Functional role of the conserved i-helix residue I346 in CYP5A1–Nanodiscs

Meling, Daryl D.; Zelasko, Susan; Kambalyal, Amogh; Roy, Jahnabi; Das, Aditi

doi:10.1016/j.bpc.2015.03.002

Cited by 6 publications

(6 citation statements)

References 35 publications

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“…While the functional significance of these I-helix features of CYP20A1 is unknown, it is notable that the presence of an Ile residue rather than Thr at the 5 th position of the I-helix motif also occurs in human CYP5A1 (thromboxane synthase). Reverse-engineering experiments with human CYP5A1 have demonstrated a specific role for the Ile residue in keeping rates of product formation by the enzyme at low levels (Meling et al, 2015). The tight control of catalysis by CYP5A1 suggests that the Ile in CYP20A1 could perhaps act similarly to limit product formation by this enzyme.…”

Section: Discussionmentioning

confidence: 99%

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

Lemaire

Kubota

O'Meara

et al. 2016

Toxicology and Applied Pharmacology

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Cytochrome P450 (CYP) enzymes for which there is no functional information are considered “orphan” CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including liver, heart, gonads, spleen and brain, as well as eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to “deorphanization”, that is, identifying CYP20A1 functions and its roles in health and disease.

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Section: Discussionmentioning

confidence: 99%

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

Lemaire

Kubota

O'Meara

et al. 2016

Toxicology and Applied Pharmacology

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“…371 The effect of mutations at position I346 on the I-helix, which corresponds to highly conserved threonine (T252) in the widely studied bacterial CYP101A1, was also probed using I346T and I346S mutants, and variations in the spectral response and in binding affinity were reported. Cyanide binding was also used with CYP5A1, as with CYP2J2, to probe substrate binding.…”

Section: Application Of Nanodiscs In Functional Studies Of Membranmentioning

confidence: 99%

“…417 A nonclassical cytochrome P450, the human thromboxane synthase, CYP5A1, was also incorporated in nanodiscs and the binding of two substrate analogs were studied using optical spectroscopy. 418 The effect of mutations at position I346 on the I-helix, which corresponds to highly conserved threonine (T252) in the widely studied bacterial CYP101A1, was also probed using I346T and I346S mutants, and variations in the spectral response and in binding affinity were reported. Cyanide binding was also used with CYP5A1, as with CYP2J2, to probe substrate binding.…”

Section: Chemical Reviewsmentioning

confidence: 99%

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Nanodiscs in Membrane Biochemistry and Biophysics

2017

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Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system which provides control over size, composition and specific functional modifications on nanometer scale. In this review we attempted to combine a comprehensive list of various applications of Nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by Nanodiscs for structural and mechanistic studies of membrane proteins.

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“…Common lipid compositions chosen for nanodiscs include: solely the zwitterionic phospholipid POPC, a mixture of POPC and the anionic phospholipid POPS, or a mixture of POPC and the anionic phospholipid POPG . In an investigation of the receptor histidine kinase AgrC, Wang et al .…”

Section: Biophysical Studies Of Membrane Proteins In Nanodiscsmentioning

confidence: 99%

Recent advances in nanodisc technology for membrane protein studies (2012–2017)

et al. 2017

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Historically, progress in membrane protein research has been hindered due to solubility issues. The introduction of biomembrane mimetics has since stimulated the field’s momentum. One mimetic, the nanodisc, has proved to be an exceptional system for solubilizing membrane proteins. Herein, we critically evaluate the advantages and imperfections from employing nanodiscs in biophysical and biochemical studies. Specifically, we examine the techniques that have been modified to study membrane proteins in nanodiscs. Techniques discussed include fluorescence microscopy, solution state/solid state NMR, electron microscopy, SAXS, and several mass spectroscopy methods. Newer techniques such as SPR, charge sensitive optical detection, and scintillation proximity assays are also reviewed. Lastly, we cover nanodiscs advancing nanotechnology through nanoplasmonic biosensing, lipoprotein-nanoplatelets, and sortase-mediated labeling of nanodiscs.

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Functional role of the conserved i-helix residue I346 in CYP5A1–Nanodiscs

Cited by 6 publications

References 35 publications

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

Nanodiscs in Membrane Biochemistry and Biophysics

Recent advances in nanodisc technology for membrane protein studies (2012–2017)

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