Substitution of lysine with glutamic acid at position 193 in bovine CYP11A1 significantly affects protein oligomerization and solubility but not enzymatic activity

Janocha, Simon; Bichet, Andreas; Zöllner, Andy; Bernhardt, Rita

doi:10.1016/j.bbapap.2010.06.002

Cited by 13 publications

(10 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The heterologous expression of CYP11A1 in E coli has been reported to be in the range between 45-55 nmol l 21 culture [27,36], which is rather low. Harnastai et al were able to enhance recombinant CYP11A1 expression up to 430 nmol l 21 culture coexpressing HemA [37].…”

Section: Optimization Of the Expression Of Cyp11a1 In E Colimentioning

confidence: 98%

“…The interaction of Adx and CYP11A1, was assayed with a Biacore3000 system as described previously [27]. The formation of CYP11A1 was injected in concentrations between 5 nM and 7.5 nM in HBS-EP buffer at least three times for each concentration.…”

Section: Optical Biosensor Measurementsmentioning

confidence: 99%

“…The subsequent CYP11A1 expression was performed as described elsewhere [27]. The obtained cell pellet was suspended in 5 ml/g wet mass buffer, consisting of 50 mM potassium phosphate, pH 7.4, 500 mM sodium acetate, 20% glycerol, 1.5% sodium cholate, 1.5% Tween20, 0.1 mM EDTA, 0.1 mM DTT, 0.1 mM PMSF and 10 mM cholesterol.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

See 2 more Smart Citations

Dehydroepiandrosterone Sulfate (DHEAS) Stimulates the First Step in the Biosynthesis of Steroid Hormones

Neunzig

Bernhardt

2014

PLoS ONE

Self Cite

View full text Add to dashboard Cite

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant circulating steroid in human, with the highest concentrations between age 20 and 30, but displaying a significant decrease with age. Many beneficial functions are ascribed to DHEAS. Nevertheless, long-term studies are very scarce concerning the intake of DHEAS over several years, and molecular investigations on DHEAS action are missing so far. In this study, the role of DHEAS on the first and rate-limiting step of steroid hormone biosynthesis was analyzed in a reconstituted in vitro system, consisting of purified CYP11A1, adrenodoxin and adrenodoxin reductase. DHEAS enhances the conversion of cholesterol by 26%. Detailed analyses of the mechanism of DHEAS action revealed increased binding affinity of cholesterol to CYP11A1 and enforced interaction with the electron transfer partner, adrenodoxin. Difference spectroscopy showed K d-values of 40±2.7 µM and 24.8±0.5 µM for CYP11A1 and cholesterol without and with addition of DHEAS, respectively. To determine the K d-value for CYP11A1 and adrenodoxin, surface plasmon resonance measurements were performed, demonstrating a K d-value of 3.0±0.35 nM (with cholesterol) and of 2.4±0.05 nM when cholesterol and DHEAS were added. Kinetic experiments showed a lower Km and a higher kcat value for CYP11A1 in the presence of DHEAS leading to an increase of the catalytic efficiency by 75%. These findings indicate that DHEAS affects steroid hormone biosynthesis on a molecular level resulting in an increased formation of pregnenolone.

show abstract

Section: Optimization Of the Expression Of Cyp11a1 In E Colimentioning

confidence: 98%

Section: Optical Biosensor Measurementsmentioning

confidence: 99%

Section: Protein Expression and Purificationmentioning

confidence: 99%

See 1 more Smart Citation

Dehydroepiandrosterone Sulfate (DHEAS) Stimulates the First Step in the Biosynthesis of Steroid Hormones

Neunzig

Bernhardt

2014

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…The initial electrostatic attraction of Cc to the CM4 Sensor Chip surface was assessed by taking into account its isoelectric point and was optimized to pH 5.8. Cc was then covalently attached to the matrix using standard amine-coupling chemistry, as previously described (36). A reference flow cell was used as a control in which the chip surface was treated as described above, but without the injection of Cc.…”

Section: Maldi-tof-ms-maldi-tof-msmentioning

confidence: 99%

Structural and Functional Analysis of Novel Human Cytochrome c Targets in Apoptosis

Martínez-Fábregas

Dı́az-Moreno

González‐Arzola

et al. 2014

Molecular & Cellular Proteomics

Self Cite

View full text Add to dashboard Cite

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivo and in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome c during apoptosis is well established, relatively little is known about its participation in signaling pathways in vivo due to its essential role during respiration. To obtain a better understanding of the role of cytochrome c in the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome c as bait in this study. In this approach, novel cytochrome c interaction partners were identified whose in vivo interaction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome c and its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome c redox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome c interacts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome c may interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals. Molecular & Cellular

show abstract

“…In directed evolution experiments, stabilized protein variants were isolated by plating the cells expressing the library of the POI in CAT fusion on solid media containing increasing concentrations of chloramphenicol. A number of successful applications of this biosensor include the screening for solubility-enhanced mitochondrial cytochrome P450 (Janocha et al 2011), stabilization of imidazole glycerol phosphate synthase (Seitz et al 2007), interleukin-15 (Behar et al 2011), (+)-δ-cadinene synthase (Yoshikuni et al 2006), and many more.…”

Section: Chloramphenicol Acetyltransferase (Cat) Reportermentioning

confidence: 99%

Selection and screening strategies in directed evolution to improve protein stability

Ren

Wen

Mencius

et al. 2019

Bioresour. Bioprocess.

View full text Add to dashboard Cite

Protein stability is not only fundamental for experimental, industrial, and therapeutic applications, but is also the baseline for evolving novel protein functions. For decades, stability engineering armed with directed evolution has continued its rapid development and inevitably poses challenges. Generally, in directed evolution, establishing a reliable link between a genotype and any interpretable phenotype is more challenging than diversifying genetic libraries. Consequently, we set forth in a small picture to emphasize the screening or selection techniques in protein stability-directed evolution to secure the link. For a more systematic review, two main branches of these techniques, namely cellular or cell-free display and stability biosensors, are expounded with informative examples.

show abstract

Substitution of lysine with glutamic acid at position 193 in bovine CYP11A1 significantly affects protein oligomerization and solubility but not enzymatic activity

Cited by 13 publications

References 27 publications

Dehydroepiandrosterone Sulfate (DHEAS) Stimulates the First Step in the Biosynthesis of Steroid Hormones

Dehydroepiandrosterone Sulfate (DHEAS) Stimulates the First Step in the Biosynthesis of Steroid Hormones

Structural and Functional Analysis of Novel Human Cytochrome c Targets in Apoptosis

Selection and screening strategies in directed evolution to improve protein stability

Contact Info

Product

Resources

About