3 validated tools for ubiquitination studies

Elongation of ubiquitin chains, regardless of the linkage, can form polyubiquitin fibrils to initiate the Autophagy pathway. A deficiency of the autophagy causes cytotoxic accumulation of ubiquitin-positive aggregates leading thus  to neurodegenerative diseases (1). A new deubiquitinating enzyme, USP36, highly expressed in human breast and lung cancers, was identified to regulate c-Myc oncoprotein stability in nucleolus. (2) Also, Itch, an E3 HECT Ubiquitin ligase, inhibits MAPK p38α activation through ubiquitylation can be exploited therapeutically to prevent chronic skin inflammation (3). A recent publication by Yumimoto K. et al. determined that expression of FBXW7, the F-box Protein of SCF Ubiquitin Ligase, can suppress cancer metastasis in either non-cell-autonomous or cell-autonomous manner. (4)

Ub proteolysis pathway

Ub and F-box proteins proteolysis pathways.

 

Immunohistochemistry staining of formalin-fixed paraffin-embedded human lung using FBXW7 monoclonal antibody (3ug/ml) (Cat. No. 157H00055294-M02).

Immunohistochemistry staining of formalin-fixed paraffin-embedded human lung using FBXW7 monoclonal antibody (3ug/ml) (Cat. No. 157H00055294-M02).

In this work, researchers used the anti FBXW7 monoclonal antibody  to perform immuno-cyto-chemical staining on breast tissues.

Other tools to study ubiquitination include the use of Tandem Ubiquitin Binding Entities (TUBES) for the isolation and detection of polyubiquitylated proteins. TUBES can be used, in combination with Anti Linear Polyubiquitin antibody to investifgate Uquitin linkages on substrates.

References

  1. Morimoto, D., et.al. (2015). Nature communications. DOI: 10.1038/ncomms7116.
  2. Sun, X.-X., et.al. (2015). PNAS. DOI: 10.1073/pnas.1411713112.
  3. Theivanthiran, B., et.al. (2015). Science. DOI: 10.1126/scisignal.2005903.
  4. Yumimoto, K., et.al. (2015) JCI. DOI: 10.172/JCI78782.

If you are studying the role of ubiquitin and ubiquitination in your model, please leave your comment!

E3 Ligases: RING- or HECT-mediated ubiquitination?

A large number of cellular processes are regulated by the reversible conjugation of Ubiquitin (UB) proteins to substrates. UB-related research tools (including E1-, E2 and E3 Ligases) are now commercially available for further studying the role of this “Ubiquitin/Proteasome” pathway. Nevertheless, the identification of UB Ligase inhibitors remains challenging (1). Such bioactive small molecules are indeed important to further characterize the actors of this pathway, and to find new compounds for therapeutic applications.

Untill now, only a few bioactive molecules were characterized as Ub – Proteasome inhibitors. For example, TAME is known as a Ub-ligase inhibitor while Thialidomide and SMER3 specifically inhibit E3 UB ligase. (2-4) [Read more…]

Proteasome and (de)ubiquitination enzyme inhibition

In 2014, the U.S. Food and Drug Administration (FDA) approved the proteasome inhibiting drug Velcade (Bortezomib) for the retreatment of adult patients with Multiple Myeloma. My colleague Philippe Fixe discussed the use of Bortezomib in his post Proteasome inhibitor approved by FDA for Myeloma retreatment.

In this post, I’d like to give you an overview of the compounds which can be used to inhibit proteasome activity and furthermore the activity of De-ubiquitinases (DUB, Isopeptidases), enzymes which catalyze the de-ubiquitination of ubiquitinated proteins and which are seen as very promising drug targets especially in cancer drug development. [Read more…]

TUBEs – efficiently detect & purify poly-ubiquitinated proteins

Traditional methods to detect and purify poly-ubiquitinated proteins require either anti ubiquitin antibodies or Ubiquitin Binding Associated domains (UBAs) which display rather low affinity for Ubiquitin and show only little (if any) specificity for specific ubiquitin linkages (e.g. K63 or K48). Furthermore, these strategies require the inclusion of inhibitors of both Deubiquinating enzymes (DUBs) and Proteasome activity to protect the integrity of poly-ubiquitylated proteins, which might alter cell physiology, which in turn may negatively impact the result or introduce experimental artifacts.

To overcome these problems, LifeSensors have developed Tandem Ubiquitin Binding Entities (TUBEs). [Read more…]

15 Ubiquitin – Proteasome inhibitors for cancer research

With more than 100 types of cancers affecting any part of the body, cancer is one of the leading causes of death in the world. It has an enormous impact on well-being, both for those with the diagnosis and for those who surround them. Understanding and eventually treating or preventing this diverse group of cell growth diseases has been, and will continue to be a major focus of the medical and research communities.

A variety of popular cancer research tools exists and can be divised into categories. We’ll try to summarize them into few posts. Today, I’d like to focus on inhibitors known to be active on the Ubiquitin/Proteasome Pathway.

[Read more…]

Proteasome inhibitor approved by FDA for Myeloma retreatment

The U.S. Food and Drug Administration (FDA) has approved Velcade (Bortezomib) for the retreatment of adult patients with Multiple Myeloma who had previously responded to Velcade therapy and relapsed at least six months following completion of prior treatment. (1)

Bortezomib (marketed as Velcade by Millennium Pharmaceuticals Inc.) is a potent and selective Proteasome inhibitor. This di-peptide (formerly called PS-341)  was the first therapeutic proteasome to be tested in humans and approved in the U.S. for treating Multiple Myeloma (2, 3). Besides Multiple Myeloma, Bortezomib was also cleared to treat patients with Mantle Cell Lymphoma.

The proteasome regulates protein expression and function by degradation of ubiquitylated proteins. By interacting with the catalytic site of the 26S proteasome, Bortezomib prevents pro-apoptotic factor degradation thus allowing the activation of apoptosis in malignant cells.

This shows that the importance of the Proteasome / Ubiquitin pathway and small bioactive molecules in Drug Discovery research programs and anti-cancer therapy arsenal.

References

  1. FDA Approves VELCADE® (bortezomib) Retreatment in Patients with Multiple Myeloma (August 2014), – Millennium (A Takeda Oncology Company).
  2. Kane R.C. et al. “Velcade®: U.S. FDA Approval for the Treatment of Multiple Myeloma Progressing on Prior Therapy” The Oncologist (2003), vol. 8 – no. 6, p508-513. doi: 10.1634/theoncologist.8-6-508.
  3. Velcade (bortezomib) is Approved for Initial Treatment of Patients with Multiple Myeloma (2008) – U.S. Food and Drug Administration (FDA)

    Bortezomib structure Velcade®). Source: Focus Biomolecules.

    Bortezomib structure (Velcade®). Source: Focus Biomolecules.

See also

A selection of ultra-pure, cell permeable and potent Proteasome Inhibitors for R&D purposes:

What about you?

Which Proteasome inhibitor are you looking for? Leave a comment on this post or share your thoughts with other readers!

3 monoclonals to discriminate free Ubiquitin from linear and polyubiquitin chains

Ubiquitin (Ub) is an 8 kDa  highly conserved polypeptide, commonly expressed in eukaryotic cells. Ub is added to lysine residues of the target proteins. This post-translational modification (known as ubiquitination) is made through the sequential action of 3 enzymes (E1 Ub activating enzyme, E2 conjugating enzyme and E3 ligase).

[Read more…]