Luciferase protection against proteolytic degradation: A key for improving signal in nano-system biology

Ataei, Farangis; Hosseinkhani, Saman; Khajeh, Khosro

doi:10.1016/j.jbiotec.2009.08.015

Cited by 11 publications

(4 citation statements)

References 38 publications

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“…The rapid decreases in luciferase activity during drug exposure could be construed as a cellular stress response in which proteolysis and amino acid release and/or selective translational inhibition is used to alter the proteome of the parasite [25,26]. Luciferase is particularly known to be susceptible to proteolytic degradation [27]. Nonetheless, the activity decrease was not affected by proteasome inhibitors, even though proteasomes are principally responsible for cytoplasmic protein turnover and homeostasis in mammalian cells [26].…”

Section: Discussionmentioning

confidence: 99%

ATP and luciferase assays to determine the rate of drug action in in vitro cultures of Plasmodium falciparum

Khan

Brummelen

Parkinson

et al. 2012

Malar J

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BackgroundKnowledge of the rate of action of compounds against cultured malaria parasites is required to determine the optimal time-points for drug mode of action studies, as well as to predict likely in vivo parasite clearance rates in order to select optimal hit compounds for further development. In this study, changes in parasite ATP levels and transgenic luciferase reporter activity were explored as means to detect drug-induced stress in cultured parasites.MethodsIn vitro cultures of Plasmodium falciparum 3D7 wild-type or firefly luciferase-expressing parasites were incubated with a panel of six anti-malarial compounds for 10 hours and parasite ATP levels or luciferase activity determined at two-hour intervals using luminescence-based reagents. For comparative purposes, parasite morphology changes were evaluated by light microscopy, as well as the extent to which parasites recover after 48 hours from a six-hour drug treatment using a parasite lactate dehydrogenase assay.ResultsChanges in parasite ATP levels displayed three phenotypes: mild or no change (chloroquine, DFMO); 2–4 fold increase (mefloquine, artemisinin); severe depletion (ritonavir, gramicidin). The respective phenotypes and the rate at which they manifested correlated closely with the extent to which parasites recovered from a six-hour drug treatment (with the exception of chloroquine) and the appearance and severity of morphological changes observed by light microscopy. Luciferase activity decreased profoundly in parasites treated with mefloquine, artemisinin and ritonavir (34-67% decrease in 2 hours), while chloroquine and DFMO produced only mild changes over 10 hours. Gramicidin yielded intermediate decreases in luciferase activity.ConclusionsATP levels and luciferase activity respond rapidly to incubation with anti-malarial drugs and provide quantitative read-outs to detect the appearance and magnitude of drug-induced stress in cultured parasites. The correlation between the observed changes and irreversible parasite toxicity is not yet sufficiently clear to predict clinical clearance rates, but may be useful for ranking compounds against each other and standard drugs vis-à-vis rate of action and for determining early time-points for drug mode of action studies.

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Section: Discussionmentioning

confidence: 99%

ATP and luciferase assays to determine the rate of drug action in in vitro cultures of Plasmodium falciparum

Khan

Brummelen

Parkinson

et al. 2012

Malar J

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“…Western blot analysis confirmed that full‐length probes can be detected as major bands in expected molecular weight (Appendix Fig S1 ). One exception that YFP‐FKBP‐Luciferase displayed few degraded forms in lower molecular weight (Appendix Fig S1 ), probably due to previously reported proteolytic degradation (Ataei et al , 2009 ). Based on the size measurement using size exclusion chromatography, full‐length β‐Gal and three truncated β‐Gal proteins form hexamer (YFP‐FKBP‐β‐Gal: 659 kDa) and dimer (YFP‐FKBP‐β‐Gals: 230 kDa; YFP‐FKBP‐β‐Galm: 262 kDa; YFP‐FKBP‐∆Nβ‐Gal: 322 kDa), respectively.…”

Section: Resultsmentioning

confidence: 87%

Actin filaments form a size‐dependent diffusion barrier around centrosomes

et al. 2022

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The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that it may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we find that centrosomes are highly accessible to soluble molecules with a Stokes radius of less than 5.8 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of this barrier is tightly regulated by branched actin filaments outside of centrosomes and it decreases during anaphase when branched actin temporally increases. The actinbased diffusion barrier gates microtubule nucleation by interfering with c-tubulin ring complex recruitment. We propose that actin filaments spatiotemporally constrain protein complexes at centrosomes in a size-dependent manner.

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“…After the incubation of the remote-loaded DPPC liposomes with CsPLA 2 (7 nM) for varying times, luciferase was added to the reaction mixture and the luminescence resulting from luciferase-luciferin raction was measured in order to follow CsPLA 2 -triggered luciferin release. The luminescent signal was only measured for a short time (5 s) at 37 C to avoid possible proteolysis of luciferase enzyme over time (Ataei et al, 2009). Unlike unloaded DPPC liposomes, which were quickly degraded by sPLA 2 (see Figs.…”

Section: Resultsmentioning

confidence: 99%