54 research outputs found
Effects of low frequency ultrasound on some properties of fibrinogen and its plasminolysis
<p>Abstract</p> <p>Background</p> <p>Pharmacological thrombolysis with streptokinase, urokinase or tissue activator of plasminogen (t-PA), and mechanical interventions are frequently used in the treatment of both arterial and venous thrombotic diseases. It has been previously reported that application of ultrasound as an adjunct to thrombolytic therapy offers unique potential to improve effectiveness. However, little is known about effects of the ultrasound on proteins of blood coagulation and fibrinolysis. Here, we investigated the effects of the ultrasound on fibrinogen on processes of coagulation and fibrinogenolysis in an <it>in vitro </it>system.</p> <p>Results</p> <p>Our study demonstrated that low frequency high intensity pulse ultrasound (25.1 kHz, 48.4 W/cm2, duty 50%) induced denaturation of plasminogen and t-PA and fibrinogen aggregates formation <it>in vitro</it>. The aggregates were characterized by the loss of clotting ability and a greater rate of plasminolysis than native fibrinogen. We investigated the effect of the ultrasound on individual proteins. In case of plasminogen and t-PA, ultrasound led to a decrease of the fibrinogenolysis rate, while it increased the fibrinogenolysis rate in case of fibrinogen. It has been shown that upon ultrasound treatment of mixture fibrinogen or fibrin with plasminogen, t-PA, or both, the rate of proteolytic digestion of fibrin(ogen) increases too. It has been shown that summary effect on the fibrin(ogen) proteolytic degradation under the conditions for combined ultrasound treatment is determined exclusively by effect on fibrin(ogen).</p> <p>Conclusions</p> <p>The data presented here suggest that among proteins of fibrinolytic systems, the fibrinogen is one of the most sensitive proteins to the action of ultrasound. It has been shown <it>in vitro </it>that ultrasound induced fibrinogen aggregates formation, characterized by the loss of clotting ability and a greater rate of plasminolysis than native fibrinogen in different model systems and under different mode of ultrasound treatment. Under ultrasound treatment of plasminogen and/or t-PA in the presence of fibrin(ogen) the stabilizing effect fibrin(ogen) on given proteins was shown. On the other hand, an increase in the rate of fibrin(ogen) lysis was observed due to both the change in the substrate structure and promoting of the protein-protein complexes formation.</p
Transcriptome Analysis in Tardigrade Species Reveals Specific Molecular Pathways for Stress Adaptations
Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant
ΠΠΈΠΊΠ΅ΡΠΎΠ½Ρ ΠΏΡΠΈΡΠΎΠ΄Π½ΠΎΠ³ΠΎ ΠΏΡΠΎΠΈΡΡ ΠΎΠΆΠ΄Π΅Π½ΠΈΡ ΠΊΠ°ΠΊ ΠΏΠΎΡΠ΅Π½ΡΠΈΠ°Π»ΡΠ½ΡΠ΅ ΠΊΠΎΠ²Π°Π»Π΅Π½ΡΠ½ΡΠ΅ Π»ΠΈΠ³Π°Π½Π΄Ρ Π±Π΅Π»ΠΊΠΎΠ² SARS-CoV-2: ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠ΅ in silico ΠΌΠ΅ΡΠΎΠ΄ΠΎΠΌ Π΄ΠΎΠΊΠΈΠ½Π³Π°
Our computer-aided protein-ligand docking test using Autodock Vina software allowed to reveal the potential of few Ξ±- and Ξ²-diketones from plants and alternative living organisms as covalent ligands for few proteins of coronavirus SARS-CoV-2 β a causative agent of COVID-19. It has been established that values for energy of binding (docking score, Ebind, kcal/mol) less than β7.5 and for distances of ligandsβ carbonyl groups to side chain nitrogens of arginine residues of some coronaviral enzymes within 0.4 nm have been true for Ξ²-diketones 6-gingerdione (Pubchem code CID162952), 8-gingerdione (CID14440537), tetrahydrocurcumine (CID124072) as well as Ξ±-diketone wallitaxane E (CID132967478). The in silico revealed interactions are interesting to be verified in vitro and they point out a possibility of investigation of the compounds and related natural materials as tools for struggle against coronaviral infections.ΠΠΎΠΌΠΏΡΡΡΠ΅ΡΠ½ΡΠΉ Π΄ΠΎΠΊΠΈΠ½Π³, ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½Π½ΡΠΉ Ρ ΠΈΡΠΏΠΎΠ»ΡΠ·ΠΎΠ²Π°Π½ΠΈΠ΅ΠΌ ΠΏΡΠΎΠ³ΡΠ°ΠΌΠΌΡ Autodock Vina, ΠΏΠΎΠ·Π²ΠΎΠ»ΠΈΠ» Π²ΡΡΠ²ΠΈΡΡ ΠΏΠΎΡΠ΅Π½ΡΠΈΠ°Π» Π½Π΅ΡΠΊΠΎΠ»ΡΠΊΠΈΡ
Ξ±- ΠΈ Ξ²-Π΄ΠΈΠΊΠ΅ΡΠΎΠ½ΠΎΠ² ΡΠ°ΡΡΠ΅Π½ΠΈΠΉ ΠΈ Π΄ΡΡΠ³ΠΈΡ
ΠΏΡΠΈΡΠΎΠ΄Π½ΡΡ
ΠΎΠ±ΡΠ΅ΠΊΡΠΎΠ² Π² ΠΊΠ°ΡΠ΅ΡΡΠ²Π΅ ΠΊΠΎΠ²Π°Π»Π΅Π½ΡΠ½ΡΡ
Π»ΠΈΠ³Π°Π½Π΄ΠΎΠ² ΡΡΠ΄Π° Π±Π΅Π»ΠΊΠΎΠ² ΠΊΠΎΡΠΎΠ½Π°Π²ΠΈΡΡΡΠ° SARS-CoV-2 β Π²ΠΎΠ·Π±ΡΠ΄ΠΈΡΠ΅Π»Ρ COVID-19. ΠΡΡΠ²Π»Π΅Π½ΠΎ, ΡΡΠΎ ΡΠ½Π΅ΡΠ³ΠΈΠ΅ΠΉ ΡΠ²ΡΠ·ΡΠ²Π°Π½ΠΈΡ (docking score, Ebind, ΠΊΠΊΠ°Π»/ΠΌΠΎΠ») ΠΌΠ΅Π½Π΅Π΅ β7,5 Ρ ΠΊΠΎΠ»ΠΎΠΊΠΎΠ»ΠΈΠ·Π°ΡΠΈΠ΅ΠΉ ΠΊΠ°ΡΠ±ΠΎΠ½ΠΈΠ»ΡΠ½ΡΡ
Π³ΡΡΠΏΠΏ Π½Π° ΡΠ°ΡΡΡΠΎΡΠ½ΠΈΠΈ Π½Π΅ Π±ΠΎΠ»Π΅Π΅ 0,4 Π½ΠΌ ΠΎΡ Π°ΡΠΎΠΌΠΎΠ² Π°Π·ΠΎΡΠ° Π±ΠΎΠΊΠΎΠ²ΠΎΠΉ ΡΠ΅ΠΏΠΈ ΠΎΡΡΠ°ΡΠΊΠΎΠ² Π°ΡΠ³ΠΈΠ½ΠΈΠ½Π° Π±Π΅Π»ΠΊΠΎΠ² ΠΊΠΎΡΠΎΠ½Π°Π²ΠΈΡΡΡΠ°. Ξ²-ΠΠΈΠΊΠ΅ΡΠΎΠ½Ρ 6-Π³ΠΈΠ½Π³Π΅ΡΠ΄ΠΈΠΎΠ½ (ΠΊΠΎΠ΄ ΡΡΡΡΠΊΡΡΡΡ ΠΏΠΎ Π±Π°Π·Π΅ Π΄Π°Π½Π½ΡΡ
Pubchem CID162952), 8-Π³ΠΈΠ½Π³Π΅ΡΠ΄ΠΈΠΎΠ½ (CID14440537), ΡΠ΅ΡΡΠ°Π³ΠΈΠ΄ΡΠΎΠΊΡΡΠΊΡΠΌΠΈΠ½ (CID124072), Π° ΡΠ°ΠΊΠΆΠ΅ Ξ±-Π΄ΠΈΠΊΠ΅ΡΠΎΠ½ Π²Π°Π»Π»ΠΈΡΠ°ΠΊΡΠ°Π½ E (CID132967478) ΠΎΠ±Π»Π°Π΄Π°Π»ΠΈ ΡΠ°ΠΊΠΈΠΌΠΈ ΡΠ²ΠΎΠΉΡΡΠ²Π°ΠΌΠΈ. ΠΡΡΠ²Π»Π΅Π½Π½ΡΠ΅ in silico Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ ΡΠΊΠ°Π·ΡΠ²Π°ΡΡ Π½Π° Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΎΠ±Π½Π°ΡΡΠΆΠ΅Π½ΠΈΡ ΠΈΡ
Π² ΡΠΊΡΠΏΠ΅ΡΠΈΠΌΠ΅Π½ΡΠ΅ ΠΈ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ ΡΡΠΈΡ
Π²Π΅ΡΠ΅ΡΡΠ² ΠΈΠ»ΠΈ ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΡ
ΠΈΡ
ΠΏΡΠΈΡΠΎΠ΄Π½ΡΡ
ΠΌΠ°ΡΠ΅ΡΠΈΠ°Π»ΠΎΠ² ΠΊΠ°ΠΊ ΡΡΠ΅Π΄ΡΡΠ² Π±ΠΎΡΡΠ±Ρ Ρ ΠΊΠΎΡΠΎΠ½ΠΎΠ²ΠΈΡΡΡΠ½ΠΎΠΉ ΠΈΠ½ΡΠ΅ΠΊΡΠΈΠ΅ΠΉ
Multiβscale ensemble properties of the Escherichia coli RNA degradosome
Abstract: In organisms from all domains of life, multiβenzyme assemblies play central roles in defining transcript lifetimes and facilitating RNAβmediated regulation of gene expression. An assembly dedicated to such roles, known as the RNA degradosome, is found amongst bacteria from highly diverse lineages. About a fifth of the assembly mass of the degradosome of Escherichia coli and related species is predicted to be intrinsically disordered β a property that has been sustained for over a billion years of bacterial molecular history and stands in marked contrast to the high degree of sequence variation of that same region. Here, we characterize the conformational dynamics of the degradosome using a hybrid structural biology approach that combines solution scattering with ad hoc ensemble modelling, cryoβelectron microscopy, and other biophysical methods. The E. coli degradosome can form punctate bodies in vivo that may facilitate its functional activities, and based on our results, we propose an electrostatic switch model to account for the propensity of the degradosome to undergo programmable puncta formation
Π‘ΠΈΠ½ΡΠ΅Π· Π½ΠΎΠ²ΡΡ ΡΠΈΠ°Π·ΠΎΠ»ΠΎ[3,2-Π°]ΠΏΠΈΡΠΈΠΌΠΈΠ΄ΠΈΠ½ΠΎΠ² ΠΈ in silico Π°Π½Π°Π»ΠΈΠ· ΠΈΡ Π±ΠΈΠΎΠ°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ
An effective method of synthesis thiazolo[3,2-a]pyrimidine derivatives was developed and the compounds with n-pentyl or Ξ²-acetoxycyclopropyl as well as fluorescent benzo[f]coumarin substituents were obtained with yields 60 % and more. Using computational (in silico) approaches we demonstrated the ability of the obtained compounds to permeate lipid bilayer as well as their affinity to some protein kinases (compounds 4 and 6 bind with a protein kinase AKT1 with PDB code 3ΠΎ96; Autodock Vina-computed energy of binding (Ebind) values were -10.9 and -10.6 kcal/mol, respectively), acethylcholine esterase and some human cytochromes P450 (for P450 3A4, pdb 5vcd, Ebind -12.3 kcal/mol).Π Π°Π·ΡΠ°Π±ΠΎΡΠ°Π½ ΡΡΡΠ΅ΠΊΡΠΈΠ²Π½ΡΠΉ ΠΌΠ΅ΡΠΎΠ΄ ΠΏΠΎΠ»ΡΡΠ΅Π½ΠΈΡ ΠΈ ΡΠΈΠ½ΡΠ΅Π·ΠΈΡΠΎΠ²Π°Π½Ρ Π½ΠΎΠ²ΡΠ΅ ΡΠΈΠ°Π·ΠΎΠ»ΠΎ[3,2-a]ΠΏΠΈΡΠΈΠΌΠΈΠ΄ΠΈΠ½Ρ, ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΠ΅ Π½-ΠΏΠ΅Π½ΡΠ°Π½ΠΎΠ²ΡΠΉ ΠΈΠ»ΠΈ Ξ²-Π³ΠΈΠ΄ΡΠΎΠΊΡΠΈΡΠΈΠΊΠ»ΠΎΠΏΡΠΎΠΏΠ°Π½ΠΎΠ²ΡΠΉ, Π° ΡΠ°ΠΊΠΆΠ΅ ΡΠ»ΡΠΎΡΠ΅ΡΡΠΈΡΡΡΡΠΈΠΉ Π±Π΅Π½Π·ΠΎ[f]ΠΊΡΠΌΠ°ΡΠΈΠ½ΠΎΠ²ΡΠΉ Π·Π°ΠΌΠ΅ΡΡΠΈΡΠ΅Π»ΠΈ Ρ Π²ΡΡ
ΠΎΠ΄Π°ΠΌΠΈ Π±ΠΎΠ»Π΅Π΅ 60 %. ΠΠΎΠΌΠΏΡΡΡΠ΅ΡΠ½ΡΠΌΠΈ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌΠΈ (in silico) ΠΏΠΎΠΊΠ°Π·Π°Π½Π° ΡΠΏΠΎΡΠΎΠ±Π½ΠΎΡΡΡ ΠΏΠΎΠ»ΡΡΠ΅Π½Π½ΡΡ
ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΠΉ ΠΏΡΠΎΠ½ΠΈΠΊΠ°ΡΡ ΡΠ΅ΡΠ΅Π· Π»ΠΈΠΏΠΈΠ΄Π½ΡΠΉ Π±ΠΈΡΠ»ΠΎΠΉ ΠΈ ΠΎΡΠ΅Π½Π΅Π½ΠΎ ΠΈΡ
ΡΡΠΎΠ΄ΡΡΠ²ΠΎ ΠΊ ΡΡΠ΄Ρ ΠΏΡΠΎΡΠ΅ΠΈΠ½ΠΊΠΈΠ½Π°Π· (ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΡ 4 ΠΈ 6 ΡΠ²ΡΠ·ΡΠ²Π°ΡΡΡΡ Ρ ΠΏΡΠΎΡΠ΅ΠΈΠ½ΠΊΠΈΠ½Π°Π·ΠΎΠΉ AKT1 Ρ ΠΊΠΎΠ΄ΠΎΠΌ PDB 3ΠΎ96; Π²Π΅Π»ΠΈΡΠΈΠ½Ρ, ΡΠ°ΡΡΡΠΈΡΡΠ²Π°Π΅ΠΌΡΠ΅ ΠΏΡΠΎΠ³ΡΠ°ΠΌΠΌΠΎΠΉ Autodock Vina ΡΠ½Π΅ΡΠ³ΠΈΠΈ ΡΠ²ΡΠ·ΡΠ²Π°Π½ΠΈΡ (Ebind), ΡΠΎΡΡΠ°Π²ΠΈΠ»ΠΈ: -10,9 ΠΈ -10,6 ΠΊΠΊΠ°Π»/ΠΌΠΎΠ»Ρ), Π°ΡΠ΅ΡΠΈΠ»Ρ
ΠΎΠ»ΠΈΠ½ΡΡΡΠ΅ΡΠ°Π·Π΅ ΠΈ ΡΠΈΡΠΎΡ
ΡΠΎΠΌΠ°ΠΌ P450 ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° (Π΄Π»Ρ ΡΠΈΡΠΎΡ
ΡΠΎΠΌΠ° P450 3A4, pdb 5vcd, Ebind -12,3 ΠΊΠΊΠ°Π»/ΠΌΠΎΠ»Ρ)
Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.
The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome-comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing
In silico Π°Π½Π°Π»ΠΈΠ· Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΠΉ, ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΡ ΡΠΎΡΠΎΠ°ΠΊΡΠΈΠ²ΠΈΡΡΠ΅ΠΌΡΠ΅ Π³ΡΡΠΏΠΏΡ, Ρ ΡΠ΅ΡΠΌΠ΅Π½ΡΠ°ΠΌΠΈ CYP7 ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ°
In silico analysis of βprotein-ligandβ complexes of human CYP7 enzymes with modified borondipyrrome-tene (BODIPY) and steroids, containing photo-activated crosslinking groups, wasperformed in order to identify structural peculiarities of their interaction. It was found that BODIPY molecules and DHEA derivative with diazirine group are able to bind tightly with human steroid-hydroxylases. Binding affinity is comparable with corresponding values for essential ligands of the enzymes. Binding mode of the modified steroid corresponds to the binding mode of essential CYP7 ligands, so formation of hydroxylated products is possible. It was found that presence of both diazirine and NBD groups in a molecule significantly increases affinity of the compound in case of CYP7A1 and, especially, CYP7B1. Amino acid residues, located in a close proximity with photo-activated groups were detected, that can form covalent adducts with them. The obtained results can shed light on the mechanism of interaction of the compounds with recombinant human CYP7 enzymes in vitro. The results can also be used for the identification of modified amino acids of the proteins that are formed under photoactivation of the compounds in vitro.Π‘ ΡΠ΅Π»ΡΡ Π²ΡΡΠ²Π»Π΅Π½ΠΈΡ ΡΡΡΡΠΊΡΡΡΠ½ΡΡ
ΠΎΡΠΎΠ±Π΅Π½Π½ΠΎΡΡΠ΅ΠΉ Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ ΡΠ΅ΡΠΌΠ΅Π½ΡΠΎΠ² CYP7 ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° Ρ ΠΏΡΠΎΠΈΠ·Π²ΠΎΠ΄Π½ΡΠΌΠΈ Π±ΠΎΡΠ΄ΠΈΠΏΠΈΡΠΎΠΌΠ΅ΡΠ΅Π½Π° (BODIPY) ΠΈ ΡΡΠ΅ΡΠΎΠΈΠ΄ΠΎΠ², ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΡ
ΡΠΎΡΠΎΠ°ΠΊΡΠΈΠ²ΠΈΡΡΠ΅ΠΌΡΠ΅ ΡΡΠΈΠ²Π°ΡΡΠΈΠ΅ Π³ΡΡΠΏΠΏΡ, ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½ in silico Π°Π½Π°Π»ΠΈΠ· ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠ² Β«Π±Π΅Π»ΠΎΠΊ-Π»ΠΈΠ³Π°Π½Π΄Β». ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ ΠΌΠΎΠ΄ΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΡΠ΅ ΠΌΠΎΠ»Π΅ΠΊΡΠ»Ρ BODIPY, Π° ΡΠ°ΠΊΠΆΠ΅ ΠΏΡΠΎΠΈΠ·Π²ΠΎΠ΄Π½ΠΎΠ΅ Π΄Π΅Π³ΠΈΠ΄ΡΠΎΡΠΏΠΈΠ°Π½Π΄ΡΠΎΡΡΠ΅ΡΠΎΠ½Π° Ρ Π΄ΠΈΠ°Π·ΠΈΡΠΈΠ½ΠΎΠ²ΠΎΠΉ Π³ΡΡΠΏΠΏΠΎΠΉ ΡΠΏΠΎΡΠΎΠ±Π½Ρ ΡΠ²ΡΠ·ΡΠ²Π°ΡΡΡΡ Π² Π°ΠΊΡΠΈΠ²Π½ΠΎΠΌ ΡΠ΅Π½ΡΡΠ΅ ΡΡΠ΅ΡΠΎΠΈΠ΄-Π³ΠΈΠ΄ΡΠΎΠΊΡΠΈΠ»Π°Π· ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° Ρ Π°ΡΡΠΈΠ½Π½ΠΎΡΡΡΡ, ΡΡΠ°Π²Π½ΠΈΠΌΠΎΠΉ Ρ ΡΠΎΠΎΡΠ²Π΅ΡΡΡΠ²ΡΡΡΠΈΠΌΠΈ Π²Π΅Π»ΠΈΡΠΈΠ½Π°ΠΌΠΈ, ΡΠ°ΡΡΡΠΈΡΠ°Π½Π½ΡΠΌΠΈ Π΄Π»Ρ ΠΏΡΠΈΡΠΎΠ΄Π½ΡΡ
ΡΡΠ±ΡΡΡΠ°ΡΠΎΠ² ΡΡΠΈΡ
ΡΠ΅ΡΠΌΠ΅Π½ΡΠΎΠ². ΠΡΠΈ ΡΡΠΎΠΌ Π³Π΅ΠΎΠΌΠ΅ΡΡΠΈΡ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠ° Β«ΡΠ΅ΡΠΌΠ΅Π½Ρ-Π»ΠΈΠ³Π°Π½Π΄Β» Π΄Π»Ρ ΠΌΠΎΠ΄ΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Π½ΠΎΠ³ΠΎ ΡΡΠ΅ΡΠΎΠΈΠ΄Π° Π² Π°ΠΊΡΠΈΠ²Π½ΠΎΠΌ ΡΠ΅Π½ΡΡΠ΅ ΡΠ°ΠΊΠΆΠ΅ ΡΠΎΠΎΡΠ²Π΅ΡΡΡΠ²ΡΠ΅Ρ Π³Π΅ΠΎΠΌΠ΅ΡΡΠΈΠΈ ΠΊΠΎΠΌΠΏΠ»Π΅ΠΊΡΠΎΠ² CYP7 ΡΠΎ ΡΠ²ΠΎΠΈΠΌΠΈ ΠΏΡΠΈΡΠΎΠ΄Π½ΡΠΌΠΈ Π»ΠΈΠ³Π°Π½Π΄Π°ΠΌΠΈ, ΡΡΠΎ ΡΠΊΠ°Π·ΡΠ²Π°Π΅Ρ Π½Π° Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΎΠ±ΡΠ°Π·ΠΎΠ²Π°Π½ΠΈΡ Π³ΠΈΠ΄ΡΠΎΠΊΡΠΈΠ»ΠΈΡΠΎΠ²Π°Π½Π½ΡΡ
ΠΏΡΠΎΠ΄ΡΠΊΡΠΎΠ² ΡΠ΅Π°ΠΊΡΠΈΠΈ - ΠΌΠ΅ΡΠ΅Π½ΡΡ
Π°Π½Π°Π»ΠΎΠ³ΠΎΠ² Π±ΠΈΠΎΠΌΠ΅ΡΠ°Π±ΠΎΠ»ΠΈΡΠΎΠ². ΠΠΎΠΊΠ°Π·Π°Π½ΠΎ, ΡΡΠΎ Π½Π°Π»ΠΈΡΠΈΠ΅ ΠΎΠ΄Π½ΠΎΠ²ΡΠ΅ΠΌΠ΅Π½Π½ΠΎ Π΄ΠΈΠ°Π·ΠΈΡΠΈΠ½ΠΎΠ²ΠΎΠΉ ΠΈ 7-Π½ΠΈΡΡΠΎΠ±Π΅Π½Π·ΠΎΡΡΡΠ°Π·Π°Π½ΠΎΠ²ΠΎΠΉ Π³ΡΡΠΏΠΏ Π·Π½Π°ΡΠΈΡΠ΅Π»ΡΠ½ΠΎ ΡΠ½ΠΈΠΆΠ°Π΅Ρ ΡΡΠΎΠ΄ΡΡΠ²ΠΎ Π»ΠΈΠ³Π°Π½Π΄Π° ΠΊ Π°ΠΊΡΠΈΠ²Π½ΠΎΠΌΡ ΡΠ΅Π½ΡΡΡ CYP7A1 ΠΈ, Π² ΠΎΡΠΎΠ±Π΅Π½Π½ΠΎΡΡΠΈ, CYP7B1. ΠΠ΄Π΅Π½ΡΠΈΡΠΈΡΠΈΡΠΎΠ²Π°Π½Ρ Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡΠ½ΡΠ΅ ΠΎΡΡΠ°ΡΠΊΠΈ, ΡΠ°ΡΠΏΠΎΠ»ΠΎΠΆΠ΅Π½Π½ΡΠ΅ Π²Π±Π»ΠΈΠ·ΠΈ ΡΠΎΡΠΎΠ°ΠΊΡΠΈΠ²ΠΈΡΡΠ΅ΠΌΡΡ
Π³ΡΡΠΏΠΏ ΠΈ ΡΠΏΠΎΡΠΎΠ±Π½ΡΠ΅ ΠΎΠ±ΡΠ°Π·ΠΎΠ²ΡΠ²Π°ΡΡ Ρ Π½ΠΈΠΌΠΈ ΠΊΠΎΠ²Π°Π»Π΅Π½ΡΠ½ΡΠ΅ Π°Π΄Π΄ΡΠΊΡΡ. ΠΠΎΠ»ΡΡΠ΅Π½Π½ΡΠ΅ ΡΠ΅Π·ΡΠ»ΡΡΠ°ΡΡ ΠΏΡΠ΅Π΄ΡΡΠ°Π²Π»ΡΡΡ ΠΈΠ½ΡΠ΅ΡΠ΅Ρ Π΄Π»Ρ ΠΎΠ±ΡΡΡΠ½Π΅Π½ΠΈΡ ΠΌΠ΅Ρ
Π°Π½ΠΈΠ·ΠΌΠ° Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΠΉ, ΡΠΎΠ΄Π΅ΡΠΆΠ°ΡΠΈΡ
ΡΠΎΡΠΎΠ°ΠΊΡΠΈΠ²ΠΈΡΡΠ΅ΠΌΡΠ΅ ΡΡΠΈΠ²Π°ΡΡΠΈΠ΅ Π³ΡΡΠΏΠΏΡ Ρ ΡΠ΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½ΡΠ½ΡΠΌΠΈ ΡΠ΅ΡΠΌΠ΅Π½ΡΠ°ΠΌΠΈ CYP7 ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° in vitro, Π° ΡΠ°ΠΊΠΆΠ΅ Π΄Π»Ρ ΠΈΠ΄Π΅Π½ΡΠΈΡΠΈΠΊΠ°ΡΠΈΠΈ ΠΏΡΠΎΠ΄ΡΠΊΡΠΎΠ² ΠΊΠΎΠ²Π°Π»Π΅Π½ΡΠ½ΠΎΠΉ ΠΌΠΎΠ΄ΠΈΡΠΈΠΊΠ°ΡΠΈΠΈ Π°ΠΌΠΈΠ½ΠΎΠΊΠΈΡΠ»ΠΎΡΠ½ΡΡ
ΠΎΡΡΠ°ΡΠΊΠΎΠ² Π±Π΅Π»ΠΊΠ°, ΠΎΠ±ΡΠ°Π·ΡΡΡΠΈΡ
ΡΡ ΠΏΡΠΈ ΡΠΎΡΠΎΠ°ΠΊΡΠΈΠ²Π°ΡΠΈΠΈ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½Π½ΡΡ
ΠΌΠΎΠ»Π΅ΠΊΡΠ»
A homologue of the Parkinson's disease-associated protein LRRK2 undergoes a monomer-dimer transition during GTP turnover.
Mutations in LRRK2 are a common cause of genetic Parkinson's disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2
Insights into the Molecular Activation Mechanism of the RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF
PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via GΞ±(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation
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