Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.
- Klíčová slova
- ASK1, MAP3K, MAPK signaling, biochemistry, chemical biology, human, molecular biophysics, protein kinase, structural biology, thioredoxin,
- MeSH
- apoptóza MeSH
- biofyzika MeSH
- elektronová kryomikroskopie MeSH
- MAP kinasa-kinasa-kinasa 5 * MeSH
- thioredoxiny * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- MAP kinasa-kinasa-kinasa 5 * MeSH
- thioredoxiny * MeSH
Procaspase-2 phosphorylation at several residues prevents its activation and blocks apoptosis. This process involves procaspase-2 phosphorylation at S164 and its binding to the scaffolding protein 14-3-3. However, bioinformatics analysis has suggested that a second phosphoserine-containing motif may also be required for 14-3-3 binding. In this study, we show that human procaspase-2 interaction with 14-3-3 is governed by phosphorylation at both S139 and S164. Using biochemical and biophysical approaches, we show that doubly phosphorylated procaspase-2 and 14-3-3 form an equimolar complex with a dissociation constant in the nanomolar range. Furthermore, our data indicate that other regions of procaspase-2, in addition to phosphorylation motifs, may be involved in the interaction with 14-3-3.
- Klíčová slova
- 14-3-3, Caspase-2, Phosphorylation, Procaspase-2, Protein-protein interaction,
- MeSH
- fosforylace MeSH
- kaspasa 2 chemie metabolismus MeSH
- lidé MeSH
- proteinové domény MeSH
- proteiny 14-3-3 metabolismus MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kaspasa 2 MeSH
- proteiny 14-3-3 MeSH
- rekombinantní proteiny MeSH
Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) activates p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase in response to proinflammatory and stress signals. In nonstress conditions, ASK1 is inhibited by association with thioredoxin (TRX) which binds to the TRX-binding domain (ASK1-TBD) at the N terminus of ASK1. TRX dissociates in response to oxidative stress allowing the ASK1 activation. However, the molecular basis for the ASK1:TRX1 complex dissociation is still not fully understood. Here, the role of cysteine residues on the interaction between TRX1 and ASK1-TBD in both reducing and oxidizing conditions was investigated. We show that from the two catalytic cysteines of TRX1 the residue C32 is responsible for the high-affinity binding of TRX1 to ASK1-TBD in reducing conditions. The disulfide bond formation between C32 and C35 within the active site of TRX1 is the main factor responsible for the TRX1 dissociation upon its oxidation as the formation of the second disulfide bond between noncatalytic cysteines C62 and C69 did not have any additional effect. ASK1-TBD contains seven conserved cysteine residues which differ in solvent accessibility with the residue C250 being the only cysteine which is both solvent exposed and essential for TRX1 binding in reducing conditions. Furthermore, our data show that the catalytic site of TRX1 interacts with ASK1-TBD region containing cysteine C200 and that the oxidative stress induces intramolecular disulfide bond formation within ASK1-TBD and affects its structure in regions directly involved and/or important for TRX1 binding.
- Klíčová slova
- TRX, ASK1, cysteine, disulfide bond, mass spectrometry,
- MeSH
- cystein chemie MeSH
- kinetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- MAP kinasa-kinasa-kinasa 5 chemie genetika metabolismus MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- peptidové fragmenty chemie genetika metabolismus MeSH
- proteinové domény MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- substituce aminokyselin MeSH
- thioredoxiny chemie genetika metabolismus MeSH
- vazebná místa genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cystein MeSH
- MAP kinasa-kinasa-kinasa 5 MeSH
- MAP3K5 protein, human MeSH Prohlížeč
- peptidové fragmenty MeSH
- rekombinantní proteiny MeSH
- thioredoxiny MeSH
- TXN protein, human MeSH Prohlížeč
Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, regulates diverse physiological processes. The activity of ASK1 is triggered by various stress stimuli and is involved in the pathogenesis of cancer, neurodegeneration, inflammation, and diabetes. ASK1 forms a high molecular mass complex whose activity is, under non-stress conditions, suppressed through interaction with thioredoxin and the scaffolding protein 14-3-3. The 14-3-3 protein binds to the phosphorylated Ser-966 motif downstream of the ASK1 kinase domain. The role of 14-3-3 in the inhibition of ASK1 has yet to be elucidated. In this study we performed structural analysis of the complex between the ASK1 kinase domain phosphorylated at Ser-966 (pASK1-CD) and the 14-3-3ζ protein. Small angle x-ray scattering (SAXS) measurements and chemical cross-linking revealed that the pASK1-CD·14-3-3ζ complex is dynamic and conformationally heterogeneous. In addition, structural analysis coupled with the results of phosphorus NMR and time-resolved tryptophan fluorescence measurements suggest that 14-3-3ζ interacts with the kinase domain of ASK1 in close proximity to its active site, thus indicating this interaction might block its accessibility and/or affect its conformation.
- Klíčová slova
- 14-3-3 protein, apoptosis signal-regulating kinase 1 (ASK1), fluorescence, nuclear magnetic resonance (NMR), protein cross-linking, small-angle x-ray scattering (SAXS),
- MeSH
- difrakce rentgenového záření MeSH
- fosforylace MeSH
- katalytická doména MeSH
- lidé MeSH
- maloúhlový rozptyl MeSH
- MAP kinasa-kinasa-kinasa 5 antagonisté a inhibitory chemie genetika metabolismus MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- MAP kinasa-kinasa-kinasa 5 MeSH
- MAP3K5 protein, human MeSH Prohlížeč
- proteiny 14-3-3 MeSH
- YWHAE protein, human MeSH Prohlížeč
Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.
- Klíčová slova
- 14-3-3 protein, fluorescence, hydrogen-deuterium exchange, phosducin, protein complex, protein phosphorylation, small-angle x-ray scattering (SAXS),
- MeSH
- fosfoproteiny chemie genetika metabolismus MeSH
- fosforylace MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- molekulární modely MeSH
- oční proteiny chemie genetika metabolismus MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- proteiny vázající GTP - regulátory chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfoproteiny MeSH
- oční proteiny MeSH
- phosducin MeSH Prohlížeč
- proteiny 14-3-3 MeSH
- proteiny vázající GTP - regulátory MeSH
- YWHAZ protein, human MeSH Prohlížeč
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp(31) being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.
- Klíčová slova
- Analytical Ultracentrifugation, Apoptosis Signal-regulating Kinase 1 (ASK1), Circular Dichroism (CD), Fluorescence, Small-angle X-ray Scattering (SAXS), Thioredoxin,
- MeSH
- biofyzika MeSH
- cirkulární dichroismus MeSH
- fluorescenční spektrometrie MeSH
- konformace proteinů MeSH
- MAP kinasa-kinasa-kinasa 5 metabolismus MeSH
- oxidace-redukce MeSH
- thioredoxiny metabolismus MeSH
- ultracentrifugace MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- MAP kinasa-kinasa-kinasa 5 MeSH
- thioredoxiny MeSH
Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.
- Klíčová slova
- 14–3-3, Bmh, Calcium, Enzyme Mechanisms, H/D Exchange, Mass Spectrometry (MS), Neutral Trehalase, Protein Cross-linking, Protein Structure, SAXS,
- MeSH
- aktivace enzymů MeSH
- katalytická doména MeSH
- molekulární modely MeSH
- motivy EF-ruky * MeSH
- proteiny 14-3-3 chemie metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
- Saccharomyces cerevisiae enzymologie MeSH
- sekvence aminokyselin MeSH
- trehalasa chemie metabolismus MeSH
- vápník metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- BMH1 protein, S cerevisiae MeSH Prohlížeč
- proteiny 14-3-3 MeSH
- Saccharomyces cerevisiae - proteiny MeSH
- trehalasa MeSH
- vápník MeSH
Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.
- MeSH
- buněčná adheze MeSH
- buněčný cyklus MeSH
- dipeptidasy genetika metabolismus MeSH
- dipeptidylpeptidasa 4 genetika metabolismus MeSH
- dipeptidylpeptidasy a tripeptidylpeptidasy genetika metabolismus MeSH
- gliom enzymologie genetika MeSH
- imunomagnetická separace MeSH
- lidé MeSH
- mutace MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- pohyb buněk MeSH
- primární buněčná kultura MeSH
- proliferace buněk MeSH
- regulace genové exprese u nádorů * MeSH
- signální transdukce genetika MeSH
- stanovení celkové genové exprese MeSH
- transfekce MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dipeptidasy MeSH
- dipeptidylpeptidasa 4 MeSH
- dipeptidylpeptidasy a tripeptidylpeptidasy MeSH
- DPP4 protein, human MeSH Prohlížeč
- DPP8 protein, human MeSH Prohlížeč
- DPP9 protein, human MeSH Prohlížeč
