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.
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).
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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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.
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.
The specificity of antibodies to histone modifications (or any antigen for that matter) is crucial for obtaining meaningful experimental data. Cross-reactivity to similar modifications at other residues (e.g. H3K27Me3 vs H3K9Me3) or off-target recognition of other modification isoforms (e.g. H3K27Me1 vs H3K27Me3) can seriously compromise the integrity of any study using these types of reagents.
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.
Protein citrullination (a.k.a. deimination) is a novel arginine-directed post-translational modification (PTM) that results in a permanent change in the targeted protein. PeptidylArginine Deiminases (PADs) mediate the calcium-dependent deimination of the guanidino group of Arginine side chains to form an ureido group and the non-standard amino acid citrulline.
Some biologically relevant proteins known to be citrullinated include Keratin, Filaggrin, Trichohyalin, Vimentin, Myelin Basic Protein (MBP), Histones, alpha-Enolase, Fibrinogen, Fibrins, Collagen type I and II, beta-Actin, and Tubulin 9-11… It is noteworthy that several of these proteins are part of the cytoskeleton and/or are structural in nature.
In their October newsletter, Cytoskeleton Inc. presents an overview about the consequences of citrullination, especially referring to cytoskeleton proteins such as Vimentin.
Interested in this exciting new PTM mechanism?
Download your free copy of the review:
Citrullination: Taking the Charge out of Arg
In the August 2014 edition of their newsletter, Cytoskeleton Inc. bring us an overview of SUMO activation and deconjugation processes, together with their role on cytoskeletal proteins (actin, tubulin…).
For the record, SUMO (for Small Ubiquitin-like Modifiers) is a family of small proteins that is covalently attached to (or detached from) cellular proteins. Such post-translational modification (called SUMOylation) modulates cellular architecture and numerous activities, including response to stress, progression through the cell cycle, transport, mobility…
Interested in reading more about SUMOylation of cytoskeletal proteins?
Download your free copy of the review “SUMOylation: A Post-translational Modification Targeting Cytoskeletal Protein“!
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).