Perturbation of Dopamine Metabolism by 3-Amino-2-(4Ј- halophenyl)propenes Leads to Increased Oxidative Stress and Apoptotic SH-SY5Y Cell Death

ABSTRACT We have recently characterized a series of 3-amino-2-phenylpropene (APP) derivatives as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) that have been previously characterized as potent irreversible dopamine-␤-monooxygenase (D␤M) and monoamine oxidase (MAO) inhibitors. Halogen substitution on the 4Ј-position of the aromatic ring gradually increases VMAT inhibition potency from 4Ј-F to 4Ј-I, parallel to the hydrophobicity of the halogen. We show that these derivatives are taken up into both neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently through passive diffusion. Uptake rates increased according to the hydrophobicity of the 4Ј-substituent. More importantly, these derivatives are highly toxic to human neuroblastoma SH-SY5Y but not toxic to M-1, Hep G2, or human embryonic kidney 293 non-neuronal cells at similar concentrations. They drastically perturb dopamine (DA) uptake and metabolism in SH-SY5Y cells under sublethal conditions and are able to deplete both vesicular and cytosolic catecholamines in a manner similar to that of amphetamines. In addition, 4Ј-IAPP treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants ␣-tocopherol, N-acetyl-L-cysteine and GSH, but not by the nonspecific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. DNA fragmentation analysis further supports that cell death is probably due to a caspase-independent ROSmediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4Ј-halo APP derivatives causes increased oxidative stress, leading to apoptotic cell death. Oxidative stress in the central and peripheral nervous systems plays a significant role in neurodegenerative disorders, aging, and the toxicity of a large number of neurotoxins Numerous studies indicate that efficient uptake, biosynthetic conversion, and storage of catecholamines in vesicles are mandatory for proper functioning of catecholaminergic neurons. The vital proteins responsible, including vesicular H ϩ -ATPase (V-H ϩ -ATPase), cytochrome b 561 (b 561 ), dopamine-␤-monooxygenase (D␤M), and vesicular monoamine transporter (VMAT), have been well characterized (Beer

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast

Acknowledgments We thank Rebecca Shapiro for creating CaLC1819, CaLC1855 and CaLC1875, Gillian Milne for help with EM, Aaron Mitchell for generously providing the transposon insertion mutant library, Jesus Pla for generously providing the hog1 hst7 mutant, and Cathy Collins for technical assistance.Peer reviewedPublisher PD

A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy

Hybridization and adaptive evolution of diverse Saccharomyces species for cellulosic biofuel production

Additional file 15. Summary of whole genome sequencing statistics

Identification of sumoylation targets, combined with inactivation of SMT3, reveals the impact of sumoylation upon growth, morphology, and stress resistance in the pathogen Candida albicans

Peer reviewedPublisher PD

Molecular and physiological basis of Saccharomyces cerevisiae tolerance to adverse lignocellulose-based process conditions

Lignocellulose-based biorefineries have been gaining increasing attention to substitute current petroleum-based refineries. Biomass processing requires a pretreatment step to break lignocellulosic biomass recalcitrant structure, which results in the release of a broad range of microbial inhibitors, mainly weak acids, furans, and phenolic compounds. Saccharomyces cerevisiae is the most commonly used organism for ethanol production; however, it can be severely distressed by these lignocellulose-derived inhibitors, in addition to other challenging conditions, such as pentose sugar utilization and the high temperatures required for an efficient simultaneous saccharification and fermentation step. Therefore, a better understanding of the yeast response and adaptation towards the presence of these multiple stresses is of crucial importance to design strategies to improve yeast robustness and bioconversion capacity from lignocellulosic biomass. This review includes an overview of the main inhibitors derived from diverse raw material resultants from different biomass pretreatments, and describes the main mechanisms of yeast response to their presence, as well as to the presence of stresses imposed by xylose utilization and high-temperature conditions, with a special emphasis on the synergistic effect of multiple inhibitors/stressors. Furthermore, successful cases of tolerance improvement of S. cerevisiae are highlighted, in particular those associated with other process-related physiologically relevant conditions. Decoding the overall yeast response mechanisms will pave the way for the integrated development of sustainable yeast cell--based biorefineries.This study was supported by the Portuguese Foundation for Science and Technology (FCT) by the strategic funding of UID/BIO/04469/2013 unit, MIT Portugal Program (Ph.D. grant PD/BD/128247/ 2016 to Joana T. Cunha), Ph.D. grant SFRH/BD/130739/2017 to Carlos E. Costa, COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004), YeasTempTation (ERA-IB-2-6/0001/2014), and MultiBiorefinery project (POCI-01-0145-FEDER-016403). Funding by the Institute for Bioengineering and Biosciences (IBB) from FCT (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) was also receiveinfo:eu-repo/semantics/publishedVersio