3 research outputs found
Π€ΠΈΠ·ΠΈΠΊΠΎ-Ρ ΠΈΠΌΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΡΠ²ΠΎΠΉΡΡΠ²Π° Ξ3β12 ΡΠΈΡΡΠ΅ΠΈΠ½-ΠΎΠ±Π΅Π΄Π½Π΅Π½Π½ΠΎΠ³ΠΎ ΡΠΈΡΠΎΡ ΡΠΎΠΌΠ° P450 3A4 Ρ Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡΠ½ΠΎΠΉ Π·Π°ΠΌΠ΅Π½ΠΎΠΉ S291C
Cytochrome P450 3A4 (3A4) is highly expressed in the human liver cells and plays a decisive role in the metabolism of xenobiotics, including more than 50 % of medical products. The activity of this enzyme can be regulated at the expression level of genes, as well as at the conformation level of the structure of the protein itself, due to changes in the molecular environment, including due to the interaction with high-molecular effectors. The understanding of the structure changes and the 3A4 dynamics in response to the environmental changes is necessary to predict the changes in the level of its activity that to a considerable extent regulates the bodyβs homeostasis. To perform in vitro experiments on the structure, dynamics, and protein-ligand/protein interactions of the enzymes by the modern spectral methods, the approach is used, in which the target protein is selectively added with cysteine residues in the given polypeptide chain loci by the protein engineering methods for subsequent labeling with specialized molecular labels. To do this, the human mutant form of membrane-bound (full length) recombinant cytochrome P450 3A4 C58A/C64M/C98A/C239T/C377A/C468S/S291C was obtained. According to the circular dichroism spectroscopy data we established that the introduced mutations do not cause significant changes in the secondary structure of the obtained form 3A4, which shows the preservation of the folding of the peptide chain. The spectral photometric measurements were made to comparatively analyze the changes in the affinity to the ligands of the active center. Moreover, we showed that the testosterone hydroxylase activity in the in vitro reconstructed system for a given mutation form of 3A4 increases many times with respect to the wild form of the enzyme.Β Π¦ΠΈΡΠΎΡ
ΡΠΎΠΌ P450 3A4 (3A4) ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΡΡΠ΅ΡΡΡ Π² ΠΊΠ»Π΅ΡΠΊΠ°Ρ
ΠΏΠ΅ΡΠ΅Π½ΠΈ ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° ΠΈ ΠΈΠ³ΡΠ°Π΅Ρ ΠΊΠ»ΡΡΠ΅Π²ΡΡ ΡΠΎΠ»Ρ Π² ΠΌΠ΅ΡΠ°Π±ΠΎΠ»ΠΈΠ·ΠΌΠ΅ ΠΊΡΠ΅Π½ΠΎΠ±ΠΈΠΎΡΠΈΠΊΠΎΠ², Π² ΡΠΎΠΌ ΡΠΈΡΠ»Π΅ ΠΈ Π±ΠΎΠ»Π΅Π΅ 50 % Π»Π΅ΠΊΠ°ΡΡΡΠ²Π΅Π½Π½ΡΡ
ΠΏΡΠ΅ΠΏΠ°ΡΠ°ΡΠΎΠ². Π Π΅Π³ΡΠ»ΡΡΠΈΡ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ Π΄Π°Π½Π½ΠΎΠ³ΠΎ ΡΠ΅ΡΠΌΠ΅Π½ΡΠ° ΠΌΠΎΠΆΠ΅Ρ ΠΏΡΠΎΠΈΡΡ
ΠΎΠ΄ΠΈΡΡ Π½Π° ΡΡΠΎΠ²Π½Π΅ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ Π³Π΅Π½ΠΎΠ², Π° ΡΠ°ΠΊΠΆΠ΅ Π½Π° ΡΡΠΎΠ²Π½Π΅ ΠΊΠΎΠ½ΡΠΎΡΠΌΠ°ΡΠΈΠΎΠ½Π½ΠΎΠ³ΠΎ ΡΠΎΡΡΠΎΡΠ½ΠΈΡ ΡΡΡΡΠΊΡΡΡΡ ΡΠ°ΠΌΠΎΠ³ΠΎ Π±Π΅Π»ΠΊΠ°, Π·Π° ΡΡΠ΅Ρ ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡ ΠΌΠΎΠ»Π΅ΠΊΡΠ»ΡΡΠ½ΠΎΠ³ΠΎ ΠΎΠΊΡΡΠΆΠ΅Π½ΠΈΡ, Π² ΡΠΎΠΌ ΡΠΈΡΠ»Π΅ Π·Π° ΡΡΠ΅Ρ Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ Ρ Π²ΡΡΠΎΠΊΠΎΠΌΠΎΠ»Π΅ΠΊΡΠ»ΡΡΠ½ΡΠΌΠΈ ΡΡΡΠ΅ΠΊΡΠΎΡΠ°ΠΌΠΈ. ΠΠΎΠ½ΠΈΠΌΠ°Π½ΠΈΠ΅ ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡ ΡΡΡΡΠΊΡΡΡΡ ΠΈ Π΄ΠΈΠ½Π°ΠΌΠΈΠΊΠΈ 3A4 Π² ΠΎΡΠ²Π΅Ρ Π½Π° ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΠ΅ ΡΡΠ»ΠΎΠ²ΠΈΠΉ ΡΡΠ΅Π΄Ρ Π½Π΅ΠΎΠ±Ρ
ΠΎΠ΄ΠΈΠΌΠΎ Π΄Π»Ρ ΠΏΡΠ΅Π΄ΡΠΊΠ°Π·Π°Π½ΠΈΡ ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡ ΡΡΠΎΠ²Π½Ρ Π΅Π³ΠΎ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ, ΠΊΠΎΡΠΎΡΡΠΉ Π² Π·Π½Π°ΡΠΈΡΠ΅Π»ΡΠ½ΠΎΠΉ ΡΡΠ΅ΠΏΠ΅Π½ΠΈ ΠΎΠ±ΡΡΠ»Π°Π²Π»ΠΈΠ²Π°Π΅Ρ Π³ΠΎΠΌΠ΅ΠΎΡΡΠ°Π· ΠΎΡΠ³Π°Π½ΠΈΠ·ΠΌΠ°. ΠΠ»Ρ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΡ in vitro ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠΎΠ² ΠΏΠΎ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ ΡΡΡΡΠΊΡΡΡΡ, Π΄ΠΈΠ½Π°ΠΌΠΈΠΊΠΈ ΠΈ Π±Π΅Π»ΠΎΠΊΠ»ΠΈΠ³Π°Π½Π΄Π½ΡΡ
/Π±Π΅Π»ΠΊΠΎΠ²ΡΡ
Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΠΉ ΡΠ΅ΡΠΌΠ΅Π½ΡΠΎΠ² ΡΠΎΠ²ΡΠ΅ΠΌΠ΅Π½Π½ΡΠΌΠΈ ΡΠΏΠ΅ΠΊΡΡΠ°Π»ΡΠ½ΡΠΌΠΈ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌΠΈ ΠΈΡΠΏΠΎΠ»ΡΠ·ΡΠ΅ΡΡΡ ΠΏΠΎΠ΄Ρ
ΠΎΠ΄, Π² ΠΊΠΎΡΠΎΡΠΎΠΌ Π² ΡΠ΅Π»Π΅Π²ΠΎΠΉ Π±Π΅Π»ΠΎΠΊ ΡΠ΅Π»Π΅ΠΊΡΠΈΠ²Π½ΠΎ Π²Π²ΠΎΠ΄ΡΡΡΡ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌΠΈ Π±Π΅Π»ΠΊΠΎΠ²ΠΎΠΉ ΠΈΠ½ΠΆΠ΅Π½Π΅ΡΠΈΠΈ ΡΠΈΡΡΠ΅ΠΈΠ½ΠΎΠ²ΡΠ΅ ΠΎΡΡΠ°ΡΠΊΠΈ Π² Π·Π°Π΄Π°Π½Π½ΡΠ΅ Π»ΠΎΠΊΡΡΡ ΠΏΠΎΠ»ΠΈΠΏΠ΅ΠΏΡΠΈΠ΄Π½ΠΎΠΉ ΡΠ΅ΠΏΠΈ Π΄Π»Ρ ΠΏΠΎΡΠ»Π΅Π΄ΡΡΡΠ΅Π³ΠΎ ΠΌΠ΅ΡΠ΅Π½ΠΈΡ ΡΠΏΠ΅ΡΠΈΠ°Π»ΠΈΠ·ΠΈΡΠΎΠ²Π°Π½Π½ΡΠΌΠΈ ΠΌΠΎΠ»Π΅ΠΊΡΠ»ΡΡΠ½ΡΠΌΠΈ ΠΌΠ΅ΡΠΊΠ°ΠΌΠΈ. ΠΠ»Ρ ΡΡΠΈΡ
ΡΠ΅Π»Π΅ΠΉ Π² Π΄Π°Π½Π½ΠΎΠΉ ΡΠ°Π±ΠΎΡΠ΅ Π±ΡΠ»Π° ΠΏΠΎΠ»ΡΡΠ΅Π½Π° ΠΌΡΡΠ°Π½ΡΠ½Π°Ρ ΡΠΎΡΠΌΠ° ΠΌΠ΅ΠΌΠ±ΡΠ°Π½ΡΠ²ΡΠ·Π°Π½Π½ΠΎΠ³ΠΎ (ΠΏΠΎΠ»Π½ΠΎΡΠ°Π·ΠΌΠ΅ΡΠ½ΠΎΠ³ΠΎ) ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΠΎΠ³ΠΎ ΡΠΈΡΠΎΡ
ΡΠΎΠΌΠ° P450 3A4 ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° C58A/C64M/C98A/C239T/C377A/C468S/S291C. ΠΠΎ Π΄Π°Π½Π½ΡΠΌ ΡΠΏΠ΅ΠΊΡΡΠΎΡΠΊΠΎΠΏΠΈΠΈ ΠΊΡΡΠ³ΠΎΠ²ΠΎΠ³ΠΎ Π΄ΠΈΡ
ΡΠΎΠΈΠ·ΠΌΠ° Π½Π°ΠΌΠΈ Π±ΡΠ»ΠΎ ΡΡΡΠ°Π½ΠΎΠ²Π»Π΅Π½ΠΎ, ΡΡΠΎ Π²Π²Π΅Π΄Π΅Π½Π½ΡΠ΅ ΠΌΡΡΠ°ΡΠΈΠΈ Π½Π΅ Π²ΡΠ·ΡΠ²Π°ΡΡ Π·Π½Π°ΡΠΈΠΌΡΡ
ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΠΉ Π²ΠΎ Π²ΡΠΎΡΠΈΡΠ½ΠΎΠΉ ΡΡΡΡΠΊΡΡΡΠ΅ ΠΏΠΎΠ»ΡΡΠ΅Π½Π½ΠΎΠΉ ΡΠΎΡΠΌΡ 3A4, ΡΡΠΎ ΡΠ²ΠΈΠ΄Π΅ΡΠ΅Π»ΡΡΡΠ²ΡΠ΅Ρ ΠΎ ΡΠΎΡ
ΡΠ°Π½Π΅Π½ΠΈΠΈ ΡΠ²Π΅ΡΠ½ΡΡΠΎΡΡΠΈ ΠΏΠΎΠ»ΠΈΠΏΠ΅ΠΏΡΠΈΠ΄Π½ΠΎΠΉ ΡΠ΅ΠΏΠΈ. ΠΡΠΎΠ²Π΅Π΄Π΅Π½Ρ ΡΠΏΠ΅ΠΊΡΡΠΎΡΠΎΡΠΎΠΌΠ΅ΡΡΠΈΡΠ΅ΡΠΊΠΈΠ΅ ΠΈΠ·ΠΌΠ΅ΡΠ΅Π½ΠΈΡ Π΄Π»Ρ ΡΡΠ°Π²Π½ΠΈΡΠ΅Π»ΡΠ½ΠΎΠ³ΠΎ Π°Π½Π°Π»ΠΈΠ·Π° ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΡ ΡΡΠΎΠ΄ΡΡΠ²Π° ΠΊ Π»ΠΈΠ³Π°Π½Π΄Π°ΠΌ Π°ΠΊΡΠΈΠ²Π½ΠΎΠ³ΠΎ ΡΠ΅Π½ΡΡΠ°. ΠΠΎΠ»Π΅Π΅ ΡΠΎΠ³ΠΎ, Π½Π°ΠΌΠΈ Π±ΡΠ»ΠΎ ΠΏΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΡΠ΅ΡΡΠΎΡΡΠ΅ΡΠΎΠ½ Π³ΠΈΠ΄ΡΠΎΠΊΡΠΈΠ»ΠΈΡΡΡΡΠ°Ρ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΡ Π² in vitro ΡΠ΅ΠΊΠΎΠ½ΡΡΡΡΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΠ΅ Π΄Π»Ρ Π΄Π°Π½Π½ΠΎΠΉ ΠΌΡΡΠ°Π½ΡΠ½ΠΎΠΉ ΡΠΎΡΠΌΡ 3A4 ΠΌΠ½ΠΎΠ³ΠΎΠΊΡΠ°ΡΠ½ΠΎ ΡΠ²Π΅Π»ΠΈΡΠΈΠ²Π°Π΅ΡΡΡ ΠΎΡΠ½ΠΎΡΠΈΡΠ΅Π»ΡΠ½ΠΎ Π΄ΠΈΠΊΠΎΠΉ ΡΠΎΡΠΌΡ ΡΠ΅ΡΠΌΠ΅Π½ΡΠ°