Discovery Peptides

UK Research Peptides

  • Peptide Substrates: Insulysin FRET substrate [Mca]/[Dnp]

    Fluorescence Resonance Energy Transfer (FRET) peptides are convenient tools for the study of peptidase specificity and enzymatic activity. The Insulysin FRET substrate [Mca]/[Dnp] is a synthetic insulysin peptide substrate that contains an N-terminal fluorescent 7-methoxycoumarin (Mca) group and a C-terminal 2, 4-dinitrophenyl (Dnp) quencher; the FRET peptide exhibits internal fluorescence quenching when intact. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence, enabling the quantitative measure of enzymatic activity.

    Insulysin, also known as Insulin-degrading enzyme (IDE), is a highly conserved zinc metallopeptidase that contains an inverted zincin motif (HXXEH), characteristic of the inverzincin family of proteases. The two histidine (H) residues chelate with the zinc ion, and the glutamate (E) is involved in catalytic water activation. Insulysin functions as a dimer and exhibits allosteric kinetics, whereby small substrate peptides and polyphosphates such as ATP can act as enzyme activators. The endopeptidase cleaves a wide variety of substrates that are diverse in length, structure and sequence and exhibits a preference for basic or bulky hydrophobic residues.

    Insulysin was initially discovered as the enzyme responsible for insulin catabolism and is ubiquitously expressed; studies have shown that it is involved in the degradation of many bioactive peptide substrates including glucagon, TGF-α, β-endorphin, dynorphins and atrial natriuretic peptide. Insulysin also selectively degrades amylin and β-amyloid amyloidogenic peptides at multiple sites. The imbalance between synthesis and clearance of β-amyloid peptides can lead to the accumulation and formation of amyloid plaques in the brain, which is one of the hallmarks of Alzheimer’s disease. Insulysin is a key component in the maintenance of proteostasis in many cellular processes, therefore the identification of novel potent and selective insulysin modulators can help advance the development of insulysin-based therapeutics for diseases such as type 2 diabetes and Alzheimer's Disease.

    Unravelling the effects of insulysin modulators on the multiplicity of insulysin targets will provide an insight of its pivotal role in interconnecting several cellular processes.

     

    Available to purchase through DISCOVERY® Peptides

    Insulysin FRET substrate [Mca]/[Dnp]

    [Mca]-GGFLRKHGQ-[Dnp]

    catalogue number: crb1100406

    http://www.discoverypeptides.com/insulysin-fret-substrate-mca-dnp

  • 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

  • Cell Penetrating Peptides: Focus on Arg9

    An Introduction to Cell Penetrating Peptides:

    Due to the hydrophilic nature of the majority of proteins and peptides, they are largely impermeable to cell membranes. Cell Penetrating Peptides (CPP’s) therefore possess a pivotal role in terms of their ability to successfully facilitate cellular uptake of molecular cargos across the cell or plasma membrane. They typically possess a high relative abundance of the positively charged amino acids Arginine and Lysine or possess an alternating pattern of polar and non-polar residues. Various mechanisms by which CPP’s traverse cell membranes have been postulated including endocytosis and direct translocation but their exact mode of action remains unclear and is, to this day, widely investigated.

    Cambridge Research Biochemicals offer a range of CPP’s through the Discovery Peptides catalogue - more information is given below

    Focus on Arg9

    Arg9 is a synthetic homopolyarginine nona-peptide (R9) cell penetrating peptide (CPP). The arginine amino acids play an important role in efficient cellular uptake. The guanidinium group is a critical structural determinant for tight and rapid interactions with cell membrane. The cationic guanidinium group can form electrostatic interactions with anionic cell membranes components such as phospholipids and sulphated proteoglycans. This interaction can trigger the activation of specific intracellular signalling and cell internalization via various pathways.

    The hydrophilic arginine amino acids destabilize and disrupt the phospholipid bilayer and nucleate transient pores for passive diffusion across the membrane. In addition, the extended 3 carbon linear chain of arginine facilitates insertion and cell permeation. In the presence of different types of phospholipids Arg9 exhibits structural polymorphism with no interfacial properties or specific secondary structure, which implicates endocytosis as another mechanism of cellular uptake. However, the specific endocytosis pathways utilized is dependent on multiple factors such as peptide concentration, environmental conditions and membrane composition. Cell viability and membrane integrity studies of Arg9 peptide intracellular delivery were shown to have low cytotoxicity, no leakage or liposome bilayer entrapment.

    Oligo-arginine peptides have been used to deliver a variety of functionally active cargos such as peptides, small interfering RNA, oligonucleotides, plasmid DNA and liposomes into mammalian cells and plant cells with high translocation efficiency. Non-covalent and covalent strategies have been used to assess cargo delivery. Click Arg9 can be used to form chemical linkages with the cargo and act as a carrier peptide. Understanding the translocation mechanisms of Arg9 can provide an insight in the development of efficient molecular delivery vehicles of drugs and other agents into the cytosol and nucleus of cells.

    Various Arg9 peptides are available via the new Discovery Peptides Catalogue:

    Discovery Peptides

    Click on the corresponding catalogue number to be taken to the Discovery webpage:

    Standard unmodified Peptide: Arg9 - Catalogue Number: crb1000182

    5-carboxyfluoroscein labelled Arg9: [5-Fam]-Arg9 - Catalogue Number: crb1100289

    4-pentynyl "Click" functionalised Arg9: Click Arg9 - Catalogue Number: crb1000111

4 Item(s)