Apoptosis signal-regulating kinase (ASK) 1, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, modulates diverse responses to oxidative and endoplasmic reticulum (ER) stress and calcium influx. As a crucial cellular stress sensor, ASK1 activates c-Jun N-terminal kinases (JNKs) and p38 MAPKs. Their excessive and sustained activation leads to cell death, inflammation and fibrosis in various tissues and is implicated in the development of many neurological disorders, such as Alzheimer's, Parkinson's and Huntington disease and amyotrophic lateral sclerosis, in addition to cardiovascular diseases, diabetes and cancer. However, currently available inhibitors of JNK and p38 kinases either lack efficacy or have undesirable side effects. Therefore, targeted inhibition of their upstream activator, ASK1, stands out as a promising therapeutic strategy for treating such severe pathological conditions. This review summarizes recent structural findings on ASK1 regulation and its role in various diseases, highlighting prospects for ASK1 inhibition in the treatment of these pathologies.

• Klíčová slova
• 14-3-3, ASK1, MAP kinase, kinase, phosphorylation, protein–protein interaction,
• MeSH
• apoptóza fyziologie   MeSH
• fosforylace MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   MeSH
• lidé MeSH
• MAP kinasa-kinasa-kinasa 5 genetika   metabolismus   fyziologie   ultrastruktura   MeSH
• MAP kinasový signální systém MeSH
• MAP kinasy kinas (kinas) genetika   metabolismus   MeSH
• mapy interakcí proteinů genetika   fyziologie   MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• proteiny 14-3-3 metabolismus   MeSH
• proteiny regulující apoptózu metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• stres endoplazmatického retikula MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• JNK mitogenem aktivované proteinkinasy MeSH
• MAP kinasa-kinasa-kinasa 5 MeSH
• MAP kinasy kinas (kinas) MeSH
• MAP3K5 protein, human MeSH Prohlížeč
• mitogenem aktivované proteinkinasy p38 MeSH
• proteiny 14-3-3 MeSH
• proteiny regulující apoptózu MeSH

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase 5, which mediates various stress signals including oxidative stress. The catalytic activity of ASK1 is tightly controlled by oligomerization and binding of several cofactors. Among these cofactors, thioredoxin stands out as the most important ASK1 inhibitor, but only the reduced form of thioredoxin inhibits ASK1 by direct binding to its N-terminal domain. In addition, oxidation-driven thioredoxin dissociation is the key event in oxidative stress-mediated ASK1 activation. However, the structural mechanism of ASK1 regulation by thioredoxin remains unknown. Here, we report the characterization of the ASK1 domain responsible for thioredoxin binding and its complex using NMR spectroscopy and chemical cross-linking, thus providing the molecular basis for ASK1: thioredoxin complex dissociation under oxidative stress conditions. Our data reveal that the N-terminal domain of ASK1 adopts a fold resembling the thioredoxin structure while retaining substantial conformational plasticity at the thioredoxin-binding interface. Although oxidative stress induces relatively moderate structural changes in thioredoxin, the formation of intramolecular disulfide bridges leads to a considerable conformational rearrangement of the thioredoxin-binding interface on ASK1. Moreover, the cysteine residue at position 250 of ASK1 is the key element of this molecular switch. Finally, our results show that the redox-active site of thioredoxin is directly involved in ASK1 binding that is modulated by oxidative stress, thereby identifying a key target for the structure-based drug design.

• Klíčová slova
• apoptosis signal-regulating kinase 1, mitogen-activated protein kinase kinase kinase, oxidative stress, protein-protein interaction, thioredoxin,
• MeSH
• apoptóza * MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• MAP kinasa-kinasa-kinasa 5 antagonisté a inhibitory   metabolismus   MeSH
• molekulární modely MeSH
• oxidace-redukce MeSH
• oxidační stres * MeSH
• thioredoxiny chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory proteinkinas MeSH
• MAP kinasa-kinasa-kinasa 5 MeSH
• MAP3K5 protein, human MeSH Prohlížeč
• thioredoxiny MeSH

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, activates the p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase (JNK) signaling cascades in response to various stressors. ASK1 activity is tightly regulated through phosphorylation and interaction with various binding partners. However, the mechanistic details underlying the ASK1 regulation are still not fully understood. This review focuses on recent advances in structural studies of protein kinase ASK1 and on the insights they provide into its mechanism of regulation. In addition, we also discuss protein-protein interactions between ASK1 and its binding partners thioredoxin (TRX) and 14-3-3 protein.

• MeSH
• biologické modely MeSH
• fosforylace MeSH
• lidé MeSH
• MAP kinasa-kinasa-kinasa 5 genetika   metabolismus   MeSH
• mitogenem aktivované proteinkinasy p38 genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• thioredoxiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• MAP kinasa-kinasa-kinasa 5 MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• proteiny 14-3-3 MeSH
• thioredoxiny 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

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