ORCID Profile
0000-0003-0486-284X
Current Organisations
Institute for Developmental Research, Aichi Developmental Disability Center
,
Macquarie University
,
University of Technology Sydney
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The University of Notre Dame Australia - Sydney Campus Broadway
,
Illumina Inc
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Publisher: Elsevier BV
Date: 12-2007
Publisher: BMJ
Date: 06-08-2015
DOI: 10.1136/JMEDGENET-2015-103231
Abstract: Short-chain enoyl-CoA hydratase-ECHS1-catalyses many metabolic pathways, including mitochondrial short-chain fatty acid β-oxidation and branched-chain amino acid catabolic pathways however, the metabolic products essential for the diagnosis of ECHS1 deficiency have not yet been determined. The objective of this report is to characterise ECHS1 and a mild form of its deficiency biochemically, and to determine the candidate metabolic product that can be efficiently used for neonatal diagnosis. We conducted a detailed clinical, molecular genetics, biochemical and metabolic analysis of sibling patients with ECHS1 deficiency. Moreover, we purified human ECHS1, and determined the substrate specificity of ECHS1 for five substrates via different metabolic pathways. Human ECHS1 catalyses the hydration of five substrates via different metabolic pathways, with the highest specificity for crotonyl-CoA and the lowest specificity for tiglyl-CoA. The patients had relatively high (∼7%) residual ECHS1 enzyme activity for crotonyl-CoA and methacrylyl-CoA caused by the compound heterozygous mutations (c.176A>G, (p.N59S) and c.413C>T, (p.A138V)) with normal mitochondrial complex I-IV activities. Affected patients excrete large amounts of N-acetyl-S-(2-carboxypropyl)cysteine, a metabolite of methacrylyl-CoA. Laboratory data and clinical features demonstrated that the patients have a mild form of ECHS1 deficiency harbouring defective valine catabolic and β-oxidation pathways. N-Acetyl-S-(2-carboxypropyl) cysteine level was markedly high in the urine of the patients, and therefore, N-acetyl-S-(2-carboxypropyl)cysteine was regarded as a candidate metabolite for the diagnosis of ECHS1 deficiency. This metabolite is not part of current routine metabolic screening protocols, and its inclusion, therefore, holds immense potential in accurate diagnosis.
Publisher: Wiley
Date: 23-07-2008
DOI: 10.1111/J.1365-2958.2008.06310.X
Abstract: Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans. To investigate the role of phosphatidylinositol-specific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii (PLC1 and PLC2), and created Deltaplc1 and Deltaplc2 deletion mutants. In Deltaplc1, which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37 degrees C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Deltaplc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Deltaplc2, which was as virulent as WT, Deltaplc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25 degrees C, demonstrating that mechanism(s) independent of the 37 degrees C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.
Publisher: Elsevier BV
Date: 10-2020
Publisher: American Society for Microbiology
Date: 03-2006
DOI: 10.1128/EC.5.3.488-498.2006
Abstract: Lipid rafts have been identified in the membranes of mammalian cells, the yeast Saccharomyces cerevisiae , and the pathogenic fungus Candida albicans . Formed by a lateral association of sphingolipids and sterols, rafts concentrate proteins carrying a glycosylphosphatidylinositol (GPI) anchor. We report the isolation of membranes with the characteristics of rafts from the fungal pathogen Cryptococcus neoformans . These characteristics include insolubility in Triton X-100 (TX100) at 4°C, more-buoyant density within a sucrose gradient than the remaining membranes, and threefold enrichment with sterols. The virulence determinant phospholipase B1 (PLB1), a GPI-anchored protein, was highly concentrated in raft membranes and could be displaced from them by treatment with the sterol-sequestering agent methyl-β-cyclodextrin (MβCD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37°C. Treatment of viable cryptococcal cells in suspension with MβCD also released PLB1 protein and enzyme activity, consistent with localization of PLB1 in plasma membrane rafts prior to secretion. The antioxidant virulence factor Cu/Zn superoxide dismutase (SOD1) was concentrated six- to ninefold in raft membrane fractions compared with nonraft membranes, whereas the cell wall-associated virulence factor laccase was not detected in membranes. We hypothesize that raft membranes function to cluster certain virulence factors at the cell surface to allow efficient access to enzyme substrate and/or to provide rapid release to the external environment.
Publisher: Elsevier BV
Date: 05-2009
Publisher: Elsevier BV
Date: 10-2011
Publisher: American Association for Cancer Research (AACR)
Date: 11-2011
DOI: 10.1158/1535-7163.TARG-11-A67
Abstract: Iron is known to be important for cell cycle progression and intracellular iron (Fe) chelation has been shown to down-regulate the expression of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1 (p21) in MCF-7 breast cancer cells [1]. p21 has been demonstrated to have both positive and negative effects on G1/S cell cycle progression and regulate cancer cell apoptosis and senescence [2, 3]. Our preliminary studies have suggested that Fe-depletion affects p21 expression differentially depending upon the cell type. Therefore, understanding the ability of Fe chelators to regulate the expression of p21 differently in cancer cells and the biological consequences thereof may provide an insight into the mechanisms involved in the anti-tumor efficacy of these agents. The current study examined the mechanisms involved in regulating p21 expression by Fe in a number of cancer cell-types with different p53 status. RT-PCR demonstrated an increase in p21 mRNA levels in all cells in response to Fe chelation. Western blotting demonstrated that the expression of p21 after incubation with chelators differed between the cell-types. Down-regulation in p21 protein expression was observed in the MCF-7 breast cancer and SK-N-MC neuroepithelioma cells, while up-regulation of p21 protein was evident in the SK-MEL-28 melanoma, CFPAC-1 pancreatic and LNCaP prostate cancer cell-types. However, the effect of the Fe chelator on p21 expression was not correlated to the p53 status of these cells. Furthermore, we showed that nuclear expression levels of p21 in cancer cells were much higher than cytoplasmic expression after Fe chelation. It has been shown that the variety of physiological roles that p21 plays, including cell cycle arrest, rely on its nuclear localisation [4]. These results were confirmed using cytoplasmic and nuclear fractionation, western analysis and immunofluorescence studies. The roles of MDM2 and Np63 in regulating p21 in response to Fe-depletion were also assessed, as these latter molecules can modulate p21 expression in a p53-independent manner [5, 6]. Results demonstrated that after incubation with Fe chelators, the expression pattern of the p75 isoform of MDM2 (p75MDM2) closely resembled that of p21 in 4 of the 5 cell lines investigated. This indicated that MDM2 may be one pathway through which these drugs regulate p21 expression. There was no significant change in Np63 expression after incubating these cells with Fe chelators, suggesting that the alterations in p21 expression observed were independent of Np63. Further studies are focusing on the possible transcriptional regulation of p21 by Fe-depletion. This is being carried out by using p21 promoter constructs to decipher the region of the p21 promoter that is involved in the Fe-regulated expression of p21. Overall, these studies will provide significant insight into the mechanism of action of Fe chelators at the molecular level. References: 1. Fu, D. and D.R. Richardson, Blood, 2007. 110(2): p. 752–61. 2. Ogryzko, V.V., et. al Mol Cell Biol, 1997. 17(8): p. 4877–82. 3. Gartel, A.L. and S.K. Radhakrishnan, Cancer Res, 2005. 65(10): p. 3980–5. 4. Child, E.S. and D.J. Mann, Cell Cycle, 2006. 5(12): p. 1313–9. 5. Zhang, Z., et al., J Biol Chem, 2004. 279(16): p. 16000–6. 6. Yang, A., et al., Mol Cell, 1998. 2(3): p. 305–16. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics 2011 Nov 12-16 San Francisco, CA. Philadelphia (PA): AACR Mol Cancer Ther 2011 (11 Suppl):Abstract nr A67.
Publisher: American Chemical Society (ACS)
Date: 14-07-2009
DOI: 10.1021/JM900552R
Publisher: Elsevier BV
Date: 10-2006
DOI: 10.1016/J.BBAGEN.2006.07.002
Abstract: Secreted phospholipase B enzymes (PLB1) with high levels of N-linked glycosylation are proven fungal virulence determinants. We demonstrated that removal of N-linked glycans from secreted cryptococcal PLB1 leads to loss of enzyme activity. To determine if in idual N-glycan attachment sites affect secretion of active enzyme, we altered three along the entire length of the protein, by site-directed mutagenesis, namely Asn56, Asn430 and Asn550 to Ala, in wild-type PLB1 (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLB1(GPI-)) that is hypersecreted due to lack of membrane association. Alteration of Asn56 and Asn550 in both PLB1 and PLB1(GPI-) abolished enzyme secretion while alteration of Asn430 reduced secretion by 60%, following expression in Saccharomyces cerevisiae. Reduced secretion coincided with reduced enzyme in membranes and cell walls confirming a reduction in the rate of PLB1 transport to the cell surface. Deglycosylation of cryptococcal PLB1 increased its susceptibility to proteolysis suggesting that the absence of full glycosylation status leads to degradation of unstable PLB1, resulting in reduced traffic through the secretory pathway. We conclude that in idual N-linked glycans are required for optimal transport of PLB1 to the cell surface and optimal secretion of both PLB1 and PLB1(GPI-).
Publisher: Elsevier BV
Date: 09-2018
Location: Japan
Location: Australia
Location: Australia
No related grants have been discovered for Kenichiro Yamada.