Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.
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PMID:Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases. 2383 92

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. The ASB proteins function as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that specifically transfer ubiquitin to cellular proteins targeting them for degradation by the proteasome. ASB9 binds to creatine kinase (CK) and targets it for degradation; however, the way in which ASB9 interacts with CK is not yet known. We present a complete characterization of the binding of ASB9 to CK. One ASB9 molecule binds to a dimer of CK. The binding affinity of ASB9(1-252) was extremely tight, and no dissociation could be observed. Deletion of the 34 N-terminal amino acids forming ASB9(35-252) resulted in weakening of the binding, so that a binding affinity of 2.6 nM could be measured. Amide hydrogen-deuterium exchange (HDXMS) experiments showed that both ASB9(1-252) and ASB9(35-252) protected the same region of CK, residues 182-203, which forms one side of the active site. The HDXMS experiments indicated that the N-terminal disordered region and first ankyrin repeat of ASB9 are protected from exchange in the complex. Molecular docking yielded a structural model consistent with all of the data that suggested the N-terminal residues of ASB9(1-252) may lie in one CK active site. This model was corroborated by enzymatic activity assays and mutational analysis.
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PMID:How the ankyrin and SOCS box protein, ASB9, binds to creatine kinase. 2565 63