DISCOVERY Peptides

  • Peak peptides: paving the way for investigating new cancer immunotherapies.

    The field of targeted cancer immunotherapy is exciting and offers promise for greatly improving treatments for many cancers. One highly promising tool is T cell bispecific antibodies (TCB), which recognise oncogene antigens (Ags) presented on the MHC-1 molecules on the surface of cancer cells, and cell surface markers from T cells. These bi-specific antibodies therefore bring the T cells and cancer cells into close proximity, resulting in cancer cell destruction. However, this method is limited to the availability of Ags with high tumour specificity and sufficient epitope density on cancer cell surfaces.

    For this reason, the groups of Andrea Romagnani and Yvonne Nagel from Roche in Basel, Switzerland aimed to increase the abundance of Ag peptides from oncoproteins on cell surface MHC-I receptors in tumour cell lines in their recent publication. The teams used proteolysis-targeting chimeras (PROTACs) also known as degraders, which are hetero-bi-functional molecules, consisting of an E3 ligase-binding ligand, connected via a linker to a ligand specific to the target protein. Therefore degraders hijack endogenous E3 ligase bringing it into close proximity with the target protein resulting in proteasome degradation of the target protein. An increase in Ags of the target protein is then available for trafficking to the cell surface for MHC presentation.

    The researchers targeted the breakdown of the Bromo- and Extra-Terminal (BET) protein BRD4, which plays important roles in transcriptional regulation, epigenetics, and cancer; and identified five novel MHC-I binding peptide antigens from BRD4. In order to use a proteomics approach overseen by senior principal scientist Axel Ducret also at Roche, the team enlisted the help of CRB and ordered our highly purified custom PEAKTM Peptides. Our PEAKTM Peptidesare isotopically labelled with the residue of the customers choosing and are analysed for net peptide content as standard, allowing for precise peptide quantification. The team ordered PEAKTM Peptides of the two most abundant MHC-I binding peptides identified, QEFGADVRL (QEFGADV-[U-13C6,15N4-Arg]-L-acid) and AEALEKLFL (AEALE-[U-13C6,15N2-Lys]-LFL-acid), which allowed them to determine the relative peptide abundance from the ratio of light (unlabelled, experimental sample) to over heavy (isotopically labelled standard) signal using MHC I-associated peptide proteomics (MAPPs) a technique which utilisies powerful mass spectrometry.

    Using this method the researchers demonstrated that PROTACs targeting bromo- and extraterminal domain proteins increase the abundance of target-derived peptide Ags on MHC I in both liquid and solid tumours. With the help of CRB they demonstrated that targeted protein degradation is a promising strategy in immuno-oncology to enhance an immune response against specific Ags and that this can be combined with a TCB in a cancer immunotherapy setting.

    Click here to see our full range of proteomics related peptides.

    Click here to see our range of CEF peptides

    References:

    Massafra et al., (2021). Proteolysis-Targeting Chimeras Enhance T Cell Bispecific Antibody-Driven T Cell Activation and Effector Function through Increased MHC Class I Antigen Presentation in Cancer Cells. J Immunol. PMID: 34215653

    Zengerle et al., (2015). Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4. ACS Chemical Biology. 10(8): 1770-7. PMID: 26035625

  • Flg22, the small elicitor with multiple roles

    Flagellin is the main structural protein of the bacterial flagella and acts as both a virulence factor helping bacteria invade host cells and is also recognised by the host to induce a potent immune response. Flagellin 22 (flg22) is the stretch of flagellin amino acids most conserved across bacterial species and is the minimal ligand recognised by the plant Toll-like receptor 5 (TLR5) homologue FLAGELLIN SENSITIVE 2 (FLS2). FLS2 is a membrane bound leucine-rich repeat-receptor kinase which activates downstream signalling resulting in pattern-triggered immunity (PTI). Flg22 is therefore an extremely useful peptide, widely used in plant defence studies, for example to elicit an immune response in order to discover new plant immunity genes or to investigate different immune responses in mutant backgrounds; as the team led by Jozef Šamaj did (Takáč et al., 2021).

    Recognition of flg22 by FLS2 results in receptor heterodimerization with BAK1 and the cytoplasmic-kinase BIK1 and the binding of the SMALL UBIQUITIN-LIKE MODIFIER (SUMO) protein to FLS2. Binding of SUMO to the receptor complex culminates in the activation of downstream signalling leading to PTI. In addition to offering highly active flg22 peptides, we also offer two highly specific antibodies to Arabidopsis SUMO available in our discovery antibodies catalogue as part of our growing plant pathology range.

    Following heterodimerisation and receptor internalisation there are a plethora of rapid responses from the plant including: reactive oxygen species (ROS) generation; mitogen‐activated protein kinase (MPK3, MPK6, MPK4, and MPK11) activation; WRKY transcription factor activation, expression of PATHOGENESIS RELATED (PR) genes, upregulation of peroxidases, chitinases and glutathione S-transferases (GSTs), reorganization of the actin cytoskeleton and deposition of callose at the plant cell wall; ultimately resulting in the inhibition of plant growth and increased resistance to pathogens.

    Bacterial flagellin is also recognized by the FLS3 receptor in certain solanaceous plants, such as tomato, potato and pepper. Tomato also contains an active FLS2 receptor which is activated by a truncated flagellin 22 (flg22-Δ2) which represents amino acids 1-20 of flg22 and is also available from our discovery peptides catalogue.

    References:

    Chinchilla et al., (2006). The Arabidopsis Receptor Kinase FLS2 Binds flg22 and Determines the Specificity of Flagellin Perception. Plant Cell. 18(2): 465. PMID: 16377758

    Felix et al., (1999). Plants have a sensitive perception system for the most conserved domain of bacterial Flagellin. Plant J. 18(3): 265. PMID: 10377992

    Orosa et al., (2018). SUMO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity Nature Communications. 9,: 5185.

    Citation:

    Takáč et al., (2021). TALEN-Based HvMPK3 Knock-Out Attenuates Proteome and Root Hair Phenotypic Responses to flg22 in Barley. Front Plant Sci. 12: 666229. PMID: 33995462

  • Histone proteins and their role in chromatin organisation

    DISCOVERY peptide LL13-37 DISCOVERY peptides from Cambridge Research Biochemicals

    Histone proteins help compact and organise the DNA of the nucleus and provide it with structural support, they also play key roles in orchestrating gene expression. The four core histones, H2A, H2B, H3 and H4 form a complex comprising an H3-H4 tetramer and two H2A-H2B dimers, known as the histone octamer core. Around this core, DNA is wound forming a DNA/histone complex known as the nucleosome. Between the nucleosome is the internucleosomal DNA which is stabilised by the linker histone H1.

    The tail region of the four core histones is able to undergo modifications such as acetylation, methylation, phosphorylation, citrullination, SUMOylation and ubiquitination. These modifications can alter the properties of the chromatin regions in which they occur. For example acetylation generally results in the more open euchromatin state which allows more active transcription such as in H4 K5Ac, K8Ac, K12Ac K16Ac fluorescently labelled peptide. Methylation generally has the opposite effect and results in more closed heterochromatin and transcriptional repression such as H3 K23 trimethylation which has been associated with meiotic cell divisions. Hypermethylation is also seen in some cancer cells and can result in unchecked cell growth.

    The modifications of core histones are carried out by histone modifying enzymes such as histone acetyltransferase (HATs) and histone deacetylases (HDACs), histone methyltransferases (HMTs) and lysine demethylases (KDMs) and kinases and phosphatases.  These histone modifications can occur in different complex combinations; making up what is known as the “histone code”.  Alterations in the histone modification patterns, often due to aberrant expression of histone modifying enzymes, have been seen in diseases such as cancers and autoimmune, cardiovascular and neurodegenerative disorders.

    In addition to these histone modifications, the four core histones can be replaced in certain cells by histone variants which can further alter the nature of the chromatin and affect its level of compaction. Examples of such variants include: histone H3.3, often associated with pericentromeric or telomeric regions for which we have also raised antibodieshistone H2AB1 which creates an unconventional chromatin structure associated with active transcription and; γH2Ax which is associated with DNA double strand breaks, such as during meiosis for which we have also raised an antibody.

    Here are some of our popular histone peptides and antibodies:

    Histone H2A:
    Histone H2A (1-20)-GGK(Biotin): crb1000863
    Histone H2A (78-86): crb1001395
    Histone H2AB1 (22-30) Light: crb1001329

    Histone H2B:
    Anti-Histone H2B antibody crb2005165

    Histone H3:
    Histone H3 (1-20) K4Me3, K9Ac, pS10-GG-[Lys(5- FAM)]: crb1101265
    Biotin-Histone H3 (14-34) pT22 K23Me3: crb1000344
    Histone H3 (20-36) K27Me2 Heavy: crb1300455

    Histone H4:
    Histone H4 (1-21) R3Me2: crb1000907
    Acetyl-Histone H4 (1-23) K5Ac, K8Ac, K12Ac, K16AcGG-[Lys(5-FAM)]: crb1101268
    H4 peptide (16-23):  crb1001167

    To discover our full histone peptides range please click here

    Visit Discovery Antibodies to view our range of histone antibodies

  • Anti-Microbial Peptides: Focus on LL-37

    Cathelicidins form a distinct class of proteins that are key components of the innate and adaptive immune response. They also play a role in apoptosis, inflammation, phagocytosis and angiogenesis. The hallmark of the cathelicidin family of proteins is the presence of a highly conserved cathelin domain. Cathelicidins are expressed as inactive precursor proteins and their proteolytic cleavage leads to the release of active mature peptides ranging from 12 to 88 amino acids. The dominant mature cathelicidin antimicrobial peptide (CAMP) is LL-37 in humans, and cathelicidin-related antimicrobial peptide (CRAMP) in rodents.

    The LL-37 peptide is a 37-residue with two leucine residues at the N-terminus. LL-37 contains an N-terminal helical structure and a flexible C-terminal region. LL-37 belongs to the class of α-helical antimicrobial peptide (AMPs) and possesses amphipathic properties. The bioactive peptides exhibit a broad-spectrum of antimicrobial activity against bacteria, viruses, and fungi. LL-37 can interact with invading pathogens, permeabilise the membranes and subsequently neutralise the activities of endotoxins, such as lipopolysaccharide.

    Cathelicidin peptides can act directly and indirectly to regulate the activity of various cells in the immune system. LL-37 is a multifunctional signalling peptide that possesses various immunomodulatory activities. Depending on the environment and the stage of disease pathogenesis, LL-37 can exhibit both pro-and anti-inflammatory activity. A multitude of receptors recognize LL-37, where it can stabilize and trigger their activity and regulate downstream immunoregulatory mediators.  LL-37 can also induce cytokines and chemokines release and acts as a potent chemoattractant and an adjuvant.

    Available to order from our Discovery® catalogue:

    LL-37 acid (catalogue number: crb1000007)

    LL-37 amide (catalogue number: crb1000864)

    Labelled LL-37 peptides available soon

    Biotin-LL-37 (catalogue number: crb1000836)

    [5-FAM]-LL-37 (catalogue number: crb1000837)

    Fragments

    LL13-37 (catalogue number: crb1000034)

    LL17-29 (catalogue number: crb1000035)

    LL17-32 (catalogue number: crb1000036)

    Mouse

    CRAMP (1-39) (catalogue number: crb1000262)

  • 10% Discount On Your First DISCOVERY® Purchase

    We are offering a 10% discount off your first purchase from DISCOVERY® Peptides & Antibodiesusing the code : ENDEAVOUR10

    The code is valid across all products on the DISCOVERY® Peptides site and also on our sister site DISCOVERY® Antibodies.

  • Peptide Substrates: Focus on LRRKtide

    Mutations in the Leucine-rich repeat kinase-2 (LRRK2) gene have been associated with familial and sporadic Parkinson’s disease (PD), which is the second most common neurodegenerative disorder worldwide after Alzheimer’s. LRRK2 is a multi-domain protein that contains an LRR (leucine rich repeats) motif, a COR (C-terminal of Ras of Complex) domain, a WD40 (Trp-Asp 40) motif and two distinct functional domains; a GTPase domain and a protein kinase domain. The majority of pathological mutations in LRRK2 are clustered within the three domains that form the enzymatic core. The most prevalent mutant form of LRRK2 is the Gly2019Ser mutation, which up-regulates LRRK2 kinase activity. Gly2019 is located within the Asp-Tyr-Gly-Mg2+-binding motif of the kinase domain.

    Moesin was identified as a physiological substrate of LRRK2 in a kinase substrate tracking and elucidation (KESTREL) screen using rat brain extracts. Belonging to the ezrin/redixin/moesin ERM family of proteins, Moesin functions as an anchor between actin-based cytoskeletons and plasma membranes. Analysis of radiolabelled phosphopeptides, derived from LRRK2 phosphorylated Moesin, mapped Thr558 as a phosphorylation site. LRRK2 was also shown to phosphorylate ezrin (Thr567) and radixin (Thr564) at residues equivalent to Moesin Thr558; consequently, the LRRKtide peptide (RLGRDKYKTLRQIRQ) from Moesin that encompasses the Thr558 was derived.  Although LRRKtide contains both threonine and tyrosine residues, LRRK2 acts on LRRKtide predominately as a serine/threonine kinase and not as a tyrosine kinase.

    LRRKtide is widely used for the enzymatic characterization of LRRK2 kinase activity and inhibition studies and is considered to be a more efficient substrate for quantitative assays, compared to measuring LRRK2 auto-phosphorylation. Multiple studies have used LRRKtide to assess the effects of pathogenic mutations on LRRK2 kinase activity and as LRRK2 is clinically linked to PD, LRRK2 kinase has been considered as a therapeutic target. In addition, LRRKtide has been used for comparing the efficiencies of Mg2+ and Mn2+ divalent metal ions as ATP cofactors, to support LRRK2 kinase activity.

    Novel LRRK2 kinase inhibitors have been identified and evaluated from a library of compounds using high-throughput screening that detects LRRKtide phosphorylation using mass spectrometry. The diverse use of LRRKtide has contributed to a better understanding of the role of LRRK2 in Parkinson’s Disease, and provides further possibilities for future investigations into combating the neurodegenerative disorder.

     

    LRRKtide

    http://www.discoverypeptides.com/lrrktide

    crb1000328

     

    LRRKtide amide

    http://www.discoverypeptides.com/lrrktide-amide

    crb1000258

     

    Phosphorylated LRRKtide

    http://www.discoverypeptides.com/phosphorylated-lrrktide

    crb1000466

  • Focus on Myelin Oligodendrocyte Glycoprotein (MOG)

    Myelin oligodendrocyte glycoprotein (MOG) is a type I integral membrane protein on the extracellular surface of oligodendrocytes in the outermost lamellae of the myelin sheath. MOG can exist as monomeric and dimeric species. Its extracellular localisation facilitates its functions as a homophilic adhesion receptor, where it plays a role in the completion, compaction and maintenance of the myelin.

    Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disorder of the central nervous system (CNS) characterized by myelin destruction and axonal degeneration. MOG has been identified as a key autoantigen for demyelination in MS and experimental autoimmune encephalomyelitis (EAE), an animal model that resembles MS. Although MOG is a minor component of the CNS, it is highly immunogenic and can stimulate the activation of T-cell and B-cell responses.

    Immunization of rodents with native or recombinant MOG or synthetic MOG derived peptides induces an inflammatory response and initiates an immune response against myelin, causing damage and degeneration of the CNS. A number of MOG peptide fragments have shown to be encephalitogenic determinants, including MOG (1-22), MOG (35-55), MOG (92-106). Crystal structure studies of the MOG extracellular domain in a homodimer complex reveal that residues within the MOG 1-22, 35-55 and 92-106 map onto the face of the β-sheet and participate in the dimerization interface. This suggests a link between the dimeric form of MOG and a failure of immunological tolerance to MOG seen in MS.

    Studies using various MOG derived peptides show that the MOG (35-55) fragment is the most potent encephalitogen and the immunodominant epitope for T cell response.  MOG (35-55) induced EAE models can be used to recapitulate all three MS subtypes, which are relapsing-remitting MS (RRMS), primary progressive MS (PPMS) and secondary progressive MS (SPMS). Depending on the MOG (35-55) dose, immunized mice are presented with varying degrees of neuropathogical impairment, immune infiltration, ascending paralysis, demyelinating lesions, axon loss and gliosis in the spinal cord and brain. MOG (35-55) induced EAE models can provide an insight into elucidating the immunopathological mechanism of MS progression and facilitate in the development of novel therapeutics.

     

    MOG (35-55) acid Mouse, Rat

    crb1000205

    http://www.discoverypeptides.com/mog-35-55-mouse-rat

     

    MOG (35-55) amide Mouse, Rat

    crb1000379

    http://www.discoverypeptides.com/mog-35-55-amide-mouse-rat

     

    MOG (92-106) Mouse, Rat

    crb1000330

    http://www.discoverypeptides.com/mog-92-106-mouse-rat

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