17 research outputs found
ΠΠΎΠ²ΡΠ΅ Π»ΠΈΠΏΠΎΡΠΈΠ»ΡΠ½ΡΠ΅ ΠΊΠΎΠ½ΡΡΠ³Π°ΡΡ ΡΠ»ΡΠΎΡΠ΅ΡΡΠΈΡΡΡΡΠ΅Π³ΠΎ NBD-ΠΏΠΈΠΏΠ΅ΡΠ°Π·ΠΈΠ½Π°: ΡΠΈΠ½ΡΠ΅Π·, in silico Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΠ΅ Ρ Π»ΠΈΠΏΠΈΠ΄Π½ΡΠΌ Π±ΠΈΡΠ»ΠΎΠ΅ΠΌ ΠΈ ΡΠΈΡΠΎΡ ΡΠΎΠΌΠ°ΠΌΠΈ P450
Two new fluorescent NBD-piperazine derivatives with lipophilic substituents of formylcholesterol and hexanoic acid (compounds 2 and 3, respectively) were synthesized. Using in silico calculations, their abilities to permeate through lipid bilayer and to be bound affinely with some cytochromes P450 of human (2 with lanosterol 14Ξ±-demethylase CYP51, 3 with drug-metabolizing CYP1A1, CYP2D6, CYP3A4; energy of binding Ebind from β14.4 to β10 kcal/mol) and of mycobacteria (2 with sterol 27-hydroxylase CYP125, 3 with βorphanβ CYP164; Ebind from β13.3 to β9.1 kcal/mol) were demonstrated. The synthesized compounds and calculated computational data will contribute to prioritization of in vitro investigations aiming to revealing properties of the proteins.Π‘ΠΈΠ½ΡΠ΅Π·ΠΈΡΠΎΠ²Π°Π½Ρ Π΄Π²Π° Π½ΠΎΠ²ΡΡ
ΡΠ»ΡΠΎΡΠ΅ΡΡΠΈΡΡΡΡΠΈΡ
ΠΏΡΠΎΠΈΠ·Π²ΠΎΠ΄Π½ΡΡ
NBD-ΠΏΠΈΠΏΠ΅ΡΠ°Π·ΠΈΠ½Π° Ρ Π»ΠΈΠΏΠΎΡΠΈΠ»ΡΠ½ΡΠΌΠΈ ΠΎΡΡΠ°ΡΠΊΠ°ΠΌΠΈ ΡΠΎΡΠΌΠΈΠ»Ρ
ΠΎΠ»Π΅ΡΡΠ΅ΡΠΎΠ»Π° ΠΈ Π³Π΅ΠΊΡΠ°Π½ΠΎΠ²ΠΎΠΉ ΠΊΠΈΡΠ»ΠΎΡΡ (ΡΠΎΠ΅Π΄ΠΈΠ½Π΅Π½ΠΈΡ 2 ΠΈ 3 ΡΠΎΠΎΡΠ²Π΅ΡΡΡΠ²Π΅Π½Π½ΠΎ). Π Π°ΡΡΠ΅ΡΠ½ΠΎ-ΡΠ΅ΠΎΡΠ΅ΡΠΈΡΠ΅ΡΠΊΠΈΠΌΠΈ ΠΌΠ΅ΡΠΎΠ΄Π°ΠΌΠΈ ΠΏΠΎΠΊΠ°Π·Π°Π½Π° ΠΈΡ
Π²ΠΎΠ·ΠΌΠΎΠΆΠ½ΠΎΡΡΡ ΠΏΡΠΎΠ½ΠΈΠΊΠ°ΡΡ ΡΠ΅ΡΠ΅Π· Π»ΠΈΠΏΠΈΠ΄Π½ΡΠΉ Π±ΠΈΡΠ»ΠΎΠΉ, Π° ΡΠ°ΠΊΠΆΠ΅ Π°ΡΡΠΈΠ½Π½ΠΎ ΡΠ²ΡΠ·ΡΠ²Π°ΡΡΡΡ Ρ Π½Π΅ΠΊΠΎΡΠΎΡΡΠΌΠΈ ΡΠΈΡΠΎΡ
ΡΠΎΠΌΠ°ΠΌΠΈ P450 ΡΠ΅Π»ΠΎΠ²Π΅ΠΊΠ° (2 Ρ Π»Π°Π½ΠΎΡΡΠ΅ΡΠΎΠ» 14Ξ±-Π΄Π΅ΠΌΠ΅ΡΠΈΠ»Π°Π·ΠΎΠΉ CYP51, 3 c Π»Π΅ΠΊΠ°ΡΡΡΠ²ΠΎ-ΠΏΡΠ΅Π²ΡΠ°ΡΠ°ΡΡΠΈΠΌΠΈ CYP1A1, CYP2D6, CYP3A4; ΡΠ½Π΅ΡΠ³ΠΈΡ ΡΠ²ΡΠ·ΡΠ²Π°Π½ΠΈΡ Ebind Π² Π΄ΠΈΠ°ΠΏΠ°Π·ΠΎΠ½Π΅ β14,4β¦β10 ΠΊΠΊΠ°Π»/ΠΌΠΎΠ»Ρ) ΠΈ ΠΌΠΈΠΊΠΎΠ±Π°ΠΊΡΠ΅ΡΠΈΠΉ (2 ΡΠΎ ΡΡΠ΅ΡΠΎΠ»-27 Π³ΠΈΠ΄ΡΠΎΠΊΡΠΈΠ»Π°Π·ΠΎΠΉ CYP125, 3 Ρ Β«ΠΎΡΡΠ°Π½Π½ΡΠΌΒ» CYP164; Ebind Π² Π΄ΠΈΠ°ΠΏΠ°Π·ΠΎΠ½Π΅ β13,3β¦β9,1 ΠΊΠΊΠ°Π»/ΠΌΠΎΠ»Ρ). ΠΠΎΠ»ΡΡΠ΅Π½Π½ΡΠ΅ Π²Π΅ΡΠ΅ΡΡΠ²Π° ΠΈ ΡΠΎΠΎΡΠ²Π΅ΡΡΡΠ²ΡΡΡΠΈΠ΅ ΡΠ°ΡΡΠ΅ΡΠ½ΠΎ-ΡΠ΅ΠΎΡΠ΅ΡΠΈΡΠ΅ΡΠΊΠΈΠ΅ Π΄Π°Π½Π½ΡΠ΅ Π±ΡΠ΄ΡΡ ΡΠΏΠΎΡΠΎΠ±ΡΡΠ²ΠΎΠ²Π°ΡΡ ΠΏΡΠΈΠΎΡΠΈΡΠ΅Π·Π°ΡΠΈΠΈ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠΉ in vitro Π΄Π»Ρ ΡΠ΅Π»Π΅ΠΉ ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡ ΡΡΠ½ΠΊΡΠΈΠΉ ΡΡΠΈΡ
Π±Π΅Π»ΠΊΠΎΠ²
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
Ligand-Binding Properties and Conformational Dynamics of Autolysin Repeat Domains in Staphylococcal Cell Wall Recognition
The bifunctional major autolysin Atl plays a key role in staphylococcal cell separation. Processing of Atl yields catalytically active amidase (AM) and glucosaminidase (GL) domains that are each fused to repeating units. The two repeats of AM (R1 and R2) target the enzyme to the septum, where it cleaves murein between dividing cells. We have determined the crystal structure of R2, which reveals that each repeat folds into two half-open Ξ²-barrel subunits. We further demonstrate that lipoteichoic acid serves as a receptor for the repeats and that this interaction depends on conserved surfaces in each subunit. Small-angle X-ray scattering of the mature amidase reveals the presence of flexible linkers separating the AM, R1, and R2 units. Different levels of flexibility for each linker provide mechanistic insights into the conformational dynamics of the full-length protein and the roles of its components in cell wall association and catalysis. Our analysis supports a model in which the repeats direct the catalytic AM domain to the septum, where it can optimally perform the final step of cell division
New Photochemical Properties of Azidoaniline and Ciprofloxacin
There is a trend to find new ways of using photocatalysis in order to synthesize valuable products or to control or track live processes with special fluorescence-based molecular probes. The paper presents some results concerning new photochemical properties of azidoaniline, its 7-nitrobenzofurazan (NBD) derivative and ciprofloxacin derivatives
Allosteric competitive inactivation of hematopoietic CSF-1 signaling by the viral decoy receptor BARF1
Hematopoietic human colony-stimulating factor 1 (hCSF-1) is essential for innate and adaptive immunity against viral and microbial infections and cancer. The human pathogen Epstein-Barr virus secretes the lytic-cycle protein BARF1 that neutralizes hCSF-1 to achieve immunomodulation. Here we show that BARF1 binds the dimer interface of hCSF-1 with picomolar affinity, away from the cognate receptor-binding site, to establish a long-lived complex featuring three hCSF-1 at the periphery of the BARF1 toroid. BARF1 locks dimeric hCSF-1 into an inactive conformation, rendering it unable to signal via its cognate receptor on human monocytes. This reveals a new functional role for hCSF-1 cooperativity in signaling. We propose a new viral strategy paradigm featuring an allosteric decoy receptor of the competitive type, which couples efficient sequestration and inactivation of the host growth factor to abrogate cooperative assembly of the cognate signaling complex
Atomic Force Microscopy Study of ProteinβProtein Interactions in the Cytochrome CYP11A1 (P450scc)-Containing Steroid Hydroxylase System
Atomic force microscopy (AFM) and photon correlation spectroscopy (PCS) were used for monitoring of the procedure for cytochrome CYP11A1 monomerization in solution without phospholipids. It was shown that the incubation of 100 ΞΌM CYP11A1 with 12% Emulgen 913 in 50 mM KP, pH 7.4, for 10 min at T = 22Β°C leads to dissociation of hemoprotein aggregates to monomers with the monomerization degree of (82 Β± 4)%. Following the monomerization procedure, CYP11A1 remained functionally active. AFM was employed to detect and visualize the isolated proteins as well as complexes formed between the components of the cytochrome CYP11A1-dependent steroid hydroxylase system. Both Ad and AdR were present in solution as monomers. The typical heights of the monomeric AdR, Ad and CYP11A1 images were measured by AFM and were found to correspond to the sizes 1.6 Β± 0.2 nm, 1.0 Β± 0.2 nm and 1.8 Β± 0.2 nm, respectively. The binary Ad/AdR and AdR/CYP11A1mon complexes with the heights 2.2 Β± 0.2 nm and 2.8 Β± 0.2 nm, respectively, were registered by use of AFM. The Ad/CYP11A1mon complex formation reaction was kinetically characterized based on optical biosensor data. In addition, the ternary AdR/Ad/CYP11A1 complexes with a typical height of 4 Β± 1 nm were AFM registered