mortality at 6 months. Further study should focus on determining the subpopulation that would most benefit from the potentially differential effects of erCT., (Copyright © 2022 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2022
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9. Pharmacoproteomics Identifies Kinase Pathways that Drive the Epithelial-Mesenchymal Transition and Drug Resistance in Hepatocellular Carcinoma.
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Golkowski M, Lau HT, Chan M, Kenerson H, Vidadala VN, Shoemaker A, Maly DJ, Yeung RS, Gujral TS, and Ong SE
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- Antineoplastic Agents pharmacology, Carcinoma, Hepatocellular pathology, Humans, Liver Neoplasms pathology, Proteomics, Antineoplastic Agents therapeutic use, Carcinoma, Hepatocellular drug therapy, Drug Resistance, Neoplasm drug effects, Epithelial-Mesenchymal Transition genetics, Liver Neoplasms drug therapy, Protein Kinases metabolism
- Abstract
Hepatocellular carcinoma (HCC) is a complex and deadly disease lacking druggable genetic mutations. The limited efficacy of systemic treatments for advanced HCC implies that predictive biomarkers and drug targets are urgently needed. Most HCC drugs target protein kinases, indicating that kinase-dependent signaling networks drive HCC progression. To identify HCC signaling networks that determine responses to kinase inhibitors (KIs), we apply a pharmacoproteomics approach integrating kinome activity in 17 HCC cell lines with their responses to 299 KIs, resulting in a comprehensive dataset of pathway-based drug response signatures. By profiling patient HCC samples, we identify signatures of clinical HCC drug responses in individual tumors. Our analyses reveal kinase networks promoting the epithelial-mesenchymal transition (EMT) and drug resistance, including a FZD2-AXL-NUAK1/2 signaling module, whose inhibition reverses the EMT and sensitizes HCC cells to drugs. Our approach identifies cancer drug targets and molecular signatures of drug response for personalized oncology., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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10. Parallel Chemoselective Profiling for Mapping Protein Structure.
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Potter ZE, Lau HT, Chakraborty S, Fang L, Guttman M, Ong SE, Fowler DM, and Maly DJ
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- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Binding, Competitive, Cysteine chemistry, HEK293 Cells, Humans, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, PTEN Phosphohydrolase chemistry, PTEN Phosphohydrolase genetics, PTEN Phosphohydrolase metabolism, Protein Binding, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors metabolism, Tandem Mass Spectrometry, Ubiquitin chemistry, Ubiquitin genetics, Ubiquitin metabolism, src-Family Kinases genetics, src-Family Kinases metabolism, Peptide Mapping methods, src-Family Kinases antagonists & inhibitors
- Abstract
Solution-based structural techniques complement high-resolution structural data by providing insight into the oft-missed links between protein structure and dynamics. Here, we present Parallel Chemoselective Profiling, a solution-based structural method for characterizing protein structure and dynamics. Our method utilizes deep mutational scanning saturation mutagenesis data to install amino acid residues with specific chemistries at defined positions on the solvent-exposed surface of a protein. Differences in the extent of labeling of installed mutant residues are quantified using targeted mass spectrometry, reporting on each residue's local environment and structural dynamics. Using our method, we studied how conformation-selective, ATP-competitive inhibitors affect the local and global structure and dynamics of full-length Src kinase. Our results highlight how parallel chemoselective profiling can be used to study a dynamic multi-domain protein, and suggest that our method will be a useful addition to the relatively small toolkit of existing protein footprinting techniques., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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11. A-kinase-anchoring protein 1 (dAKAP1)-based signaling complexes coordinate local protein synthesis at the mitochondrial surface.
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Gabrovsek L, Collins KB, Aggarwal S, Saunders LM, Lau HT, Suh D, Sancak Y, Trapnell C, Ong SE, Smith FD, and Scott JD
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- A Kinase Anchor Proteins genetics, Autoantigens genetics, Autoantigens metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Electron Transport Chain Complex Proteins biosynthesis, HEK293 Cells, Humans, Mitochondria genetics, Poly(A)-Binding Protein I genetics, Poly(A)-Binding Protein I metabolism, RNA-Seq, Ribonucleoproteins genetics, Ribonucleoproteins metabolism, SS-B Antigen, A Kinase Anchor Proteins metabolism, Mitochondria metabolism, Mitochondrial Membranes metabolism, Protein Biosynthesis, Second Messenger Systems
- Abstract
Compartmentalization of macromolecules is a ubiquitous molecular mechanism that drives numerous cellular functions. The appropriate organization of enzymes in space and time enables the precise transmission and integration of intracellular signals. Molecular scaffolds constrain signaling enzymes to influence the regional modulation of these physiological processes. Mitochondrial targeting of protein kinases and protein phosphatases provides a means to locally control the phosphorylation status and action of proteins on the surface of this organelle. Dual-specificity protein kinase A anchoring protein 1 (dAKAP1) is a multivalent binding protein that targets protein kinase A (PKA), RNAs, and other signaling enzymes to the outer mitochondrial membrane. Many AKAPs recruit a diverse set of binding partners that coordinate a broad range of cellular processes. Here, results of MS and biochemical analyses reveal that dAKAP1 anchors additional components, including the ribonucleoprotein granule components La-related protein 4 (LARP4) and polyadenylate-binding protein 1 (PABPC1). Local translation of mRNAs at organelles is a means to spatially control the synthesis of proteins. RNA-Seq data demonstrate that dAKAP1 binds mRNAs encoding proteins required for mitochondrial metabolism, including succinate dehydrogenase. Functional studies suggest that the loss of dAKAP1-RNA interactions reduces mitochondrial electron transport chain activity. Hence, dAKAP1 plays a previously unappreciated role as a molecular interface between second messenger signaling and local protein synthesis machinery., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Gabrovsek et al.)
- Published
- 2020
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12. N-glycosylation of α 1D -adrenergic receptor N-terminal domain is required for correct trafficking, function, and biogenesis.
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Janezic EM, Lauer SM, Williams RG, Chungyoun M, Lee KS, Navaluna E, Lau HT, Ong SE, and Hague C
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- Amino Acid Substitution, Endoplasmic Reticulum genetics, Glycosylation, HEK293 Cells, Humans, Protein Domains, Protein Transport genetics, Receptors, Adrenergic, alpha-1 genetics, Endoplasmic Reticulum metabolism, Mutation, Missense, Receptors, Adrenergic, alpha-1 metabolism
- Abstract
G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by post-translational modifications, including N-glycosylation of asparagine residues. α
1D -adrenergic receptors (α1D -ARs) - key regulators of central and autonomic nervous system function - contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α1D -AR between transmembrane domain 2 and 3. Label-free dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α1D -AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α1D- AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α1D -AR N-terminal glycosylation is required for complete translation of nascent, functional receptor.- Published
- 2020
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13. An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma.
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Turnham RE, Smith FD, Kenerson HL, Omar MH, Golkowski M, Garcia I, Bauer R, Lau HT, Sullivan KM, Langeberg LK, Ong SE, Riehle KJ, Yeung RS, and Scott JD
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- A Kinase Anchor Proteins metabolism, Animals, Cell Line, Cell Proliferation, Cyclic AMP-Dependent Protein Kinase Catalytic Subunits genetics, Fetal Proteins genetics, HSP70 Heat-Shock Proteins metabolism, Hepatocytes pathology, Humans, Mice, Models, Theoretical, Molecular Chaperones genetics, Protein Binding, Proto-Oncogene Mas, Recombinant Fusion Proteins genetics, Signal Transduction, Carcinoma, Hepatocellular physiopathology, Cyclic AMP-Dependent Protein Kinase Catalytic Subunits metabolism, Fetal Proteins metabolism, Liver Neoplasms physiopathology, Molecular Chaperones metabolism, Recombinant Fusion Proteins metabolism
- Abstract
Fibrolamellar carcinoma (FLC) is a rare liver cancer. FLCs uniquely produce DNAJ-PKAc, a chimeric enzyme consisting of a chaperonin-binding domain fused to the Cα subunit of protein kinase A. Biochemical analyses of clinical samples reveal that a unique property of this fusion enzyme is the ability to recruit heat shock protein 70 (Hsp70). This cellular chaperonin is frequently up-regulated in cancers. Gene-editing of mouse hepatocytes generated disease-relevant AML12
DNAJ-PKAc cell lines. Further analyses indicate that the proto-oncogene A-kinase anchoring protein-Lbc is up-regulated in FLC and functions to cluster DNAJ-PKAc/Hsp70 sub-complexes with a RAF-MEK-ERK kinase module. Drug screening reveals Hsp70 and MEK inhibitor combinations that selectively block proliferation of AML12DNAJ-PKAc cells. Phosphoproteomic profiling demonstrates that DNAJ-PKAc biases the signaling landscape toward ERK activation and engages downstream kinase cascades. Thus, the oncogenic action of DNAJ-PKAc involves an acquired scaffolding function that permits recruitment of Hsp70 and mobilization of local ERK signaling., Competing Interests: RT, FS, HK, MO, MG, IG, RB, HL, KS, LL, SO, KR, RY, JS No competing interests declared, (© 2019, Turnham et al.)- Published
- 2019
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14. Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins.
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Smith FD, Omar MH, Nygren PJ, Soughayer J, Hoshi N, Lau HT, Snyder CG, Branon TC, Ghosh D, Langeberg LK, Ting AY, Santana LF, Ong SE, Navedo MF, and Scott JD
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- A Kinase Anchor Proteins genetics, Cyclic AMP-Dependent Protein Kinases metabolism, Gene Expression Regulation, Enzymologic, Genome-Wide Association Study, Humans, Membrane Proteins genetics, Models, Molecular, Polymorphism, Single Nucleotide, Protein Binding, Protein Conformation, Protein Isoforms, Protein Transport, A Kinase Anchor Proteins metabolism, Cyclic AMP-Dependent Protein Kinases chemistry, Cyclic AMP-Dependent Protein Kinases genetics, Membrane Proteins metabolism
- Abstract
A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca
2+ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms., Competing Interests: The authors declare no conflict of interest.- Published
- 2018
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15. Crystal Structure of the COMPASS H3K4 Methyltransferase Catalytic Module.
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Hsu PL, Li H, Lau HT, Leonen C, Dhall A, Ong SE, Chatterjee C, and Zheng N
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- Amino Acid Sequence, Animals, Catalytic Domain, Cell Line, Crystallography, X-Ray, DNA-Binding Proteins chemistry, Humans, Insecta, Methylation, Nuclear Proteins chemistry, Protein Domains, Saccharomyces cerevisiae chemistry, Sequence Alignment, Substrate Specificity, Transcription Factors chemistry, Fungal Proteins chemistry, Histone-Lysine N-Methyltransferase chemistry, Histones chemistry, Kluyveromyces chemistry, Saccharomyces cerevisiae Proteins chemistry
- Abstract
The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family., (Published by Elsevier Inc.)
- Published
- 2018
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16. Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells.
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Beltejar MG, Lau HT, Golkowski MG, Ong SE, and Beavo JA
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- Algorithms, Humans, Jurkat Cells, Metabolic Networks and Pathways, 3',5'-Cyclic-AMP Phosphodiesterases antagonists & inhibitors, Phosphoproteins metabolism, T-Lymphocytes metabolism
- Abstract
Specific functions for different cyclic nucleotide phosphodiesterases (PDEs) have not yet been identified in most cell types. Conventional approaches to study PDE function typically rely on measurements of global cAMP, general increases in cAMP-dependent protein kinase (PKA), or the activity of exchange protein activated by cAMP (EPAC). Although newer approaches using subcellularly targeted FRET reporter sensors have helped define more compartmentalized regulation of cAMP, PKA, and EPAC, they have limited ability to link this regulation to downstream effector molecules and biological functions. To address this problem, we have begun to use an unbiased mass spectrometry-based approach coupled with treatment using PDE isozyme-selective inhibitors to characterize the phosphoproteomes of the functional pools of cAMP/PKA/EPAC that are regulated by specific cAMP-PDEs (the PDE-regulated phosphoproteomes). In Jurkat cells we find multiple, distinct PDE-regulated phosphoproteomes that can be defined by their responses to different PDE inhibitors. We also find that little phosphorylation occurs unless at least two different PDEs are concurrently inhibited in these cells. Moreover, bioinformatics analyses of these phosphoproteomes provide insight into the unique functional roles, mechanisms of action, and synergistic relationships among the different PDEs that coordinate cAMP-signaling cascades in these cells. The data strongly suggest that the phosphorylation of many different substrates contributes to cAMP-dependent regulation of these cells. The findings further suggest that the approach of using selective, inhibitor-dependent phosphoproteome analysis can provide a generalized methodology for understanding the roles of different PDEs in the regulation of cyclic nucleotide signaling., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
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17. Isolated unilateral upper alveolar numbness in silent sinus syndrome.
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Lau HT and Lim KH
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- Chronic Disease, Diagnosis, Differential, Endoscopy methods, Enophthalmos etiology, Facial Asymmetry etiology, Humans, Magnetic Resonance Imaging, Male, Maxillary Nerve injuries, Maxillary Sinus abnormalities, Maxillary Sinus diagnostic imaging, Middle Aged, Tomography, X-Ray Computed, Maxillary Sinus surgery, Maxillary Sinusitis diagnosis, Paresthesia, Syndrome
- Abstract
We present a case of a 59-year-old man with left upper alveolar numbness of 2 years' duration in the absence of sinonasal symptoms. On physical examination, he demonstrated mild left facial asymmetry and diminished sensation of his left upper alveolus from the left second upper incisor to first canine. CT imaging revealed chronic sinusitis changes of the left maxillary sinus, with reduced volume and depressed anterior wall. The patient underwent functional endoscopic sinus surgery to re-establish maxillary sinus ventilation. He was noted to have some improvement of his upper alveolar paraesthesia postoperatively. Silent sinus syndrome is part of the spectrum of chronic maxillary atelectasis. In the presented case, chronic osteitic bony sclerosis, as opposed to osteopenic change of the maxillary sinus, was seen. We postulate that bony encasement of the anterior superior alveolar nerve resulted in chronic nerve compression and the patient's unusual symptom of upper alveolar paraesthesia.
- Published
- 2017
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18. Zfp57 mutant ES cell lines directly derived from blastocysts.
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Lau HT, Liu L, and Li X
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- Animals, Blastocyst cytology, Cell Line, Chromosomes genetics, DNA Methylation, DNA-Binding Proteins metabolism, Embryoid Bodies cytology, Embryoid Bodies metabolism, Female, Genomic Imprinting, Genotype, Heterozygote, Male, Metaphase, Mice, Microscopy, Fluorescence, Mouse Embryonic Stem Cells metabolism, Mutagenesis, Reverse Transcriptase Polymerase Chain Reaction, Transcription Factors metabolism, DNA-Binding Proteins genetics, Mouse Embryonic Stem Cells cytology
- Abstract
Zfp57 is a master regulator of genomic imprinting in mouse embryos. To further test its functions, we have derived multiple Zfp57 mutant ES clones directly from mouse blastocysts. Indeed, we found DNA methylation imprint was lost at most examined imprinting control regions in these Zfp57 mutant ES clones, similar to what was observed in Zfp57 mutant embryos in the previous studies. This result indicates that these blastocyst-derived Zfp57 mutant ES clones can be employed for functional analyses of Zfp57 in genomic imprinting., (Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
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19. Derivation of hybrid ES cell lines from two different strains of mice.
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Lau HT, Liu L, Ray C, Bell FT, and Li X
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- Animals, Blastocyst cytology, Cell Line, DNA Methylation, Embryoid Bodies cytology, Embryonic Stem Cells metabolism, Genotype, Heterozygote, Mice, Mice, Inbred DBA, Microscopy, Fluorescence, Real-Time Polymerase Chain Reaction, Transcription Factors genetics, Transcription Factors metabolism, Embryonic Stem Cells cytology
- Abstract
Parental origin-dependent expression of the imprinted genes is essential for mammalian development. Zfp57 maintains genomic imprinting in mouse embryos and ES cells. To examine the allelic expression patterns of the imprinted genes in ES cells, we obtained multiple hybrid ES clones that were directly derived from the blastocysts generated from the cross between mice on two different genetic backgrounds. The blastocyst-derived ES clones displayed largely intact DNA methylation imprint at the tested imprinted regions. These hybrid ES clones will be useful for future studies to examine the allelic expression of the imprinted genes in ES cells and their differentiated progeny., (Copyright © 2016. Published by Elsevier B.V.)
- Published
- 2016
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20. Efficient translation of Dnmt1 requires cytoplasmic polyadenylation and Musashi binding elements.
- Author
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Rutledge CE, Lau HT, Mangan H, Hardy LL, Sunnotel O, Guo F, MacNicol AM, Walsh CP, and Lees-Murdock DJ
- Subjects
- Animals, Base Sequence, Blotting, Southern, Blotting, Western, Chickens, Cytoplasm metabolism, DNA (Cytosine-5-)-Methyltransferase 1, DNA (Cytosine-5-)-Methyltransferases metabolism, DNA Primers genetics, Genetic Vectors genetics, HeLa Cells, Humans, Immunoprecipitation, Mice, Molecular Sequence Data, Nerve Tissue Proteins metabolism, Oocytes growth & development, Oocytes metabolism, RNA-Binding Proteins metabolism, Rats, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Xenopus, Zebrafish, DNA (Cytosine-5-)-Methyltransferases genetics, Nerve Tissue Proteins genetics, Phylogeny, Polyadenylation genetics, Protein Biosynthesis genetics, RNA-Binding Proteins genetics
- Abstract
Regulation of DNMT1 is critical for epigenetic control of many genes and for genome stability. Using phylogenetic analysis we characterized a block of 27 nucleotides in the 3'UTR of Dnmt1 mRNA identical between humans and Xenopus and investigated the role of the individual elements contained within it. This region contains a cytoplasmic polyadenylation element (CPE) and a Musashi binding element (MBE), with CPE binding protein 1 (CPEB1) known to bind to the former in mouse oocytes. The presence of these elements usually indicates translational control by elongation and shortening of the poly(A) tail in the cytoplasm of the oocyte and in some somatic cell types. We demonstrate for the first time cytoplasmic polyadenylation of Dnmt1 during periods of oocyte growth in mouse and during oocyte activation in Xenopus. Furthermore we show by RNA immunoprecipitation that Musashi1 (MSI1) binds to the MBE and that this element is required for polyadenylation in oocytes. As well as a role in oocytes, site-directed mutagenesis and reporter assays confirm that mutation of either the MBE or CPE reduce DNMT1 translation in somatic cells, but likely act in the same pathway: deletion of the whole conserved region has more severe effects on translation in both ES and differentiated cells. In adult cells lacking MSI1 there is a greater dependency on the CPE, with depletion of CPEB1 or CPEB4 by RNAi resulting in substantially reduced levels of endogenous DNMT1 protein and concurrent upregulation of the well characterised CPEB target mRNA cyclin B1. Our findings demonstrate that CPE- and MBE-mediated translation regulate DNMT1 expression, representing a novel mechanism of post-transcriptional control for this gene.
- Published
- 2014
- Full Text
- View/download PDF
21. Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain.
- Author
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Zuo X, Sheng J, Lau HT, McDonald CM, Andrade M, Cullen DE, Bell FT, Iacovino M, Kyba M, Xu G, and Li X
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, DNA Modification Methylases genetics, Embryonic Stem Cells cytology, Mice, Mice, Mutant Strains, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Mutation, Nuclear Proteins genetics, Nuclear Proteins metabolism, Repressor Proteins genetics, Tripartite Motif-Containing Protein 28, DNA Methylation physiology, DNA Modification Methylases metabolism, Embryonic Stem Cells metabolism, Genomic Imprinting physiology, Repressor Proteins metabolism, Zinc Fingers
- Abstract
Previously, we discovered that ZFP57 is a maternal-zygotic effect gene, and it maintains DNA methylation genomic imprint at multiple imprinted regions in mouse embryos. Despite these findings, it remains elusive how DNA methyltransferases are targeted to the imprinting control regions to initiate and maintain DNA methylation imprint. To gain insights into these essential processes in genomic imprinting, we examined how ZFP57 maintains genomic DNA methylation imprint in mouse embryonic stem (ES) cells. Here we demonstrate that the loss of ZFP57 in mouse ES cells led to a complete loss of genomic DNA methylation imprint at multiple imprinted regions, similar to its role in mouse embryos. However, reintroduction of ZFP57 into Zfp57-null ES cells did not result in reacquisition of DNA methylation imprint, suggesting that the memory for genomic imprinting had been lost or altered in Zfp57-null ES cells in culture. Interestingly, ZFP57 and DNA methyltransferases could form complexes in the presence of KAP1/TRIM28/TIF1β when co-expressed in COS cells. We also found that the wild-type exogenous ZFP57 but not the mutant ZFP57 lacking the KRAB box that interacts with its co-factor KAP1/TRIM28/TIF1β could substitute for the endogenous ZFP57 in maintaining the DNA methylation imprint in ES cells. These results suggest that ZFP57 may recruit DNA methyltransferases to its target regions to maintain DNA methylation imprint, and this interaction is likely facilitated by KAP1/TRIM28/TIF1β.
- Published
- 2012
- Full Text
- View/download PDF
22. DNA methyltransferase loading, but not de novo methylation, is an oocyte-autonomous process stimulated by SCF signalling.
- Author
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Lees-Murdock DJ, Lau HT, Castrillon DH, De Felici M, and Walsh CP
- Subjects
- Animals, DNA (Cytosine-5-)-Methyltransferase 1, DNA Methylation, Female, Growth Hormone, Inhibitor of Apoptosis Proteins metabolism, Mice, Oogenesis, Ovarian Follicle metabolism, Transcription, Genetic, DNA (Cytosine-5-)-Methyltransferases metabolism, Oocytes growth & development, Signal Transduction, Stem Cell Factor metabolism
- Abstract
Epigenetic reprogramming occurs during oocyte growth in mice, a stage where a number of important events are occurring, including transcription of maternal mRNAs for storage in the mature egg, global transcriptional silencing and the acquisition of meiotic competence. Oocyte growth occurs in conjunction with follicular development over a period of many days. The signals involved in initiating different stages in oocyte and follicular development and the concurrent epigenetic changes are poorly understood. Here we examine the role of stem cell factor (SCF or Kit ligand) on the early- to mid-stages of oocyte growth and on DNA methyltransferase expression and function using a one-step in vitro culture system. Our results show that SCF promotes early oocyte growth and development to the multilaminar follicle stage. Oocyte growth is sufficient to trigger transcription of Dnmt1 and Dnmt3L from dedicated oocyte promoters, and we show that eggs undergoing growth in the absence of follicle development in Foxo3 mutants show elevated levels of Dnmt1. The methyltransferase proteins undergo sequential relocalisation in the oocyte, with DNMT1 being exported from the nucleus at the bilaminar follicle stage, while DNMT3A is transported into the nucleus at the multilaminar stage, indicating an important role for trafficking in controlling imprinting. SCF is thought to signal partly through the phophostidylinositol 3 (PI3) kinase pathway: inhibiting this path was previously shown to prevent FOXO3 nuclear export and we could show here that it also prevented DNMT1 export. Some oocytes reached full size (70 microM) in this in vitro system, but no secondary follicles were formed, most likely due to failure of the thecal layer to form properly. De novo methylation of imprinted genes was seen in some oocyte cultures, with methylation levels being highest for Snrpn and Igf2r which are methylated early in vivo, while Peg1, which is methylated late, showed little or no methylation. SCF treatment did not increase the number of cultures showing methylation. We saw no evidence for de novo methylation of IAP repeats in our cultures. These results suggest that while methyltransferase loading is triggered by oocyte growth, in which SCF plays an important role, complete methylation probably requires progression to the secondary follicle stage and is unlikely to be affected by SCF.
- Published
- 2008
- Full Text
- View/download PDF
23. FasL--too much of a good thing? Transplanted grafts of pancreatic islet cells engineered to express Fas ligand are destroyed not protected by the immune system.
- Author
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Lau HT and Stoeckert CJ
- Subjects
- Animals, Cell Transplantation, Diabetes Mellitus, Experimental immunology, Fas Ligand Protein, Genetic Engineering, Islets of Langerhans cytology, Membrane Glycoproteins genetics, Mice, Mice, Transgenic, Neutrophils immunology, Islets of Langerhans immunology, Membrane Glycoproteins immunology, Transplantation Immunology
- Published
- 1997
- Full Text
- View/download PDF
24. Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice.
- Author
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Lau HT, Yu M, Fontana A, and Stoeckert CJ Jr
- Subjects
- Animals, Apoptosis, Cell Transplantation, Coculture Techniques, Diabetes Mellitus, Experimental surgery, Fas Ligand Protein, Genetic Engineering, Graft Survival, Ligands, Mice, Mice, Inbred A, Mice, Inbred C3H, Mice, Inbred C57BL, Muscle Fibers, Skeletal transplantation, Recombinant Proteins biosynthesis, T-Lymphocytes cytology, T-Lymphocytes immunology, Transfection, Transplantation, Heterotopic, Transplantation, Homologous, Graft Rejection prevention & control, Islets of Langerhans Transplantation, Membrane Glycoproteins biosynthesis, Muscle Fibers, Skeletal cytology, Muscle Fibers, Skeletal metabolism
- Abstract
Allogeneic transplantation of islets of Langerhans was facilitated by the cotransplantation of syngeneic myoblasts genetically engineered to express the Fas ligand (FasL). Composite grafting of allogeneic islets with syngeneic myoblasts expressing FasL protected the islet graft from immune rejection and maintained normoglycemia for more than 80 days in mice with streptozotocin-induced diabetes. Graft survival was not prolonged with composite grafts of unmodified myoblasts or Fas-expressing myoblasts. Islet allografts transplanted separately from FasL-expressing myoblasts into the contralateral kidney were rejected, as were similarly transplanted third-party thyroid allografts. Thus, the FasL signal provided site- and immune-specific protection of islet allografts.
- Published
- 1996
- Full Text
- View/download PDF
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