Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma

Our plain-English summary of Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma, by P. Scalia, C. Merali, C. Barrero, et al (International Journal of Molecular Sciences, 2023).

The study

• Authors: P. Scalia, C. Merali, C. Barrero, A. Suma, V. Carnevale, S. Merali, S.J. Williams
• Journal: International Journal of Molecular Sciences
• Published: 2023;24(8)
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.

In plain English

This laboratory study used cultured cancer cells. It did not study asbestos exposure or patients. The authors looked for the cell machinery that breaks down a protein called EphB4 in mesothelioma cells when IGF-II, a growth signal the cells make themselves, is blocked. They worked mainly with one malignant mesothelioma cell line, MSTO211H, blocking IGF-II with a neutralizing antibody, and compared it with HelaS3 cells. Using protein analysis, binding tests, gene sequencing and computer modeling, they reported that four proteins (UBA1, UBE2N, DTX3 and Cdc48/p97) bound more strongly to the tail end of EphB4 when IGF-II was blocked. They also reported that blocking the activity of Cdc48/p97 interfered with EphB4 recruitment. HelaS3 cells expressed the known DTX3a form. MSTO211H cells expressed a previously unknown form, which the authors named DTX3c and which computer models predicted folds differently. The authors concluded this was new evidence of DTX3 involvement outside the Notch signaling pathway. They said further work, for example in patient-derived cells, is needed to confirm the relationship in cancer.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study of cultured cell lines using proteomics, protein pull-down, ELISA and co-immunoprecipitation, PCR cloning and sequencing, and computer 3D structure modeling
• Who was studied: Cultured human cell lines, not patients: the malignant mesothelioma cell line MSTO211H (main focus) and HelaS3 cells. HUVEC cell lysates were used as a control source of EphB4 interactors.
• Exposure: No asbestos or fiber exposure was studied. The experimental treatment was blocking the cells' own IGF-II growth signal by adding a neutralizing anti-IGF-II antibody to the culture medium, evaluated after 12 hours. Some experiments also used a proteasome inhibitor (MG132) and a Cdc48/p97 inhibitor (ML240) at 200 nM.
• Main results:
  • Protein analysis of EphB4 complexes from MSTO211H cells identified ubiquitin-related proteins among the EphB4 partners that responded to blocking IGF-II: UBA1 (E1 ligase), UBE2N (E2 ligase), DTX3 (putative E3 ligase) and the chaperone Cdc48/p97.
  • In MSTO211H cell extracts, binding of UBA1, DTX3, UBE2N and Cdc48/p97 to the last 30 amino acids of EphB4 was reported as markedly increased when the IGF-II signal was blocked.
  • ELISA and co-immunoprecipitation in MSTO211H lysates confirmed that DTX3 associated with the other complex components, and that this binding depended on IGF-II signal status.
  • In MSTO211H lysates, inhibiting Cdc48/p97 activity interfered with EphB4 recruitment after IGF-II signal deprivation. The authors reported that coprecipitation of EphB4 required a fully active Cdc48/p97.
  • HelaS3 cells expressed the known DTX3a isoform. Sequencing of the product from MSTO211H cells revealed a new N-terminal DTX3 isoform, which the authors named DTX3c and submitted to GenBank.
  • In computer models, the most common predicted structures of the DTX3a and DTX3b N-terminal regions were similar to each other (RMSD 5.5 Å). Both differed from DTX3c (RMSD 12.2 Å versus DTX3a and 12.9 Å versus DTX3b).
• The authors' conclusion: The authors concluded that their findings provide new evidence that the DTX3 E3 ligase is directly involved in a cellular pathway other than Notch signaling. They stated their findings are in line with the hypothesis that DTX3c may explain why EphB4 levels depend on the IGF-II signal in MSTO211H cells but not in HelaS3 cells.
• Limitations the authors state:
  • The authors stated that further investigation is needed to confirm the relationship in cancer, for example through screening in patient-derived cells.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma.

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