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Steven M. Smith - One of the best experts on this subject based on the ideXlab platform.
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Knockout of the two evolutionarily conserved peroxisomal 3-ketoacyl-CoA thiolases in Arabidopsis recapitulates the abnormal inflorescence meristem 1 phenotype
Journal of experimental botany, 2014Co-Authors: Andrew A. G. Wiszniewski, John D. Bussell, Rowena L. Long, Steven M. SmithAbstract:A specific function for peroxisomal β-oxidation in inflorescence development in Arabidopsis thaliana is suggested by the mutation of the ABNORMAL INFLORESCENCE MERISTEM 1 gene, which encodes one of two peroxisomal multifunctional proteins. Therefore, it should be possible to identify other β-oxidation mutants that recapitulate the aim1 phenotype. Three genes encode peroxisomal 3-ketoacyl-CoA thiolase (KAT) in Arabidopsis. KAT2 and KAT5 are present throughout angiosperms whereas KAT1 is a Brassicaceae-specific duplication of KAT2 expressed at low levels in Arabidopsis. KAT2 plays a dominant role in all known aspects of peroxisomal β-oxidation, including that of fatty acids, pro-auxins, jasmonate precursor oxophytodienoic acid, and trans-cinnamic acid. The functions of KAT1 and KAT5 are unknown. Since KAT5 is conserved throughout vascular plants and expressed strongly in flowers, kat2 KAT5 double mutants were generated. These were slow growing, had abnormally branched inflorescences, and ectopic organ growth. They made viable pollen, but produced no seed indicating that infertility was due to defective gynaecium function. These phenotypes are strikingly similar to those of aim1. KAT5 in the Brassicaceae encodes both cytosolic and peroxisomal proteins and kat2 KAT5 defects could be complemented by the re-introduction of peroxisomal (but not cytosolic) KAT5. It is concluded that peroxisomal KAT2 and KAT5 have partially redundant functions and operate downstream of AIM1 to provide β-oxidation functions essential for inflorescence development and fertility.
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Conservation of two lineages of peroxisomal (Type I) 3-ketoacyl-CoA thiolases in land plants, specialization of the genes in Brassicaceae, and characterization of their expression in Arabidopsis thaliana
Journal of experimental botany, 2012Co-Authors: Andrew A. G. Wiszniewski, Steven M. Smith, John D. BussellAbstract:Arabidopsis thaliana has three genes encoding type I 3-ketoacyl-CoA thiolases (KAT1, KAT2, and KAT5), one of which (KAT5) is alternatively transcribed to produce both peroxisomal and cytosolic proteins. To evaluate the potential importance of these four gene products, their evolutionary history in plants and their expression patterns in Arabidopsis were investigated. Land plants as a whole have gene lineages corresponding to KAT2 and KAT5, implying conservation of distinct functions for these two genes. By contrast, analysis of synteny shows that KAT1 arose by duplication of the KAT2 locus. KAT1 is found in the Brassicaceae family, including in the genera Arabidopsis, Capsella, Thellungiella (=Eutrema) and Brassica, but not in the more distantly related Caricaceae (order Brassicales), or other plants. Gene expression analysis using qRT-PCR and β-glucuronidase reporter genes showed strong expression of KAT2 during germination and in many plant tissues throughout the life cycle, consistent with its observed dominant function in fatty acid β-oxidation. KAT1 was expressed very weakly while KAT5 was most strongly expressed during flower development and in seedlings after germination. Isoform-specific qRT-PCR analysis and promoter β-glucuronidase reporters revealed that the two splicing variants of KAT5 have similar expression profiles. Alternative splicing of KAT5 to produce cytosolic and peroxisomal proteins is specific to and ubiquitous in the Brassicaceae, and possibly had an earlier origin in the order Brassicales. This implies that an additional function for KAT5 arose between 43 and 115 mybp. We speculate that this KAT5 mutation was recruited for a cytosolic function in secondary metabolism.
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Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria
Plant Molecular Biology, 2006Co-Authors: Chris Carrie, Steven M. Smith, Monika W. Murcha, A. Harvey Millar, James WhelanAbstract:The sub-cellular location of enzymes of fatty acid β-oxidation in plants is controversial. In the current debate the role and location of particular thiolases in fatty acid degradation, fatty acid synthesis and isoleucine degradation are important. The aim of this research was to determine the sub-cellular location and hence provide information about possible functions of all the putative 3-ketoacyl-CoA thiolases (KAT) and acetoacetyl-CoA thiolases (ACAT) in Arabidopsis. Arabidopsis has three genes predicted to encode KATs, one of which encodes two polypeptides that differ at the N-terminal end. Expression in Arabidopsis cells of cDNAs encoding each of these KATs fused to green fluorescent protein (GFP) at their C-termini showed that three are targeted to peroxisomes while the fourth is apparently cytosolic. The four KATs are also predicted to have mitochondrial targeting sequences, but purified mitochondria were unable to import any of the proteins in vitro. Arabidopsis also has two genes encoding a total of five different putative ACATs. One isoform is targeted to peroxisomes as a fusion with GFP, while the others display no targeting in vivo as GFP fusions, or import into isolated mitochondria. Analysis of gene co-expression clusters in Arabidopsis suggests a role for peroxisomal KAT2 in β-oxidation, while KAT5 co-expresses with genes of the flavonoid biosynthesis pathway and cytosolic ACAT2 clearly co-expresses with genes of the cytosolic mevalonate biosynthesis pathway. We conclude that KATs and ACATs are present in the cytosol and peroxisome, but are not found in mitochondria. The implications for fatty acid β-oxidation and for isoleucine degradation in mitochondria are discussed.
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Requirement for 3‐ketoacyl‐CoA thiolase‐2 in peroxisome development, fatty acid β‐oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings
The Plant journal : for cell and molecular biology, 2001Co-Authors: Veronique Germain, Elizabeth L Rylott, Tony R Larson, Sarah M Sherson, Nicole Bechtold, Jean Pierre Carde, James H Bryce, Ian A Graham, Steven M. SmithAbstract:3-ketoacyl-CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid β-oxidation. Expression of the Arabidopsis thaliana KAT gene on chromosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in early seedling growth. We identified a T-DNA insertion in this gene which abolishes its expression and eliminates most of the thiolase activity in seedlings. In the homozygous kat2 mutant, seedling growth is dependent upon exogenous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and some peroxisomes contain unusual membrane inclusions. The size and number of plastids and mitochondria are also modified. Long-chain (C16 to C20) fatty acyl-CoAs accumulate in kat2 seedlings, indicating that the mutant lacks long-chain thiolase activity. In addition, extracts from kat2 seedlings have significantly decreased activity with aceto-acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at significant levels during germination and seedling growth, indicating that KAT2 has broad substrate specificity. The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by the CaMV 35S promoter, showing that these enzymes are functionally equivalent, but that expression of the KAT5 gene in seedlings is too low for effective catabolism of TAG. By comparison with glyoxylate cycle mutants, it is concluded that while gluconeogenesis from fatty acids is not absolutely required to support Arabidopsis seedling growth, peroxisomal β-oxidation is essential, which is in turn required for breakdown of TAG in lipid bodies.
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requirement for 3 ketoacyl coa thiolase 2 in peroxisome development fatty acid β oxidation and breakdown of triacylglycerol in lipid bodies of arabidopsis seedlings
Plant Journal, 2001Co-Authors: Veronique Germain, Elizabeth L Rylott, Tony R Larson, Sarah M Sherson, Nicole Bechtold, Jean Pierre Carde, James H Bryce, Ian A Graham, Steven M. SmithAbstract:3-ketoacyl-CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid β-oxidation. Expression of the Arabidopsis thaliana KAT gene on chromosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in early seedling growth. We identified a T-DNA insertion in this gene which abolishes its expression and eliminates most of the thiolase activity in seedlings. In the homozygous kat2 mutant, seedling growth is dependent upon exogenous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and some peroxisomes contain unusual membrane inclusions. The size and number of plastids and mitochondria are also modified. Long-chain (C16 to C20) fatty acyl-CoAs accumulate in kat2 seedlings, indicating that the mutant lacks long-chain thiolase activity. In addition, extracts from kat2 seedlings have significantly decreased activity with aceto-acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at significant levels during germination and seedling growth, indicating that KAT2 has broad substrate specificity. The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by the CaMV 35S promoter, showing that these enzymes are functionally equivalent, but that expression of the KAT5 gene in seedlings is too low for effective catabolism of TAG. By comparison with glyoxylate cycle mutants, it is concluded that while gluconeogenesis from fatty acids is not absolutely required to support Arabidopsis seedling growth, peroxisomal β-oxidation is essential, which is in turn required for breakdown of TAG in lipid bodies.
Michael J. Kerin - One of the best experts on this subject based on the ideXlab platform.
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Quantifying Tip60 (KAT5) stratifies breast cancer
Scientific reports, 2019Co-Authors: Andrew Mcguire, Emer Bourke, Maire-caitlin Casey, Aliaa Shalaby, Olga Kalinina, Catherine Curran, Mark Webber, Grace Callagy, Emma Holian, Michael J. KerinAbstract:Breast cancer is stratified into four distinct clinical subtypes, using three key biomarkers (Her2/Neu gene status, Estrogen and Progesterone receptor status). However, each subtype is a heterogeneous group, displaying significant variation in survival rates and treatment response. New biomarkers are required to provide more precise stratification of breast cancer cohorts to inform personalised treatment options/predict outcomes. Tip60 is a member of the MYST sub-family of histone acetyltransferases (HATs), and is directly involved in genome maintenance, gene regulation and DNA damage response/repair pathways (key chemotherapeutic influencing mechanisms). We aimed to determine if quantifying Tip60 staining patterns improved breast cancer stratification. We defined Tip60 protein in vivo, quantifying location (cytoplasmic, nuclear), percent of cells and staining intensity in a breast cancer tissue microarray (n = 337). A significant association of specific Tip60 staining patterns with breast cancer subtype, ER or PR status and Tumour grade was found. Importantly, low Tip60 mRNA expression correlated with poor overall survival and relapse free survival. We found Tip60 is a biomarker able to stratify breast cancer patients, and low Tip60 expression is a significant risk factor indicating a higher chance of disease reoccurrence. This work highlights Tip60 regulation as a key factor influencing the development of breast cancer.
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Targeting cancer using KAT inhibitors to mimic lethal knockouts.
Biochemical Society transactions, 2016Co-Authors: James A. Brown, Emer Bourke, Leif A. Eriksson, Michael J. KerinAbstract:Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (KAT5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies.
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Irish Area Section (Protein Interactions in Biology) Targeting cancer using KAT inhibitors to mimic lethal knockouts
2016Co-Authors: Emer Bourke, Leif A. Eriksson, Michael J. KerinAbstract:Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (KAT5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies.
Jurg Utzinger - One of the best experts on this subject based on the ideXlab platform.
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estimating sensitivity of the kato katz technique for the diagnosis of schistosoma mansoni and hookworm in relation to infection intensity
PLOS Neglected Tropical Diseases, 2017Co-Authors: Eliezer K Ngoran, Jurg Utzinger, Oliver Barenbold, Jean T Coulibaly, Penelope Vounatsou, Giovanna RasoAbstract:The Kato-Katz technique is the most widely used diagnostic method in epidemiologic surveys and drug efficacy trials pertaining to intestinal schistosomiasis and soil-transmitted helminthiasis. However, the sensitivity of the technique is low, particularly for the detection of light-intensity helminth infections. Examination of multiple stool samples reduces the diagnostic error; yet, most studies rely on a single Kato-Katz thick smear, thus underestimating infection prevalence. We present a model which estimates the sensitivity of the Kato-Katz technique in Schistosoma mansoni and hookworm, as a function of infection intensity for repeated stool sampling and provide estimates of the age-dependent 'true' prevalence. We find that the sensitivity for S. mansoni diagnosis is dominated by missed light infections, which have a low probability to be diagnosed correctly even through repeated sampling. The overall sensitivity strongly depends on the mean infection intensity. In particular at an intensity of 100 eggs per gram of stool (EPG), we estimate a sensitivity of 50% and 80% for one and two samples, respectively. At an infection intensity of 300 EPG, we estimate a sensitivity of 62% for one sample and 90% for two samples. The sensitivity for hookworm diagnosis is dominated by day-to-day variation with typical values for one, two, three, and four samples equal to 50%, 75%, 85%, and 95%, respectively, while it is only weakly dependent on the mean infection intensity in the population. We recommend taking at least two samples and estimate the 'true' prevalence of S. mansoni considering the dependence of the sensitivity on the mean infection intensity and the 'true' hookworm prevalence by taking into account the sensitivity given in the current study.
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quality control in the diagnosis of trichuris trichiura and ascaris lumbricoides using the kato katz technique experience from three randomised controlled trials
Parasites & Vectors, 2015Co-Authors: Marco Albonico, Jurg Utzinger, Jennifer Keiser, Benjamin SpeichAbstract:Background An accurate diagnosis of soil-transmitted helminthiasis is important for individual patient management, for drug efficacy evaluation and for monitoring control programmes. The Kato-Katz technique is the most widely used method detecting soil-transmitted helminth eggs in faecal samples. However, detailed analyses of quality control, including false-positive and faecal egg count (FEC) estimates, have received little attention.
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evaluation of banked urine samples for the detection of circulating anodic and cathodic antigens in schistosoma mekongi and s japonicum infections a proof of concept study
Acta Tropica, 2015Co-Authors: Govert J Van Dam, Jurg Utzinger, Peter Odermatt, Luz P Acosta, Robert Bergquist, Claudia J De Dood, Dieuwke Kornelis, Sinuon MuthAbstract:In Asia, Schistosoma japonicum is the predominant schistosome species, while Schistosoma mekongi is confined to limited foci in Cambodia and Lao People's Democratic Republic. While the People's Republic of China has been successful in controlling schistosomiasis, the disease remains a major public health issue in other areas. In order to prioritise intervention areas, not only accurate diagnosis is important but also other factors, such as practicality, time-efficiency and cost-effectiveness, since they strongly influence the success of control programmes. To evaluate the highly specific urine-based assays for the schistosome circulating cathodic antigen (CCA) and the circulating anodic antigen (CAA), banked urine samples from Cambodia (n=106) and the Philippines (n=43) were examined by the upconverted phosphor lateral flow (UCP-LF) CAA assay and the point-of-care (POC)-CCA urine assay. Based on 250 μl urine samples, UCP-LF CAA sensitivity outcomes surpassed a single stool examination by the Kato-Katz technique. The banked urine samples in the current study did not allow the evaluation of larger volumes, which conceivably should deliver considerably higher readings. The sensitivity of a single urine POC-CCA was in the same order as that of a single Kato-Katz thick smear examination, while the sensitivity approached that of triplicate Kato-Katz when a combination of both CAA and CCA assays was used. The promising results from the current proof-of-concept study call for larger investigations that will determine the accuracy of the urine-based CCA and CAA assays for S. mekongi and S. japonicum diagnosis.
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effect of sampling and diagnostic effort on the assessment of schistosomiasis and soil transmitted helminthiasis and drug efficacy a meta analysis of six drug efficacy trials and one epidemiological survey
Parasitology, 2014Co-Authors: Bruno Levecke, Jurg Utzinger, Stefanie Knopp, Simon Brooker, Russell J Stothard, Jose C Sousafigueiredo, Peter Steinmann, Jozef VercruysseAbstract:It is generally recommended to perform multiple stool examinations in order to improve the diagnostic accuracy when assessing the impact of mass drug administration programmes to control human intestinal worm infections and determining efficacy of the drugs administered. However, the collection and diagnostic work-up of multiple stool samples increases costs and workload. It has been hypothesized that these increased efforts provide more accurate results when infection and drug efficacy are summarized by prevalence (proportion of subjects infected) and cure rate (CR, proportion of infected subjects that become egg-negative after drug administration), respectively, but not when these indicators are expressed in terms of infection intensity and egg reduction rate (ERR). We performed a meta-analysis of six drug efficacy trials and one epidemiological survey. We compared prevalence and intensity of infection, CR and ERR based on collection of one or two stool samples that were processed with single or duplicate Kato-Katz thick smears. We found that the accuracy of prevalence estimates and CR was lowest with the minimal sampling effort, but that this was not the case for estimating infection intensity and ERR. Hence, a single Kato-Katz thick smear is sufficient for reporting infection intensity and ERR following drug treatment.
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accuracy of the kato katz method and formalin ether concentration technique for the diagnosis of clonorchis sinensis and implication for assessing drug efficacy
Parasites & Vectors, 2013Co-Authors: Menbao Qian, Peiling Yap, Yichao Yang, Hai Liang, Zhihua Jiang, Jurg Utzinger, Xiaonong Zhou, Jennifer KeiserAbstract:Background: Clonorchiasis is a chronic neglected disease caused by a liver fluke, Clonorchis sinensis. Chemotherapy is the mainstay of control and treatment efficacy is usually determined by microscopic examination of fecal samples. We assessed the diagnostic accuracy of the Kato-Katz method and the formalin-ether concentration technique (FECT) for C. sinensis diagnosis, and studied the effect of diagnostic approach on drug efficacy evaluation. Methods: Overall, 74 individuals aged ≥18 years with a parasitological confirmed C. sinensis infection at baseline were re-examined 3 weeks after treatment. Before and after treatment, two stool samples were obtained from each participant and each sample was subjected to triplicate Kato-Katz thick smears and a single FECT examination. Results: Thirty-eight individuals were still positive for C. sinensis according to our diagnostic ‘gold’ standard (six Kato-Katz thick smears plus two FECT). Two FECT had a significantly lower sensitivity than six Kato-Katz thick smears (44.7% versus 92.1%; p <0.001). Examination of single Kato-Katz and single FECT considerably overestimated cure rates. Conclusions: In settings where molecular diagnostic assays are absent, multiple Kato-Katz thick smears should be examined for an accurate diagnosis of C. sinensis infection and for assessing drug efficacy against this liver fluke infection.
Emer Bourke - One of the best experts on this subject based on the ideXlab platform.
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Quantifying Tip60 (KAT5) stratifies breast cancer
Scientific reports, 2019Co-Authors: Andrew Mcguire, Emer Bourke, Maire-caitlin Casey, Aliaa Shalaby, Olga Kalinina, Catherine Curran, Mark Webber, Grace Callagy, Emma Holian, Michael J. KerinAbstract:Breast cancer is stratified into four distinct clinical subtypes, using three key biomarkers (Her2/Neu gene status, Estrogen and Progesterone receptor status). However, each subtype is a heterogeneous group, displaying significant variation in survival rates and treatment response. New biomarkers are required to provide more precise stratification of breast cancer cohorts to inform personalised treatment options/predict outcomes. Tip60 is a member of the MYST sub-family of histone acetyltransferases (HATs), and is directly involved in genome maintenance, gene regulation and DNA damage response/repair pathways (key chemotherapeutic influencing mechanisms). We aimed to determine if quantifying Tip60 staining patterns improved breast cancer stratification. We defined Tip60 protein in vivo, quantifying location (cytoplasmic, nuclear), percent of cells and staining intensity in a breast cancer tissue microarray (n = 337). A significant association of specific Tip60 staining patterns with breast cancer subtype, ER or PR status and Tumour grade was found. Importantly, low Tip60 mRNA expression correlated with poor overall survival and relapse free survival. We found Tip60 is a biomarker able to stratify breast cancer patients, and low Tip60 expression is a significant risk factor indicating a higher chance of disease reoccurrence. This work highlights Tip60 regulation as a key factor influencing the development of breast cancer.
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Targeting cancer using KAT inhibitors to mimic lethal knockouts.
Biochemical Society transactions, 2016Co-Authors: James A. Brown, Emer Bourke, Leif A. Eriksson, Michael J. KerinAbstract:Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (KAT5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies.
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Irish Area Section (Protein Interactions in Biology) Targeting cancer using KAT inhibitors to mimic lethal knockouts
2016Co-Authors: Emer Bourke, Leif A. Eriksson, Michael J. KerinAbstract:Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (KAT5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies.
John D. Bussell - One of the best experts on this subject based on the ideXlab platform.
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Knockout of the two evolutionarily conserved peroxisomal 3-ketoacyl-CoA thiolases in Arabidopsis recapitulates the abnormal inflorescence meristem 1 phenotype
Journal of experimental botany, 2014Co-Authors: Andrew A. G. Wiszniewski, John D. Bussell, Rowena L. Long, Steven M. SmithAbstract:A specific function for peroxisomal β-oxidation in inflorescence development in Arabidopsis thaliana is suggested by the mutation of the ABNORMAL INFLORESCENCE MERISTEM 1 gene, which encodes one of two peroxisomal multifunctional proteins. Therefore, it should be possible to identify other β-oxidation mutants that recapitulate the aim1 phenotype. Three genes encode peroxisomal 3-ketoacyl-CoA thiolase (KAT) in Arabidopsis. KAT2 and KAT5 are present throughout angiosperms whereas KAT1 is a Brassicaceae-specific duplication of KAT2 expressed at low levels in Arabidopsis. KAT2 plays a dominant role in all known aspects of peroxisomal β-oxidation, including that of fatty acids, pro-auxins, jasmonate precursor oxophytodienoic acid, and trans-cinnamic acid. The functions of KAT1 and KAT5 are unknown. Since KAT5 is conserved throughout vascular plants and expressed strongly in flowers, kat2 KAT5 double mutants were generated. These were slow growing, had abnormally branched inflorescences, and ectopic organ growth. They made viable pollen, but produced no seed indicating that infertility was due to defective gynaecium function. These phenotypes are strikingly similar to those of aim1. KAT5 in the Brassicaceae encodes both cytosolic and peroxisomal proteins and kat2 KAT5 defects could be complemented by the re-introduction of peroxisomal (but not cytosolic) KAT5. It is concluded that peroxisomal KAT2 and KAT5 have partially redundant functions and operate downstream of AIM1 to provide β-oxidation functions essential for inflorescence development and fertility.
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Conservation of two lineages of peroxisomal (Type I) 3-ketoacyl-CoA thiolases in land plants, specialization of the genes in Brassicaceae, and characterization of their expression in Arabidopsis thaliana
Journal of experimental botany, 2012Co-Authors: Andrew A. G. Wiszniewski, Steven M. Smith, John D. BussellAbstract:Arabidopsis thaliana has three genes encoding type I 3-ketoacyl-CoA thiolases (KAT1, KAT2, and KAT5), one of which (KAT5) is alternatively transcribed to produce both peroxisomal and cytosolic proteins. To evaluate the potential importance of these four gene products, their evolutionary history in plants and their expression patterns in Arabidopsis were investigated. Land plants as a whole have gene lineages corresponding to KAT2 and KAT5, implying conservation of distinct functions for these two genes. By contrast, analysis of synteny shows that KAT1 arose by duplication of the KAT2 locus. KAT1 is found in the Brassicaceae family, including in the genera Arabidopsis, Capsella, Thellungiella (=Eutrema) and Brassica, but not in the more distantly related Caricaceae (order Brassicales), or other plants. Gene expression analysis using qRT-PCR and β-glucuronidase reporter genes showed strong expression of KAT2 during germination and in many plant tissues throughout the life cycle, consistent with its observed dominant function in fatty acid β-oxidation. KAT1 was expressed very weakly while KAT5 was most strongly expressed during flower development and in seedlings after germination. Isoform-specific qRT-PCR analysis and promoter β-glucuronidase reporters revealed that the two splicing variants of KAT5 have similar expression profiles. Alternative splicing of KAT5 to produce cytosolic and peroxisomal proteins is specific to and ubiquitous in the Brassicaceae, and possibly had an earlier origin in the order Brassicales. This implies that an additional function for KAT5 arose between 43 and 115 mybp. We speculate that this KAT5 mutation was recruited for a cytosolic function in secondary metabolism.