Ubiquitination

How to activate or inhibit autophagy?

10/11/2016 By Ali El Baya, PhD Leave a Comment

The term Autophagy was introduced by Christian de Duve during the Ciba Foundation Symposium on Lysosomes – which was held in London in February 1963. In 1974 he was honoured with the Nobel price in Physiology or Medicine for his pioneering research about peroxisomes and lysosomes. In 2016, once more, a pioneer in the field of autophagy research won the Nobel price: Yoshinori Ohsumi, a Japanese researcher, whose findings “led to a new paradigm in our understanding of how the cell recycles its content”.

Fig 1: The process of autophagy

Autophagy (Autophagocytosis) describes the fundamental catabolic mechanism during which cells degrade dysfunctional and unnecessary cellular components. This process is driven by the action of lysosomes and promotes survival during starvation periods as the cellular energy level can thus be maintained. [Read more…]

Check whether your protein is ubiquitylated!

11/10/2016 By Ali El Baya, PhD Leave a Comment

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…]

Post-Translational Modifications (PTM) of Tubulin

18/08/2016 By Ali El Baya, PhD Leave a Comment

In this post, I’d like to take a look at the current understanding of tubulin PTMs, that include tyrosination/detyrosination, Δ2-tubulin formation, acetylation, phosphorylation, ubiquitination, glutamylation, and glycylation. This is inspired by contribution provided by Cytoskeleton Inc., who are experts in this domain.

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Easily customizable E3-ubiquitin ligase assay kits

08/02/2016 By Ali El Baya, PhD Leave a Comment

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?

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TUBEs: study M1 (linear) polyubiquitylated proteins

05/02/2016 By Elodie Monin Leave a Comment

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.

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Monoclonal antibodies validated for PTM studies

01/09/2015 By Ana Arraztio Leave a Comment

Knowing the Human genome better has allowed major advancements in Personalised Medicine. Nowadays, we can know (if we want) the likelihood to develop a given disease and/or how we will react to different pharmacological treatments. Examples of this include diseases like breast cancer (for diagnosis or estimation of likelihood) and lung cancer (for response to treatment), to name just a few.

That said, our genotype does not have the last word. Research in the last couple of decades has shown the power of other regulatory mechanisms, that may enhance or diminish the effect that our genotype will have on our health. Starting from basic healthy life styles, to other more subtle mechanisms, our genotype defines us, but not completely. Above genetics, we have epigenetics… and everything at the protein level. This post will focus on Post-Translational Modifications (PTM), because, after all, it’s the proteins that are the final effectors of a given response to a treatment or to an environmental stimulus.

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3 validated tools for ubiquitination studies

24/07/2015 By Ana Arraztio Leave a Comment

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…]

Measuring total ubiquitinated proteins in cell lysates – made easy!

16/06/2015 By Ali El Baya, PhD Leave a Comment

To date, detecting changes in the ubiquitylation of specific substrate proteins in response to external stimuli, e.g. stress, cytokine exposure, drug candidate treatment, etc., has been a long, labor-intensive process involving immunoprecipitation followed by gel electrophoresis and Western blot analysis. This method is low through-put, resource intensive, and only semi-quantitative at best.

ELISA like method to measure all ubiquitinated proteins in cell lysates

Fig. 1.: Decrease in LDLR ubiquitylation following treatment of transfected cells with an IDOL inhibitor.

To enable researchers to avoid the above mentioned limitations,� LifeSensors has developed the UbiQuantTM S kit as a facile, robust, and quantitative alternative to IP/WB analysis. [Read more…]

Post-translational modifications regulate Ral GTPases

21/04/2015 By Ali El Baya, PhD Leave a Comment

RalA and RalB GTPases regulate cell motility, morphology, signaling, vesicular trafficking, and endo/exocytosis. The regulation of these functions is critical for the development and spread of cancer, implicating Ral in oncogenesis and metastasis. Both isoforms are integral for Ras-mediated tumorigenesis, metastasis, and invasion. Despite sharing 82% amino acid sequence identity, effectors, and structural/biochemical properties, RalA and RalB have their own unique functions in oncogenesis due to distinct subcellular localization and differential effector interactions. Ral localization, binding partners, and function are regulated by post-translational modifications (PTMs).

In their recent newsletter, Cytoskeleton Inc. summarize recent findings about the relevance of geranylgeranylation, carboxymethylation, palmitoylation, phosphorylation, and ubiquitination in regulating Ral activity, subcellular localization, effector binding, and ultimately, function.

You can download a copy of this newsletter, or if you have any questions or comments, don’t hesitate to get in contact through the form below.

Kits to measure RalA activation

If you’d like to get an overview about what’s available in the small G protein field, take a look at this� Small GTPase product guide.

How to manipulate and measure Autophagy?

20/04/2015 By Ali El Baya, PhD Leave a Comment

In this post, I’d like to give you an overview of autophagy and its implication in cell biology, and tools to manipulate and detect autophagy in cells. [Read more…]