Ali El Baya, PhD

Designer Nucleosomes – the next generation of epigenetic substrates!

10/06/2016 By Ali El Baya, PhD Leave a Comment

Fig. 1: Mononucleosome structure

In eukaryotic cells DNA is packaged in nucleosome units called mono-nucleosomes which consist of a segment of DNA called core DNA (147 bp in length) wound around a histone octamer (Fig 1.). Histone octamers are assembled from 2 copies of the the core histones H2A, H2B, H3, and H4. These mono-nucleosomes are connected by linker 80bp-long DNA. A fifth histone type of protein, the so-called linker histone H1, binds to the linker DNA close to the entry and exit of the core DNA and is involved in chromatin compaction (Fig 1.).

Substrates for epigenetic enzyme assays and inhibitor screenings

In the past, native or recombinant nucleosomes, single histone proteins, or histone derived peptides have been available for assays with epigenetic enzymes such as histone methyltransferases or histone acetylases and related screenings for inhibitors of the respective enzymes (for an overview about the products available, you might like to read my recent post Find the best epigenetic enzyme substrate for your needs).

Table 1: Specific histone modifications can be linked to certain diseases and effector proteins (provided by Epicypher).

As some epigenetic enzymes require highly specific substrates, Epicypher has now launched a new product line – Designer Nucleosomes (dNuc). dNUCs are semi-synthetic nucleosomes incorporating specific histone post-translational modifications. These reagents represent a powerful new technology – critical in understanding chromatin biology and for the development of novel drug targets and precision therapeutics. It is known that highly specific histone modifications can be linked to certain diseases (see Table 1). Table 1 liste the dNUCs which are already available, for further information have a look at our list of Designer Nucleosomes. dNUCs serving as substrates for the most relevant epigenetic enzymes will be added to our catalog in the coming months. If you have specific modifications in mind, which which are know to be optimal for your enzyme of interest, please let me know through the form below. Epicypher might already have this designer nucleosome in their pipeline – or might be able to produce it on a customized basis.

To get more insight into the dNuc manufacturing process Epicypher applies, you can download the white paper Not All Designer Nucleosomes are Created Equal: A Tale of Two Cysteines.

The paper compares the two currently used synthetic methods to produce dNUCs, native chemical ligation (NCL) and methyl lysine analog (MLA). The paper shows that NCL – the method used by Epicypher – yields superior nucleosome preparations.

Any questions or comments? Please feel free to contact me with the form below!

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Have a closer look at GPCR mediated signaling!

02/06/2016 By Ali El Baya, PhD 2 Comments

Cell-based calcium assays have been a mainstay of GPCR (G protein coupled receptor) drug discovery and signal transduction research since the mid 1990s. However, a calcium readout alone might be ambiguous, because many cellular processes produce calcium. Furthermore, it does not differentiate between signalling pathways routed through the different types of heterotrimeric G proteins. While effects on GPCRs which exhibit effects on Gs proteins lead to an elevated level of the second messaeger cAMP by activating the activity of adenylate cyclase, signalling through Gi proteins inhibits adenylate cyclase and thus leads to a decrease of cAMP. Finally signalling through Gq proteins leads to phospolipase C activation which cleaves Phosphatidylinositol 4,5-bisphosphate (PIP2) to Diacetlyglycerol (DAG) and Inositol trisphosphate (IP3) which leads to an increase in Ca2+ level (for an overview see reference 1).

Dye based methods to measure Ca2+ are very well established, whereas there are no dyes available for most of the other second messengers in these signalling pathways.

Genetically encoded fluorescent biosensors for live cell discovery

That’s why Montana Molecular developed genetically encoded fluorescent biosensors for second messengers such as cAMP, DAG, PIP2, and Ca2+, references 2, 3). [Read more…]

IL-17A pathway – a key target for immunotherapies

12/05/2016 By Ali El Baya, PhD Leave a Comment

Fig 1: Function of IL-17 in diverse processes

Interleukin-17 (IL-17) is a family of 6 closely related cytokines, designated IL-17A-F, that play a central role in mediating inflammation, autoimmunity, and host defense. IL-17 is mainly secreted by a specific subset of T helper cells known as T_H17 cells (for an overview see Fig 1). The IL-17 cytokines mediate their biological functions via surface receptors on target cells. IL-17A binds to IL-17 receptor A (IL-17RA), which stimulates the production of other pro-inflammatory cytokines including IL-6 and IL-8. IL-17A and its receptor play a pathogenic role in many inflammatory and autoimmune diseases such as rheumatoid arthritis. IL-17 signaling is also involved in mucosal immunity and host defense against extracellular bacterial and fungal infections (Staph, Candida, Pneumonia, etc.). [Read more…]

Protein PTMs analysis with Signal-Seeker kits

06/05/2016 By Ali El Baya, PhD Leave a Comment

Signal-Seeker assay kits enable the relevant and simple characterization of the key protein post-modifications (PTMs). In this post, I invite you to look at experimental results obtained with these unique assays developed by Cytoskeleton Inc. for protein Tyrosine phosphorylation and Ubiquitination.

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Protein post-translational modification analysis

28/04/2016 By Ali El Baya, PhD 1 Comment

Protein post-translational modifications (PTMs) are part of a complex regulatory network that controls physiological and pathological cellular processes. In this post, new user-friendly assays are introduced to help Life Scientists gain a deeper understanding of these mechanisms involved in protein biology.

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Find the best epigenetic enzyme substrate for your needs

20/04/2016 By Ali El Baya, PhD 1 Comment

Fig. 1: Nucleosome structure

Nucleosomes are basis units of DNA packaging in eukaryotic cells. A core particle (mono-nucleosome) consists of a segment of DNA called core DNA (147 bp in length) wound around a histone octamer (Fig 1.). Histone octamers are made up of 2 copies of the the core histones H2A, H2B, H3, and H4. These mono-nucleosomes are connected by linker 80bp-long DNA. H1 histone, the so-called linker histone, binds to the linker DNA close to the entry and exit of the core DNA and is involved in chromatin compaction (Fig 1.). [Read more…]

CBR-5884 – a Serine biosynthesis inhibitor for cancer research

15/04/2016 By Ali El Baya, PhD Leave a Comment

Metabolic pathways are upregulated in cancer cells to promote their growth and proliferation. The evidence pointing to the requirement of the amino acid serine in tumorigenesis is overwhelming. Serine is required for a large number of anabolic processes including amino acid and lipid biosynthesis. The enzyme 3-phosphoglycerate dehydrogenase (PHGDH) catalyzes the first committed step in cellular serine biosynthesis and is overexpressed in cancer cell lines and tumors.

CBR-5884 – a selective small molecule inhibitor of PHGDH recently discovered

CBR-5884 - focus biomolecules and tebu-bio

CBR-5884 chemical structure

Very recently, in a joint effort, the laboratories of Cantley, Lyssiotis and Lairson reported the discovery of a selective small molecule inhibitor of PHGDH designated CBR-5884 (1).

This compound inhibits serine biosynthesis in cancer cells and displays selective cytotoxicity to tumor cell lines overexpressing PHGDH. The identification of CBR-5884 as a drug-like inhibitor of serine biosynthesis is a breakthrough in targeting metabolism in cancer drug development. It is an important compound which provides proof of concept that PHGDH is a viable target, and may represent a lead molecule for the development of more potent analogs. In the mean time it serves as an important new tool compound for the study of cellular serine metabolism.

CBR-5884 is available as a research tool from Focus Biomolecules and tebu-bio

Focus Biomolecules has been the first company to make this promising compound available to the market for cancer research studies.

CBR-5884 (cat. nr 10-1588) can be purchased in Europe through tebu-bio. The compound is available in standard sizes (5 mg & 25 mg) or in bulk amounts (upon request).

Interested in testing CBR-5884 in your experimental setup ?

Please contact me through the form below!

Reference:

(1) Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers. E Mullarky et al. Proc. Acad. Natl. Sci. USA 2016 113:1778

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Versatile colorimetric method to measure Hyaluronic acid (HA)

13/04/2016 By Ali El Baya, PhD 1 Comment

Fig 1: Structure of HA

Hyaluronic acid (Hyaluronan) belongs to the group of glycosaminoglycans (GAGs), but unlike other GAGs like Heparan sulfate, Chondroitin sulfate or Keratan sulfate, it cannot be found as a proteoglycan. It represents a non-sulfated polysaccharide consisting of alternating residues of D-glucuronic acid and N-acetylglucosamine (Fig 1). In the human umbilical cord and synovial fluid, the size of HA is reported to be about 3,000,000 Da (a flexible chain of 4000 disaccharide units).

HA is one of the major components of the extracellular matrix and it absorbs high amounts of water thus giving tissues the ability to resist compression. Furthermore, Hyaluronan contributes significantly to cell proliferation and cell migration. As HA levels often correlate with malignancy and poor prognosis of certain cancers, it can be used as a tumor marker. As Hyaluronan plays a role in skin healing, it’s a widely used ingredient of skin-care products. [Read more…]

Cancer Immunotherapy: Generate your PD-L1 target cell line

06/04/2016 By Ali El Baya, PhD Leave a Comment

Fig 1: Immuno checkpoint proteins on T cell interact with ligands on APCs or cancer cells

In a recent post, I invited you to discover a cell line provided by BPS Bioscience over-expressing the checkpoint receptor PD-1. They have now just released a new kit which allows to generate a PD-L1 and TCR activator over-expressing target cell line. [Read more…]

Measure the anti-oxidative potential of your sample

25/03/2016 By Ali El Baya, PhD 4 Comments

Fig 1: Oxidative stress and related diseases

Oxidative stress can lead to diverse pathophysiological changes in the body. Neurodegenerative diseases such as Parkinson and Alzheimer are linked to oxidative stress, as well as several cancers and diseases such as chronic fatigue syndrome, fragile X syndrome, heart and blood vessel disorders, heart attack, heart failure, and atherosclerosis (for an overview see Fig 1). [Read more…]