Mambalgin 1 – Blocking ASIC channels in pain pathways

Acid-Sensing Ion Channels (ASICs) are neuronal voltage-insensitive cationic channels which are activated by extracellular protons. They belong to the ENaC/Deg superfamily of ion channels. Up to now 6 members of the ASIC family have been identified: ASIC1 – ASIC 4, and the splice variants ASIC1a, 1b and 2a, 2b. The ASIC family members are trimeric and can be made up of different combinations of subunits. All ASICs are expressed in the peripheral nervous system. ASIC1a, 2a, 2b and 4 are expressed in the central nervous system. ASICs are promising drug targets for treating a wide variety of conditions linked to both the CNS and PNS especially implied in pain pathways.

Structure of Mambalgin-1

Schwarze Mamba

Fig. 1: Dendroaspis polylepis polylepis

Mambalgin-1 (from Smartox, now available through tebu-bio) was initially isolated by Sylvie Diochot and collaborators from the venom of the black mamba (Dendroaspis polylepis polylepis, Fig. 1).

Mambalgin-1 belongs to the family of three-finger toxins (Fig. 2) and has no sequence/structural homology with either PcTx1 or APETx2. Mambalgin-1 differs from mambalgin-2 by one amino acid. Both have demonstrated a similar activity.

Mambalgin 1 – a blocker of ASIC1 channels implied in pain pathways

It has been shown that Mambalgin-1 is a potent and selective blocker of ASICs implied in pain pathways (1, 2). Mambalgin-1 rapidly and reversibly inhibits recombinant homomeric ASIC1a (IC50=55 nM) and heteromeric ASIC1a+ASIC2a (IC50=246 nM) or ASIC1a+ASIC2b channels (IC50=61 nM) but also human channels hASIC1b (IC50=192 nM) and   hASIC1a+hASIC1b (IC50=72nM).

Mambalgin-1 has no effect on ASIC2a, ASIC3, ASIC1a+ASIC3 and ASIC1b+ASIC3 channels, as well as on TRPV1, P2X2, 5-HT3A, Nav1.8, Cav3.2 and Kv1.2 channels. Thus Mambalgin-1 can be used as a selective inhibitor for the above mentioned homomeric and heteromeric ASICs.

Interested in further information? Please contact me through the form below.

If you’d like an overview of the complete venomous toxins available, take a look to our special page: Synthetic peptide toxins ideal for studying ion channels

References:

(1) S, Dichot et al., Black mamba venom peptides target acid-sensing ion channels to abolish pain, Nature. 490 (7421), pages 552-5 (2012)

(2) Wen M., et al., Site-specific fluorescence spectrum detection and characterization of hASIC1a channels upon toxin mambalgin-1 binding in live mammalian cells. Chem Commun. 51 (38), pages 8153-6 (2015)

Keen to work with the best quality nucleosomes?

Histone Structure- tebu-bio

Fig. 1: Structure of a mono-nucleosome.

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…]

Focus on Actin detection and Actin binding proteins

Actin can exist in two forms: Globular subunit (G-actin) and Filamentous polymer (F-actin). Both forms of actin interact with a plethora of proteins in the cell. To date there are over 50 distinct classes of Actin-Binding Proteins (ABPs), and the inventory is still far from complete. Actin Binding Proteins allow the actin cytoskeleton to respond rapidly to cellular and extracellular signals and are integral to cytoskeletal involvement in many cellular processes. These include cell shape and motility, muscle contraction, intracellular trafficking, cell pathogenesis and signal transduction.

In the coming weeks I’d like to give you an overview of methods in actin research with validated R&D products and kits (actin polymerisation, and G-F actin ratio detection in cells); I also invite you to take a look at a post recently released about actin visualization: Focus on Actin staining and visualization.

In today’s post, let’s concentrate on a method which allows measuring actin binding capabilities of proteins of interest. But it’s not only about the simple fact that a given protein is binding to actin, with the method presented here, you’re also able to get an idea of the functionality of the protein – be it F-actin bundling activity, F-actin severing activity or G-actin binding activity. [Read more…]

Focus on Actin staining and visualization

Actin serves as one of the major cytoskeleton structures. It is a crucial component involved in a plethora of processes in cell biology:  stabilizing the cell shape, cell movements (e.g. cell migration)  and intracellular movements and transport mechanisms.

Actin is a 43 kDa protein that is very highly conserved between species. Actin has three main isotypes (α-actin, β-actin and γ-actin), which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group.

A brief reminder: G-actin polymerizes to form F-actin

Untitled

Fig. 1: Double-helical structure of actin filaments (provided by Cytoskeleton Inc.)

Globular-actin (G-actin) readily polymerizes under physiological conditions to form Filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. F-actin is a double-helical filament (Fig. 1).  Actin can polymerize from both ends in vitro. However, the rate of polymerization is not equal. This results in an intrinsic polarity in the actin filament. It has therefore become the convention to term the rapidly polymerizing end the plus-end or barbed-end (+) while the slow growing end is called the minus-end or pointed-end (-).

In the coming weeks I will give you an overview about methods in actin research with validated R&D products and kits (actin binding and actin binding proteins, actin polymerisation, and G-F actin ratio detection in cells).

 

Today I will focus on methods to visualize actin in fixed or living cells – which belong to basic experimental set-ups in Cell Biology.

Actin staining of fixed cells

Actistain photo

Fig. 2: Swiss 3T3 fibroblasts stained with ActiStain 488 (green), Dapi (blue) and Anti Vinuclin (orange). Provided by Cytoskeleton Inc.

Often fluorescent phalloidins are used to stain actin in fixed cells. Phalloidin belongs to the group of phallotoxins produced by the mushroom Amanita phalloides (death cap mushroom). The natural toxicity of Phalloidin is due to its stabilizing effect on F actin in cells. Based on its affinity for F-actin and coupled to a fluorescent dye, it can be used to visualize F-actin.

Cytoskeleton Inc. offers a set of phalloidin based stains (Acti-Stains) coupled to a number of different fluorophores compatible with popular filter sets such as FITC, TRITC and Cy5. The stains are exceptionally bright and stable and are indeed offered at very economical prices compared to other phalloidin based stains coupled to fluorophores of similar stability.

Results of staining of Swiss 3T3 cells with ActiStain 488 are shown in Fig. 2.

Live-cell imaging of Actin

Live-cell imaging of actin has been quite tricky as far as actin labelling is concerned – either cells had to be transfected with vectors carrying the genetic information for fluorescently tagged actin or actin binding proteins or, labelled actin had to be micro-injected to single cells.

Actin

Fig. 3: 3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts. Provided by Spirochrome.

Together with Spirochrome, tebu-bio launched in Europe the first tool to directly label actin in living cells with no need to transfect or micro-inject anything.

SiR-Actin is a cell permeable compound which stains F-actin in living cells. The stain is composed of a photostable silicon rhodamine-like (SiR) dye which can be used with standard Cy5 settings and a component (Jasplakinolide) which specifically binds to F-actin (Fig. 3). SiR-actin is compatible with Super-Resolution Microscopy like Stimulated Emission Depletion [STED] and Structured Illumination Microscopy [SIM].

f you would like to get an overview about the results SiR-actin users got so far, please have a look at my recent blog: User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging.

 

 

 

Interested in our Phalloidin and/or SiR-based stains?

Leave your comment or request in the form below.

 

9 pathway-specific screening assays in Immunotherapy

The immune system is a system of cells and organs whose function is to defend an organism from foreign pathogens. With the ability to mount a response against virtually any foreign material and return to a quiescent state following neutralization of the threat, this fascinating organ system displays remarkable specificity and plasticity. To achieve this, there is a multifaceted balancing act between the many activators and suppressors which maintains homeostasis of the body’s perhaps most complex organ system.

[Read more…]

A simple way to measure Elastin

Recently I reported about methods to measure:

Today, I invite you to take a closer look at another component of the extacellular matrix (ECM) – Elastin.

Visual representation of skin changes over a lifetime.

Visual representation of skin changes over a lifetime.

Elastin is a highly elastic protein in connective tissue and enables tissues in the body to resume their shape after contracting or stretching. Elastin helps skin to return to its original position when it is pinched or poked. During aging, elastin appearance decreases. The ELN gene encodes a protein which is rich in hydrophobic amino acids like glycine and proline, which form mobile hydrophobic regions bounded by crosslinks between lysine residues. A number of transcript variants encoding different isoforms are know for this gene. Together with the elastic microfibril (consisting of proteins such as microfibrillar-associated glycoproteins, fibrillin, fibullin, and the elastin receptor) elastin forms so called elastic fibers in the ECM of connective tissues.

Elastin and Diseases

Deletions and mutations in the ELN gene are associated with Supravalvular aortic stenosis (SVAS) and the autosomal dominant cutis laxa (or Chalazoderma or Dermatochalasia). Further elastin-related defects include Marfan syndrome, emphysema, atherosclerosis, Buschke-Ollendorff syndrome, Menkes syndrome, pseudoxanthoma elasticum, and Williams syndrome.

Detection of Elastin

Often elastin is detected with either ELISA or immunohistochemistry based methods. Our partner Biocolor developed a quantitative dye-binding method for the analysis of elastins released into tissue culture medium and extracted from biological materials (see the Fastin Flowchart in Fig.1)

Fastin flowchart

Fig. 1: Flowchart of the Fastin assay to measure elastin (click to view full size)

Which elastin forms can be measured by the Fastin Assay?

  • soluble tropoelastins
  • athyrogenic elastins
  • insoluble elastins (following solubilization to elastin polypeptides [α-elastin, κ-elastin])

    Fastin results

    Fig. 2: Extraction of elastin from mouse tissue by hot oxalic acid digestion. The extracts shown were pooled and expressed as μg elastin per 10 mg wet tissue.

The dye reagent (5,10,15,20-tetraphenyl-21 H, 23 H-porphine tetra-sulfonate, TPPS) binds to the ‘basic’ and ‘non-polar’ amino acid sequences found in mammalian elastins. The elastin-Dye complex can be read with a

microplate reader, with a suitable colour filter (absorbance peak of dye occurs at 513 nm).
Typical results of elastin measurements (in mouse tissue) are shown in Fig. 2.
Interested in measuring Elastin – or any other components of the extracellular matrix – in biological samples? Don’t hesitate to get in touch through the form below!

Pharmacological agents which alleviate Pain

Chronic pain of varying types including inflammatory pain, cancer pain and neuropathic pain is an increasing health problem for which there are inadequate therapeutic options.
Many new targets for analgesic therapeutics have been elucidated, these include TRP channels, the endocannabinoid system, sodium channels and various signaling pathways just to name a few.

Many new pharmacological agents which alleviate pain have been identified. In this post, I’d like to introduce some of them which have been recently released by Focus Biomolecules to support pain related research activities. [Read more…]

Measuring total ubiquitinated proteins in cell lysates – made easy!

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

Ubiquant S results 1

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. It is built on LifeSensors UbiQuant platform, in which ubiquitinated proteins in cell lysates are captured in the wells of a precoated microtiter plate using a proprietary ubiquitin binding reagent and then detected by an antibody against the protein of interest (either native or tagged) to quantitate the amount of the protein bound. Another version of this platform – the Ubiquitin Ubiquant ELISA – opens the possibility to measure the concentration of total free ubiquitin (poly- mono-) in biological samples including cell lysates, tissue homogenates, and plasma.

Fig. 1 shows a typical experiment using the UbiquantTM S assay. Decrease in LDLR ubiquitination following treatment of transfected cells with an IDOL (E3 ubiquitin ligase) inhibitor.  Cells are treated with different concentrations of an inhibitor of the E3 ubiquitin ligase IDOL and subsequently the decreasing ubiquitination of Low density lipoprotein receptor (LDLR) is measured with the UbiquantTM S assay. More detailed results can be found in an application note recently relased by LifeSenors.

Please note that if you want to order and use this kit and intend to use a tagged-substrate, you will have to specify the epitope tag in order to receive the appropriate antibody for detection with the kit(e.g. myc, HA, FLAG®, V5, GST, etc.  Unfortunately, His6 cannot be used for this assay.) Of course you can also use your own antibodies directed against your protein of interest.

Interested to apply this method to detect the total amount of ubiquitinated proteins in your sample? Get in touch through the form below, for advice on selecting the correct anti-tag antibody you might need.

TET1 – the first DNA demethylase

Graph DM

Fig. 1: Example of a DNA methyltransferase assay: DNMT1 enzyme activity, measured using the DNMT1 Assay Kit.

Methylation of DNA plays a crucial role in transcriptional regulation, imprinting, and chromatin structure. DNA methyltransferases add a methyl group to cytosine or adenine DNA nucleotides. DNA methylation is a dynamic and reversible process and alters the expression of genes during cell division and differentiation from embryonic stem cells to specific tissues. The process is usually permanent, preventing cells e.g. from re-differentiation. During zygote formation, methyl groups are removed and subsequently re-established during development.

The reducing effect of gene expression is mediated by methylation at the 5 position of cytosine and it is present on all vertebrates. In adult somatic cells methylation is seen in a CgG context, while non-CpG methylation is current in embryogenic stem cells. Enzymes that catalyze DNA methylation are well characterized and already used as target enzymes for drug discovery screenings. BPS Biosciences offers a range of active DNA methyltransferases and DNA methyltransferase assays for inhibitor screenings (see Fig.1). [Read more…]

Direct localization of MMP activity in 3D tumor invasion model

Recently, the non-FRET EnSens technology was launched for  in vitro assessment of specific protease activities (Enzium, Inc.). Up to date, the EnSens substates were validated for the detection of protease activity in microplate assays. Now, Enzium has extended the applicability of their Ensens method to 3 D live-cell imaging. On the top of this, they announce that the experimental procedure is easy and non-toxic for cell cultures and co-cultures. So how does live-cell imaging help you locate protease activities in in vitro tumor invasion assays ?

[Read more…]