Targeted regulation of protein levels is an important tool to investigate the role of proteins essential for cell function and development. In recent years, methods based on the Escherichia coli dihydrofolate reductase destabilization domain (ecDHFR DD) have been established and used in various cell types. ecDHFR DD destabilizes the fused protein of interest and causes its degradation by proteasomes, unless it is stabilized by a specific ligand, trimethoprim. In this work we developed an inducible protein stabilization system in Leishmania mexicana based on ecDHFR DD.

• Klíčová slova
• Leishmania mexicana, Protein stabilization, Trimethoprim, ecDHFR,
• MeSH
• aktivace transkripce * MeSH
• dihydrofolátreduktasa genetika   metabolismus   MeSH
• Escherichia coli enzymologie   genetika   MeSH
• Leishmania mexicana genetika   metabolismus   MeSH
• molekulární biologie metody   MeSH
• parazitologie metody   MeSH
• regulace genové exprese * MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• trimethoprim metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dihydrofolátreduktasa MeSH
• rekombinantní proteiny MeSH
• trimethoprim MeSH

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates several key physiological and pathophysiological processes, and its dysregulation has been implicated in obesity, diabetes, and cancer. CaMKK2 is inhibited through phosphorylation in a process involving binding to the scaffolding 14-3-3 protein, which maintains CaMKK2 in the phosphorylation-mediated inhibited state. The previously reported structure of the N-terminal CaMKK2 14-3-3-binding motif bound to 14-3-3 suggested that the interaction between 14-3-3 and CaMKK2 could be stabilized by small-molecule compounds. Thus, we investigated the stabilization of interactions between CaMKK2 and 14-3-3γ by Fusicoccin A and other fusicoccanes-diterpene glycosides that bind at the interface between the 14-3-3 ligand binding groove and the 14-3-3 binding motif of the client protein. Our data reveal that two of five tested fusicoccanes considerably increase the binding of phosphopeptide representing the 14-3-3 binding motif of CaMKK2 to 14-3-3γ. Crystal structures of two ternary complexes suggest that the steric contacts between the C-terminal part of the CaMKK2 14-3-3 binding motif and the adjacent fusicoccane molecule are responsible for differences in stabilization potency between the study compounds. Moreover, our data also show that fusicoccanes enhance the binding affinity of phosphorylated full-length CaMKK2 to 14-3-3γ, which in turn slows down CaMKK2 dephosphorylation, thus keeping this protein in its phosphorylation-mediated inhibited state. Therefore, targeting the fusicoccin binding cavity of 14-3-3 by small-molecule compounds may offer an alternative strategy to suppress CaMKK2 activity by stabilizing its phosphorylation-mediated inhibited state.

• MeSH
• fosforylace účinky léků   MeSH
• glykosidy chemie   farmakologie   MeSH
• kinasa proteinkinasy závislé na vápníku a kalmodulinu chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• mapy interakcí proteinů účinky léků   MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• vazba proteinů účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CAMKK2 protein, human MeSH Prohlížeč
• fusicoccin MeSH Prohlížeč
• glykosidy MeSH
• kinasa proteinkinasy závislé na vápníku a kalmodulinu MeSH
• proteiny 14-3-3 MeSH

Modern computational tools can predict the mutational effects on protein stability, sometimes at the expense of activity or solubility. Here, we investigate two homologous computationally stabilized haloalkane dehalogenases: (i) the soluble thermostable DhaA115 (Tmapp = 74 °C) and (ii) the poorly soluble and aggregating thermostable LinB116 (Tmapp = 65 °C), together with their respective wild-type variants. The intriguing difference in the solubility of these highly homologous proteins has remained unexplained for three decades. We combined experimental and in-silico techniques and examined the effects of stabilization on solubility and aggregation propensity. A detailed analysis of the unfolding mechanisms in the context of aggregation explained the negative consequences of stabilization observed in LinB116. With the aid of molecular dynamics simulations, we identified regions exposed during the unfolding of LinB116 that were later found to exhibit aggregation propensity. Our analysis identified cryptic aggregation-prone regions and increased surface hydrophobicity as key factors contributing to the reduced solubility of LinB116. This study reveals novel molecular mechanisms of unfolding for hyperstabilized dehalogenases and highlights the importance of contextual information in protein engineering to avoid the negative effects of stabilizing mutations on protein solubility.

• MeSH
• hydrofobní a hydrofilní interakce MeSH
• hydrolasy * chemie   metabolismus   genetika   MeSH
• proteinové agregáty * MeSH
• rozbalení proteinů * MeSH
• rozpustnost MeSH
• simulace molekulární dynamiky MeSH
• stabilita proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy * MeSH
• proteinové agregáty * MeSH

Integral membrane proteins carry out essential functions in the cell, and their activities are often modulated by specific protein-lipid interactions in the membrane. Here, we elucidate the intricate role of cardiolipin (CDL), a regulatory lipid, as a stabilizer of membrane proteins and their complexes. Using the in silico-designed model protein TMHC4_R (ROCKET) as a scaffold, we employ a combination of molecular dynamics simulations and native mass spectrometry to explore the protein features that facilitate preferential lipid interactions and mediate stabilization. We find that the spatial arrangement of positively charged residues as well as local conformational flexibility are factors that distinguish stabilizing from non-stabilizing CDL interactions. However, we also find that even in this controlled, artificial system, a clear-cut distinction between binding and stabilization is difficult to attain, revealing that overlapping lipid contacts can partially compensate for the effects of binding site mutations. Extending our insights to naturally occurring proteins, we identify a stabilizing CDL site within the E. coli rhomboid intramembrane protease GlpG and uncover its regulatory influence on enzyme substrate preference. In this work, we establish a framework for engineering functional lipid interactions, paving the way for the design of proteins with membrane-specific properties or functions.

• Klíčová slova
• E. coli, lipid binding, mass spectrometry, membrane protein, molecular biophysics, structural biology,
• MeSH
• DNA vazebné proteiny MeSH
• endopeptidasy metabolismus   chemie   genetika   MeSH
• Escherichia coli metabolismus   genetika   MeSH
• kardiolipiny * metabolismus   chemie   MeSH
• membránové proteiny * metabolismus   chemie   genetika   MeSH
• proteinové inženýrství * MeSH
• proteiny z Escherichia coli * metabolismus   chemie   genetika   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• endopeptidasy MeSH
• GlpG protein, E coli MeSH Prohlížeč
• kardiolipiny * MeSH
• membránové proteiny * MeSH
• proteiny z Escherichia coli * MeSH

Proline-tryptophan complexes derived from experimental structures are investigated by quantum chemical procedures known to properly describe the London dispersion energy. We study two geometrical arrangements: the "L-shaped", stabilized by an H-bond, and the "stacked-like", where the two residues are in parallel orientation without any H-bond. Interestingly, the interaction energies in both cases are comparable and very large ( approximately 7 kcal mol(-1)). The strength of stabilization in the stacked arrangement is rather surprising considering the fact that only one partner has an aromatic character. The interaction energy decomposition using the SAPT method further demonstrates the very important role of dispersion energy in such arrangement. To elucidate the structural features responsible for this unexpectedly large stabilization we examined the role of the nitrogen heteroatom and the importance of the cyclicity of the proline residue. We show that the electrostatic interaction due to the presence of the dipole, caused by the nitrogen heteroatom, contributes largely to the strength of the interaction. Nevertheless, the cyclic arrangement of proline, which allows for the largest amount of dispersive contact with the aromatic partner, also has a notable-effect. Geometry optimizations carried out for the "stacked-like" complexes show that the arrangements derived from protein structure are close to their gas phase optimum geometry, suggesting that the environment has only a minor effect on the geometry of the interaction. We conclude that the strength of proline non-covalent interactions, combined with this residue's rigidity, might be the explanation for its prominent role in protein stabilization and recognition processes.

• MeSH
• chemické modely MeSH
• fyzikální chemie metody   MeSH
• konformace proteinů MeSH
• ligandy MeSH
• mapování interakce mezi proteiny MeSH
• molekulární konformace MeSH
• prolin chemie   MeSH
• proteiny chemie   MeSH
• statická elektřina MeSH
• terciární struktura proteinů MeSH
• tryptofan chemie   MeSH
• vazba proteinů MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ligandy MeSH
• prolin MeSH
• proteiny MeSH
• tryptofan MeSH

In protein-based formulations, conformational distortions and attractive interactions may cause insoluble and undesired aggregates. In the case of ionic peptides, including cationic or anionic, commonly electrostatic interactions are the main factors that control structure assembling. In this study, it was proposed that grafting of chitosan (CS) to γ-polyglutamic acid (γ-PGA) might exhibit much strong inhibiting effect on the formation of protein aggregates due to multiple amino groups and hydrophilic properties. To guarantee stable and safe biopharmaceutical formulation, the potency of a variety of stabilizers including sugars (glucose, sucrose), polyols (sorbitol, glycerol), surfactant (Tween 20), salting-out salt (PBS), and also different pH values have been evaluated on stabilizing or destabilizing the native state of CS-g-PGA copolymer using FTIR, CD, DLS, and SDS-PAGE. The comparable analysis revealed that the stability of CS-g-PGA was strongly dependent on pH owing to the polyelectrolyte characteristics of the polymers. Altogether these results implied that CS at optimized conditions might be an important precursor for the pharmaceutical industry and function as a new polymer for aggregation suppression and protein stabilization.

• Klíčová slova
• Chemical stabilization, Chitosan, Protein folding, pH depending structure, γ-polyglutamic acid,
• MeSH
• chitosan * chemie   MeSH
• koncentrace vodíkových iontů MeSH
• kyselina polyglutamová * chemie   analogy a deriváty   MeSH
• rozpouštědla chemie   MeSH
• stabilita proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chitosan * MeSH
• kyselina polyglutamová * MeSH
• poly(gamma-glutamic acid) MeSH Prohlížeč
• rozpouštědla MeSH

Viruses have evolved mechanisms to manipulate microtubules (MTs) for the efficient realization of their replication programs. Studying the mechanisms of replication of mouse polyomavirus (MPyV), we observed previously that in the late phase of infection, a considerable amount of the main structural protein, VP1, remains in the cytoplasm associated with hyperacetylated microtubules. VP1-microtubule interactions resulted in blocking the cell cycle in the G2/M phase. We are interested in the mechanism leading to microtubule hyperacetylation and stabilization and the roles of tubulin acetyltransferase 1 (αTAT1) and deacetylase histone deacetylase 6 (HDAC6) and VP1 in this mechanism. Therefore, HDAC6 inhibition assays, αTAT1 knock out cell infections, in situ cell fractionation, and confocal and TIRF microscopy were used. The experiments revealed that the direct interaction of isolated microtubules and VP1 results in MT stabilization and a restriction of their dynamics. VP1 leads to an increase in polymerized tubulin in cells, thus favoring αTAT1 activity. The acetylation status of MTs did not affect MPyV infection. However, the stabilization of MTs by VP1 in the late phase of infection may compensate for the previously described cytoskeleton destabilization by MPyV early gene products and is important for the observed inhibition of the G2→M transition of infected cells to prolong the S phase.

• Klíčová slova
• VP1, histone deacetylase 6, microtubule acetylation, microtubule stabilization, microtubules, mouse polyomavirus, α-tubulin acetyltransferase 1,
• MeSH
• acetylace MeSH
• acetyltransferasy genetika   metabolismus   MeSH
• buněčné linie MeSH
• buněčný cyklus MeSH
• cytoplazma metabolismus   MeSH
• fibroblasty virologie   MeSH
• histondeacetylasa 6 genetika   metabolismus   MeSH
• interakce mikroorganismu a hostitele * MeSH
• mikrotubuly metabolismus   virologie   MeSH
• myši MeSH
• Polyomavirus genetika   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• tubulin metabolismus   MeSH
• virové plášťové proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetyltransferasy MeSH
• Hdac6 protein, mouse MeSH Prohlížeč
• histondeacetylasa 6 MeSH
• tubulin MeSH
• virové plášťové proteiny MeSH
• VP1 protein, polyomavirus MeSH Prohlížeč

Microtubules, polymers of the heterodimeric protein αβ-tubulin, are indispensable for many cellular activities such as maintenance of cell shape, division, migration, and ordered vesicle transport. In vitro assays to study microtubule functions and their regulation by associated proteins require the availability of assembly-competent purified tubulin. However, tubulin is a thermolabile protein that rapidly converts into non-polymerizing state. For this reason it is usually stored at -80 °C to preserve its conformation and polymerization properties. In this chapter we describe a method for freeze-drying of assembly-competent tubulin in the presence of nonreducing sugar trehalose and methods enabling evaluation of tubulin functions in rehydrated samples.

• MeSH
• lyofilizace * MeSH
• proteiny chemie   MeSH
• trehalosa chemie   MeSH
• tubulin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny MeSH
• trehalosa MeSH
• tubulin MeSH

Several signaling pathways that monitor the dynamic state of the cell converge on the tumor suppressor p53. The ability of p53 to process these signals and exert a dynamic downstream response in the form of cell cycle arrest and/or apoptosis is crucial for preventing tumor development. This p53 function is abrogated by p53 gene mutations leading to alteration of protein conformation. Hsp90 has been implicated in regulating both wild-type and mutant p53 conformations, and Hsp90 antagonists are effective for the therapy of some human tumors. Using cell lines that contain human tumor-derived temperature-sensitive p53 mutants we show that Hsp90 is required for both stabilization and reactivation of mutated p53 at the permissive temperature. A temperature decrease to 32 degrees C causes conversion to a protein conformation that is capable of inducing expression of MDM2, leading to reduction of reactivated p53 levels by negative feedback. Mutant reactivation is enhanced by simultaneous treatment with agents that stabilize the reactivated protein and is blocked by geldanamycin, a specific inhibitor of Hsp90 activity, indicating that Hsp90 antagonist therapy and therapies that act to reactivate mutant p53 will be incompatible. In contrast, Hsp90 is not required for maintaining wild-type p53 or for stabilizing wild-type p53 after treatment with chemotherapeutic agents, indicating that Hsp90 therapy might synergize with conventional therapies in patients with wild-type p53. Our data demonstrate the importance of the precise characterization of the interaction between p53 mutants and stress proteins, which may shed valuable information for fighting cancer via the p53 tumor suppressor pathway.

• MeSH
• genetická transkripce MeSH
• jaderné proteiny genetika   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mutace * MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   fyziologie   MeSH
• nádory patologie   MeSH
• proteiny tepelného šoku HSP90 metabolismus   fyziologie   MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny genetika   MeSH
• regulace genové exprese u nádorů MeSH
• teplota * MeSH
• zpětná vazba fyziologická MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• proteiny tepelného šoku HSP90 MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH

p53 missense mutant proteins commonly show increased stability compared to wild-type p53, which is thought to depend largely on the inability of mutant p53 to induce the ubiquitin ligase MDM2. However, recent work using mouse models has shown that the accumulation of mutant p53 occurs only in tumour cells, indicating that stabilization requires additional factors. To clarify the stabilization of p53 mutants in tumours, we analysed factors that affect their folding and degradation. Although all missense mutants that we studied are more stable than wild-type p53, the levels correlate with individual structural characteristics, which may be reflected in different gain-of-function properties. In the absence of Hsp90 activity, the less stable unfolded p53 mutants preferentially associate in a complex with Hsp70 and CHIP (carboxy terminus of Hsp70-interacting protein), and we show that CHIP is responsible for ubiquitination and degradation of these mutants. The demonstration of a complex interplay between Hsp90, Hsp70 and CHIP that regulate the stability of different p53 mutant proteins improves our understanding of the pro-tumorigenic effects of increased Hsp90 activity during multi-stage carcinogenesis. Understanding the roles of Hsp90, Hsp70 and CHIP in cancers may also provide an important avenue through which to target p53 to enhance treatment of human cancers.

• MeSH
• benzochinony farmakologie   MeSH
• ELISA MeSH
• fibroblasty cytologie   metabolismus   MeSH
• imunoblotting MeSH
• imunoprecipitace MeSH
• konformace proteinů MeSH
• kultivované buňky MeSH
• lidé MeSH
• makrocyklické laktamy farmakologie   MeSH
• mutace genetika   MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorový supresorový protein p53 chemie   fyziologie   MeSH
• nádory genetika   metabolismus   patologie   MeSH
• proteiny tepelného šoku HSC70 genetika   metabolismus   MeSH
• proteiny tepelného šoku HSP90 antagonisté a inhibitory   genetika   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 fyziologie   MeSH
• ubikvitinace MeSH
• ubikvitinligasy genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzochinony MeSH
• makrocyklické laktamy MeSH
• MDM2 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• proteiny tepelného šoku HSC70 MeSH
• proteiny tepelného šoku HSP90 MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• STUB1 protein, human MeSH Prohlížeč
• Stub1 protein, mouse MeSH Prohlížeč
• tanespimycin MeSH Prohlížeč
• ubikvitinligasy MeSH