Inflammatory responses mediated by the transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) play key roles in immunity, autoimmune diseases, and cancer. NF-κB is directly regulated through protein-protein interactions, including those with IκB and 14-3-3 proteins. These two important regulatory proteins have been reported to interact with each other, although little is known about this interaction. We analyzed the inhibitor of nuclear factor kappa B α (IκBα)/14-3-3σ interaction via a peptide/protein-based approach. Structural data were acquired via X-ray crystallography, while binding affinities were measured with fluorescence polarization assays and time-resolved tryptophan fluorescence. A high-resolution crystal structure (1.13 Å) of the uncommon 14-3-3 interaction motif of IκBα (IκBαpS63) in a complex with 14-3-3σ was evaluated. This motif harbors a tryptophan that makes this crystal structure the first one with such a residue visible in the electron density at that position. We used this tryptophan to determine the binding affinity of the unlabeled IκBα peptide to 14-3-3 via tryptophan fluorescence decay measurements.

• Publikační typ
• časopisecké články MeSH

The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ∼230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.

• MeSH
• antigeny nádorové genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• Cajalova tělíska genetika   metabolismus   MeSH
• HeLa buňky MeSH
• jaderné proteiny metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U4-U6 genetika   metabolismus   MeSH
• malý jaderný ribonukleoprotein U5 genetika   metabolismus   MeSH
• molekulární modely * MeSH
• nádorové buněčné linie MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• ribonukleoproteiny malé jaderné genetika   metabolismus   MeSH
• RNA-helikasy genetika   metabolismus   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   metabolismus   MeSH
• vazba proteinů genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• DHX15 protein, human MeSH Prohlížeč
• jaderné proteiny MeSH
• malý jaderný ribonukleoprotein U4-U6 MeSH
• malý jaderný ribonukleoprotein U5 MeSH
• p80-coilin MeSH Prohlížeč
• prekurzory RNA MeSH
• proteiny vázající RNA MeSH
• ribonukleoproteiny malé jaderné MeSH
• RNA-helikasy MeSH
• SART3 protein, human MeSH Prohlížeč
• SNRNP200 protein, human MeSH Prohlížeč

Mammalian pyruvate kinase exists in four isoforms with characteristics tuned to specific metabolic requirements of different tissues. All of the isoforms, except the muscle isoform, exhibit typical allosteric behavior. The case of the muscle isoform is a conundrum. It is inhibited by an allosteric inhibitor, Phe, yet it has traditionally not been considered as an allosteric enzyme. In this series of study, an energetic landscape of rabbit muscle pyruvate kinase (RMPK) was established. The phenomenon of inhibition by Phe is shown to be physiological. Furthermore, the thermodynamics for the temperature fluctuation and concomitant pH change as a consequence of muscle activity were elucidated. We have shown that (1) the differential number of protons released or absorbed with regard to the various linked reactions adds another level of control to shift the binding constants and equilibrium of active <--> inactive state changes (the latter controls quantitatively the activity of RMPK); (2) ADP plays a major role in the allosteric mechanism in RMPK under physiological temperatures (depending on the temperature, ADP can assume dual and opposite roles of being an inhibitor by binding preferentially to the inactive form and a substrate); and (3) simulation of the RMPK behavior under physiological conditions shows that the net results of the 21 thermodynamic parameters involved in the regulation are well-tuned to allow the maximal response of the enzyme to even minute changes in temperature and ligand concentration.

• MeSH
• adenosindifosfát chemie   metabolismus   MeSH
• aktivace enzymů MeSH
• alosterická regulace MeSH
• chemické modely MeSH
• energetický metabolismus MeSH
• fenylalanin chemie   MeSH
• kosterní svaly enzymologie   MeSH
• králíci MeSH
• ligandy MeSH
• počítačová simulace MeSH
• pyruvátkinasa antagonisté a inhibitory   chemie   metabolismus   fyziologie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosindifosfát MeSH
• fenylalanin MeSH
• ligandy MeSH
• pyruvátkinasa MeSH

Rabbit muscle pyruvate kinase (RMPK) is an important allosteric enzyme of the glycolytic pathway catalyzing a transfer of the phosphate from phosphoenolpyruvate (PEP) to ADP. The energetic landscape of the allosteric regulatory mechanism of RMPK was characterized by isothermal titration calorimetry (ITC) in the temperature range from 4 to 45 degrees C. ITC data for RMPK binding to substrates PEP and ADP, for the allosteric inhibitor Phe, and for combination of ADP and Phe were globally analyzed. The thermodynamic parameters characterizing the linked-multiple-equilibrium system were extracted. Four novel insights were uncovered. (1) The binding preference of ADP for either the T or R state is temperature-dependent, namely, more favorable to the T and R states at high and low temperatures, respectively. This crossover of affinity toward R and T states implies that ADP plays a complex role in modulating the allosteric behavior of RMPK. Depending on the temperature, binding of ADP can regulate RMPK activity by favoring the enzyme to either the R or T state. (2) The binding of Phe is negatively coupled to that of ADP; i.e., Phe and ADP prefer not to bind to the same subunit of RMPK. (3) The release or absorption of protons linked to the various equilibria is specific to the particular reaction. As a consequence, pH will exert a complex effect on these linked equilibria, resulting in the proton being an allosteric regulatory ligand of RMPK. (4) The R <--> T equilibrium is accompanied by a significant DeltaC(p), rendering RMPK most sensitive to temperature under physiological conditions. During muscle activity, both pH and temperature fluctuations are known to happen; thus, results of this study are physiologically relevant.

• MeSH
• adenosindifosfát metabolismus   MeSH
• alosterická regulace MeSH
• chemické modely MeSH
• energetický metabolismus MeSH
• fenylalanin metabolismus   MeSH
• kalorimetrie * metody   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• kosterní svaly enzymologie   MeSH
• králíci MeSH
• ligandy MeSH
• pyruvátkinasa antagonisté a inhibitory   chemie   metabolismus   fyziologie   MeSH
• termodynamika * MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• adenosindifosfát MeSH
• fenylalanin MeSH
• ligandy MeSH
• pyruvátkinasa MeSH