Cupid Peptides presentation wjr

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Transcript of Cupid Peptides presentation wjr

  • William Jonathan Ryves

    Cardiff, U.K.

  • Applica(ons for Cupid Technology

    Cell Marker and Tracking. Real-(me, dye-less Real-(me Protein / Pep(de delivery for protein-protein interac(on and protein func(on mapping

    Drug delivery vehicle for cell-impermeable conjoined APIs

    Regenera(ve medicine e.g Safe crea(on of Stem Cells ex-vivo, Cancer diagnosis / therapy in vivo, wound / burn treatment in situ

  • Cell Penetra(ng Pep(des classified by ac(on

    Class 1 (e.g. Synthe(c Ca(onic Pep(des) Short strings of +vely charged amino acids e.g. Polyarginine, Polylysine Adhere to outer-cell membrane through charge interac(on Endocytosed into vesicles through ac(on of membrane recycling machinery

    Class 2 (e.g. Viral Pep(des) Short strings of amino acids derived from viral proteins e.g. TAT from HIV Adhere to outer-cell membrane through interac(on with receptor protein

    embedded in cell membrane Endocytosed into vesicles through ac(on of receptor recycling machinery Class 3 (Membrane-Permeable Pep(des) Short strings of amphipathic amino acids e.g. Cupid Adhere to outer-cell membrane through charge interac(on Pass directly through lipid bilayer through interac(on with both hydrophilic and

    hydrophobic parts

  • LIVE

    FIXED

    FIG. 2. Visualization of PTDGFP fusion protein import in CHO cells. The recombinant proteins were added to the cells for 5 min at 37C. The cells were washedextensively with PBS and microscopy was performed either on live unfixed cells or after methanol fixation. FITC, fluoresceinisothiocyanate filter; PC, phasecontrast.

    FIG. 3. Adherence of PTDGFP fusion proteins to prefixed CHO cells. The cells were fixed with methanol and rehydrated in PBS. Recombinant proteins wereadded for 5 min at room temperature and the cells were washed extensively with PBS prior to microscopy. FITC, fluoresceinisothiocyanate filter; PC, phasecontrast.

    ARTICLEdoi:10.1016/S1525-0016(03)00135-7

    145MOLECULAR THERAPY Vol. 8, No. 1, July 2003Copyright The American Society of Gene Therapy

    FIG. 2. Visualization of PTDGFP fusion protein import in CHO cells. The recombinant proteins were added to the cells for 5 min at 37C. The cells were washedextensively with PBS and microscopy was performed either on live unfixed cells or after methanol fixation. FITC, fluoresceinisothiocyanate filter; PC, phasecontrast.

    FIG. 3. Adherence of PTDGFP fusion proteins to prefixed CHO cells. The cells were fixed with methanol and rehydrated in PBS. Recombinant proteins wereadded for 5 min at room temperature and the cells were washed extensively with PBS prior to microscopy. FITC, fluoresceinisothiocyanate filter; PC, phasecontrast.

    ARTICLEdoi:10.1016/S1525-0016(03)00135-7

    145MOLECULAR THERAPY Vol. 8, No. 1, July 2003Copyright The American Society of Gene Therapy

    GFP alone

    VP-22 GFP

    TAT GFP

    K8 GFP

    R8 GFP

    FIG. 2. Visualization of PTDGFP fusion protein import in CHO cells. The recombinant proteins were added to the cells for 5 min at 37C. The cells were washedextensively with PBS and microscopy was performed either on live unfixed cells or after methanol fixation. FITC, fluoresceinisothiocyanate filter; PC, phasecontrast.

    FIG. 3. Adherence of PTDGFP fusion proteins to prefixed CHO cells. The cells were fixed with methanol and rehydrated in PBS. Recombinant proteins wereadded for 5 min at room temperature and the cells were washed extensively with PBS prior to microscopy. FITC, fluoresceinisothiocyanate filter; PC, phasecontrast.

    ARTICLEdoi:10.1016/S1525-0016(03)00135-7

    145MOLECULAR THERAPY Vol. 8, No. 1, July 2003Copyright The American Society of Gene Therapy

    BUT Added to cells AFTER FIXATION

    The Lundberg Revision: How different condi(ons can result in

    misinterpreta(on of CPP ac(on

    Cell surface adherence and endocytosis of protein transduc?on domains. Lundberg M, Wikstrm S, Johansson M. Mol Ther. 2003 Jul;8(1):143-50.

    Problems in deploying Class 1 and 2 CPPs

  • bind to negatively charged structures within the cells,such as DNA, which become exposed upon membranedisruption by cell fixation. The ability to of the proteins toadhere to intracellular structures results in a redistributionof protein during fixation, resulting in an apparent butnot true translocation across the cell membrane. The re-location of PTD proteins during fixation explains whyprotein import into almost all cells in a cell population isdetected within only minutes of incubation as well aswhy the import process can occur at 37 and 4C[5,18,26,27,29]. It also explains why many PTD sequencemutants, and even peptides with reversed amino acidsequences, retain the ability of protein import [18,27].The possibility of postfixation movement of proteins andpeptides thereby invalidates methods requiring fixationto study membrane translocation by PTDs. However,methods studying live cells such as flow cytometry alsorequire caution because they do not distinguish betweenprotein immobilized on the cell surface and protein thathas translocated across the cell membrane.

    The binding of PTDs to the cell surface and artificialimport during fixation do not exclude that a smallamount of protein, undetectable by standard imagingtechniques, is imported into cells. The notion that PTDsin fact translocate across the cell membrane is supportedby several studies on biological effects mediated by PTDfusion proteins. These studies include the functional de-livery of p16INK4 [23], p27Kip1 [21], an HIV protease-acti-

    vated caspase-3 [20], and Cre and Flp recombinases[25,4143]. A fixation artifact of protein import cannotexplain the results of these studies, since the biologicaleffects observed for the imported proteins require that thecells are viable. Based on the data presented in the presentstudy, we suggest three possible mechanisms to explainthe biological effects observed: (i) PTD proteins exert ef-fects on the cell surface, (ii) the proteins exert their effectwithin endosomes, or (iii) the PTDs are released from theendosomes into the cytosol by endosomolysis. The firstmechanism implies that the PTD peptides and proteinsadhered to the cell surface may affect cell surface recep-tors, which results in a biological effect. The membrane-translocating property of TAT was first discovered whenrecombinant TAT was added to a cell line containing anHIV long terminal repeat promoter reporter construct [3].The exogenously added TAT was shown to activate thereporter gene, which was interpreted as import of TATinto the cell nucleus and TAT-mediated activation of thepromoter. However, subsequent studies suggested thatexogenously added TAT binds to the cell surface where itactivates cell surface receptors that in turn activate tran-scription factors and induce transcription of the promoter[44,45]. Accordingly, it is possible that some of the bio-logical effects of the PTD fusion proteins are mediated bycell surface receptor activation. The second possiblemechanism of PTD action suggested is that the proteinsexert a local effect within the endosomes and lysosomes.

    FIG. 5. Endocytosis of VP22-GFP. CHO cells were incubated 5 min, 1 h, or 24 h with VP22-GFP. Microscopy was performed on live unfixed cells.

    ARTICLEdoi:10.1016/S1525-0016(03)00135-7

    147MOLECULAR THERAPY Vol. 8, No. 1, July 2003Copyright The American Society of Gene Therapy

    Class 2 CPP stuck on cell surface Class 2 CPP becomes

    trapped in vesicles

    Class 2 CPP excluded from Parts of cell e.g. nucleus

    With live imaging the class 2 CPPs can be seen to be trapped in vesicles

    Cell surface adherence and endocytosis of protein transduc?on domains. Lundberg M, Wikstrm S, Johansson M. Mol Ther. 2003 Jul;8(1):143-50.

  • Problems in deploying Class 1 and 2 CPPs

    CPPs of Class 1 and 2 have a problem exi(ng the endosoma(c pathway to meet cellular targets

    = Endocyto(c vesicle

    = Degrada(on pathway

    CPP 1

    CPP 2

    Cell

    ?

    Recycle

  • Early work with CPP3 inhibi(ng PKA in vivo

    Free living Dictyostelium amoeba Starva?on: Cells release when they begin starving. This ac(vates PKA which causes them to Aggregate within 24 hours

    CPP3 alone No treatment CPP3-PKA inhibitor

    pep(de

    Cells aggregate normally Cells fail to aggregate

    PKA inhibitor pep(de alone

    Use of a penetra?n-linked pep?de in Dictyostelium. Ryves WJ, Harwood AJ. Mol Biotechnol. 2006 Jun;33(2):123-32.

  • Success in Dictyostelium PKA inhibi(on points to new tools to inves(gate protein interac(ons

    Unlike gene(cally engineered cells, the CPP3 based research is fast and in real (me

    Unlike CPP1 and CPP2 related work, CPP3s penetrate cells quickly and directly without using receptors or vesicles.

    Early work with CPP3 inhibi(ng PKA in vivo

  • CPP3 blockade of PTEN interaction with Drebrin.

    A CPP3-linked pep(de inhibi(ng PTEN in vivo

    The interaction of PTEN with Drebrin was observed in vivo by co-expression of GFP-PTEN with mCherry-Drebin in PC12 cells. Interaction was analyzed by measuring fluorescence resonance energy transfer (FRET) using multiphoton fluorescence-lifetime imaging microscopy (FLIM) and demonstrated these proteins bound together in a complex and this complex regulates the phosphorylation state of Drebrin

  • A CPP3-linked pep(de inhibi(ng PTEN in vivo

    Phosphorylation of the actin binding protein Drebrin at S647 is regulated by neuronal activity and PTEN. Kreis P, Hendricusdottir R, Kay L, Papageorgiou IE, van Diepen M, Mack T, Ryves J, Harwood A, Leslie NR, Kann O, Parsons M, Eickholt BJ. PLoS One. 2013 Aug 5;8(8):e71957. doi: 10.1371/journal.pone.0071957.