New Publication: Cell Cited LifeTein Biotinylated Peptide Products

Pull-down assay using biotinylated peptides

Giantin, a novel conserved Golgi membrane protein, is a disulfide-linked homodimer. It was found that BFA-induced Golgi disorganization is associated with the monomerization of giantin.

The pull-down experiment was performed. The control peptide biotin-GHGTGSTGSGSMLRTLLRRRL synthesized by LifeTein was incubated with lysate and Dynabeads, as well as the lysate incubated with Dynabeads only served as a control. Dynabeads carrying MGAT1 peptide were able to pull-down giantin from the lysate of HeLa cells, however, giantin was not detected in the pull-down fraction from the lysate exposed to the Dynabeads or in combination with control peptide. It is logical to hypothesize that the MGAT1 binding domain of giantin lies within its N-terminal non-coiled-coil area.

The Dynabeads function similarly to LifeTein magnetic beads:
https://www.lifetein.com/peptide-product/amineactivated-peptide-conjugation-magnetic-beads-p-3647.html

Cells 2019, 8(12), 1631; https://doi.org/10.3390/cells8121631

Full list of Cell-Penetrating Peptides

Table 1 Selection of cell-penetrating peptides

(Reference: https://doi.org/10.1007/978-981-13-8747-0, Ülo Langel, CPP, Cell-Penetrating Peptides, 2019)

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How to form the fibrillary structure using beta-amyloid peptides?

Aβ-(1–42) was dissolved to 1 mM in 100% hexafluoroisopropanol, hexafluoroisopropanol was removed under vacuum, and the peptide was stored at −20 °C. For the aggregation protocols, the peptide was first resuspended in dry Me2SO (DMSO) to 5 mM. For oligomeric conditions, F-12 (without phenol red) culture media was added to bring the peptide to a final concentration of 100 μM, and the peptide was incubated at 4 °C for 24 h. For fibrillar conditions, 10 mM HCl was added to bring the peptide to a final concentration of 100 μM, and the peptide was incubated for 24 h at 37 °C. ADDLS, amyloid derived diffusible ligands.

Aducanumab is a human monoclonal antibody that has been studied for the treatment of Alzheimer’s disease.

A click chemistry was reported about the formation of azides from primary amines

Click chemistry for drug screening

A click chemistry was reported about the formation of azides from primary amines. This powerful tool enables the reaction of just one equivalent of a simple diazotizing species, and fluorosulfuryl azide (FSO2N3), for the preparation of over 1,200 azides on 96-well plates in a safe and practical manner. This method greatly expands the number of accessible azides and 1,2,3-triazoles because the primary amine is one of the most abundant functional groups in small compounds, proteins and antibodies.

The method opens the door for numerous applications in drug screening and discovery. The cell penetration peptides can be easily introduced to conjugate with any azide containing drugs, compounds, antibodies, or proteins.

The cell penetration peptides (CPPs) are capable of delivering biologically active cargo to the cell interior. The desired therapeutic cargo could be attached to a CPP using the copper free click chemistry and then delivered to an intracellular target, thereby overcoming the entry restrictions set by the plasma membrane.

Braftide, a 10mer peptide synthesized at LifeTein, a potent allosteric inhibitor of BRAF dimer for cancer therapy

Dabrafenib


BRAF is an RAF kinase. It is a core component of the RAS/RAF/MEK/ERK signaling cascade, known as mitogen-activated protein kinase (MAPK) pathway. It is one of the major effectors of oncogene RAS, and is often mutated in human cancer cells.


Two FDA approved drugs, Dabrafenib, and vemurafenib, effectively inhibit the most common BRAF variant V600E, a monomeric BRAF. But, the non-V600E BRAF mutations are intrinsically resistant to these drugs. These drugs may also paradoxically stimulate the pathway when the tumor cells contain wild-type BRAF and oncogenic RAS, causing secondary malignancies.
The researchers tried to tackle the dimeric BRAF. The dimeric BRAF, such as the wild type and G469A, a most prevalent non-V600E variant in lung cancer cells, hinges on dimer interface (DIF), a 20aa span near the tail end of the alpha-C helix of BRAF. The researchers designed Braftide using computational modeling, aiming to block the dimerization. They tested the functionality in vitro, in HEK263 cells and colon cancer cell lines.

LifeTein synthesized Braftide (TRHVNILLFM), Null-Braftide (THHVNILLFM), Cy3-Braftide (TRHVNILLFM-Cy3), TAT-Braftide (GRKKRRQRRRPQ-PEG-TRHVNILLFM), and TAT (GRKKRRQRRRPQ). We reviewed here some of the assays that helped support Braftide as an allosteric inhibitor of BRAF dimer and down-regulator of MAPK signaling pathway for cancer therapy.


1) Cell-free in vitro assay: dose-response curve. First of all, the researchers show that Braftide has a sub-micromolar IC50 for dimeric BRAF. Full-length dimeric BRAF-WT and BRAF-G469A (from HEK293F cells) were used for dose-response curves, and the BRAF activity was probed by pMEK production.


2) Cell-free in vitro assay: Saturation binding assay. The researchers used Cy3-labeled Braftide (Cy3-Braftide) to characterize (KD) the binding of Braftide with dimeric BRAF-WT using fluorescence quantification.


3) Cell-free in vitro assay: Immunoprecipitation (IP). The purpose of IP was to show Braftide disrupted the BRAF dimerization. Braftide was added to HEK293 cell lysate coexpressing V5- and FLAG-tagged BRAF-WT. FLAG-tagged BRAF was pulled down by FLAG antibody-conjugated resin, which was further probed for V5-tagged BRAF. Braftide indeed reduced homodimer BRAF.


4) Delivery of Braftide into HEK cell for BRAF inhibition. Braftide was tagged with cell-penetrating peptide TAT. TAT-Braftide (and its negative control TAT alone) was used to treat HEK293 cells transiently transfected with BRAF-WT and BRAF-G469A. Four hours of treatment resulted in reductions of BRAF, pMEK, MEK (i.e. the MAPK pathway), which were analyzed with respective antibodies by immunoblotting.


5) Delivery of Braftide into cancer cells for BRAF inhibition and cell proliferation inhibition. Two colon cancer cell lines (KRAS-G13D-colon carcinoma) were treated with cell-penetrating TAT-Braftide and assayed for the inhibition of BRAF activity, down-regulation of MAPK signaling, and cell proliferation. All were shown positive, while the negative control TAT alone were negative.

https://pubs.acs.org/doi/abs/10.1021/acschembio.9b00191

LifeTein’s Synthetic Scorpion Toxin Peptides Helped Scientists Unravel Chronic Pain Mechanisms

Synthetic Scorpion Toxin Peptides
Synthetic Wasabi Receptor Toxin

LifeTein’s synthetic Wasabi Receptor Toxin, Wasabi Receptor Toxin Mutants, Biotinylated Wasabi Receptor Toxin, and AlexaFluor-488 conjugated Wasabi Receptor Toxin and Mutants helped scientists unravel chronic pain mechanisms.

Researchers at the University of California, San Francisco (UCSF) have identified a scorpion toxin that targets the “wasabi receptor”. The wasabi receptor is an ion channel protein that is responsible for the sinus-clearing or eye-stinging pain experienced when eating wasabi or chopping onions.

It was found that the scorpion toxin, a peptide as the wasabi receptor toxin, or WaTx, activates the wasabi receptor TRPA1 and triggers this pain response to irritants. The WaTx peptide is a novel cell-penetrating peptide and can directly pass through the plasma membrane, without needing to traverse through channel proteins.

The WaTx peptide could be used to study chronic pain and inflammation and may lead to the development of novel non-opioid pain therapies. WaTx produces pain and pain hypersensitivity, but not neurogenic inflammation.

Reference: Lin King, J. V., Emrick, J. J., Kelly, M. J. S., Herzig, V., King, G. F., Medzihradszky, K. F., & Julius, D. (2019). A Cell-Penetrating Scorpion Toxin Enables Mode-Specific Modulation of TRPA1 and Pain. Cell. doi:10.1016/j.cell.2019.07.014

LifeTein peptide FLAG(GS)HA: DYKDDDDK-GGGGS-YPYDVPDYA-NH2 helped discover insulin-like peptide6, Dilp6, in regulating growth in fruit flies Drosophila

FLAG and HA tagged IGF1

In humans, liver-derived insulin-like growth factor (IGF1) drives postnatal growth. Early childhood infection of E. coli, Campylobacter spp., even asymptomatic, reduces IGF1 level and restricts early-childhood growth. Does the pathogen-induced Toll-like innate immune signaling contribute to growth restriction? To answer the question, the researchers examined a corresponding pathway in fruit flies.

In fruit flies, Dilps (Drosophila insulin-like peptides) drive their growth, for example, the growth rate of imaginal discs which give rise to adult structures such as wings. Dilps share homology with insulin and IGF1, and they bind to the insulin receptor. Dilp6 is produced by fat body, an organ for nutrient storage and immune functions.

The researchers found Dilp6 is a selective target of Toll signaling in the fat body, an innate immune response from bacterial infections. They also found that Toll signaling reduces Dilp6 transcripts, and dramatically suppresses circulatory Dilp6 levels, and restricts whole-body growth. Restoring Dilp6, on the other hand, rescues growth and viability in fruit flies even with active Toll signaling.

LifeTein’s peptide FLAG(GS)HA was used as a standard in ELISA to quantify Dilp6 in fruit fly hemolymph samples. Here, Dilp6 was tagged with FLAG and HA because of FLAG- and HA-tagged Dilp6HF allele from CRISPR/CAS9. In this ELISA assay, the plate wells were coated with anti-FLAG antibody, then FLAG(GS)HA or fruit fly hemolymph sample were added to the wells. FLAG(GS)HA and FLAG- and HA-tagged Dilp6 were quantified by anti-HA-Peroxidase 3F10 antibody and subsequent chromogenic reaction. For more details of the method, see the section “Hemolymph Dilp6 measurements by ELISA” in the link.

https://www.sciencedirect.com/science/article/pii/S2211124719309052

The smaller ions (F- and Cl-, 50mM NaF and NaCl solutions) tend to stabilize β-sheets

Peptide amphiphiles are composed of hydrophobic alkyl tails and peptide regions designed to self-assemble into cylindrical supramolecular nanofibers in solution. While some β-sheets are formed by hydrogen bonds between short β-strands (2 or 3 residues) others are formed by extended β-strands.

The strongly-hydrated ions (F- and Cl-) are more attracted to the positively charged lysine residues on the surface of the peptide nanofiber. When peptide residues form β-sheets, an F- or Cl- ion forms a salt bridge between the side chains of lysine residues from two neighboring peptide amphiphile chains. The salt bridge stabilizes the peptide by bringing the backbones closer, which in turn results in a transition from random coil to extended β-sheets structures. The smaller ions (F- and Cl-, 50mM NaF and NaCl solutions) tend to stabilize β-sheets slightly better compared to the larger ions (I-, Br-).

So self-assembly of peptide amphiphiles into supramolecular nanofibers can be regulated by modifying the salt solution.

Reference: doi.org/10.1021/acs.jpcb.9b05532