Recently, a new series of ready-to-use reagents for studying post-translation modifications (PTMs) of your proteins of interest have been released by Cytoskeleton, Inc. To illustrate the advantages of these research tools, this post is focuses on ubiquitination detection, which is known to be an important event in cell signalling.
Check whether your protein is ubiquitylated!
Detecting ubiquitylation
Ubitest Flow chart – LifeSensors at tebu-bio.com
Determining the linkage of polyubiquitin on target proteins is challenging. The traditional methods are either through Mass Spectrometry or immunoblot with linkage specific antibodies, which are cumbersome. LifeSensors has developed the UbiTest assay, a more definitive method for demonstrating the ubiquitylation linkage of a protein, which is to couple immunoprecipitation of polyubiquitylated protein with digestion by a linkage specific deubiquitylase prior to immunoblot analysis. An increased signal for the unmodified substrate or a decreased signal of polyubiquitylated substrate at high molecular weight after K48/K63 specific DUB treatment is a clear indication that the protein was K48/K63 ubiquitylated. [Read more…]
Easily customizable E3-ubiquitin ligase assay kits
A ready-to-use E3 ligase activity enzymatic assay, the E3LITE kit, has been recently designed enabling researchers to screen for E3 enzyme inhibitors and potentially new therapeutical drugs. What’s it for and why is it of interest?
TUBEs: study M1 (linear) polyubiquitylated proteins
Traditional methods to detect and purify poly-ubiquitinated proteins require either anti-ubiquitin antibodies or Ubiquitin Binding Associated domains (UBAs). Most of the current methods display rather low affinity for Ubiquitin (UBL) and show only little (if any) specificity for specific ubiquitin linkages (e.g. K63 or K48). To overcome these hurdles, Tandem Ubiquitin Binding Entities (TUBEs) have been designed.
IQF substrates for true isopeptidase/deubiquitinase activity measurement
Deubiquitylating enzymes (DUBs) are promising therapeutical targets, especially in oncology (see my previous post dedicated to this topic). Today, I’d like to introduce you to new innovative substrates to screen selectively DUB inhibitors.
Deubiquitylating enzymes (DUBs) as pharmaceutical targets
While it is likely that all Ubiquitin-Like proteins (UBLs) will be important for drug discovery because of these associations, Ubiquitin has been the most completely validated for this purpose by genetic, biological, and biochemical evidence. The enzymes of the Ubiquitin pathway are emerging as pioneer drug targets for many therapeutic areas.
The ubiquitin family of proteins is an important means of regulating protein trafficking, cell division, apoptosis, and metabolism in cells. Turnover of most cellular proteins is effected by ubiquitin tagging, which marks them for transport to the 26S proteasome for degradation. [Read more…]
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) [Read more…]
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…]
A selection of Proteasomal and De-ubiquitinase inhibitors
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…]