Hydrophobic Transmembrane-Peptide Lipid Conjugations Enhance Membrane Binding and Functional Activity in T-Cells

Abstract

Application of different delivery methods for therapeutic peptides has gained much attention in recent years. In this paper we conjugated a transmembrane hydrophobic peptide (core peptide; CP) derived from the T-cell antigen receptor alpha-chain sequence with either one (LP1), two (LP2), or three (LP3) palmitic acids through a Tris linkage. The effect of these lipopeptides (LPs) were compared to CP's activity both in vitro and in model membrane binding experiments using surface plasmon resonance. The influence of charged amino acids, arginine and lysine, within the CP sequence was examined by synthesizing analogues where arginine and lysine were replaced by the neutral amino acid alanine and these analogues were subsequently Tris-lipid conjugated with either one (XP1), two (XP2), or three (XP3) palmitic acids through a Tris linkage. The results indicated that the amount of irreversible binding for LPs were all greater than that of the underivatized CP in model membranes. None of the LPs could be dissociated from the liposome membranes, even after prolonged washing. Binding results for the neutral conjugates showed that only the XP1 bound to model membranes. This binding was 20% as efficient compared to LP1. In biological assays it was found that LP1 and XP1 were toxic to cells. LP3 inhibited IL-2 production more effectively than CP. Control lipopeptides (XP2, XP3) did not inhibit IL-2 production. These results demonstrate that the number of lipids conjugated to peptide, and the charged amino acids of CP, are both essential factors for peptide function and activity that can be enhanced by lipidation.

Targeting Intramembrane Protein–Protein Interactions: Novel Therapeutic Strategy of Millions Years Old

“…This finding as well as colocalization of TCR CP with TCR in the membrane of resting cells (Wang, Djordjevic, Bender, et al, 2002) directly prove the hypothesis about "pre-" rather than full dissociation state of the unstimulated TCR complex in the presence of TCRα CP, whereas upon stimulation, the affected signaling subunits, ζζ and CD3εδ, become physically disconnected from the remaining receptor complex. It should be noted that the proposed SCHOOL mechanism is the only mechanism consistent with all experimental and clinical data reported up to date for transmembrane peptides of TCR and other MIRRs as well as for lipid and/or sugar conjugates of these peptides (Ali, Amon, Bender, & Manolios, 2005;Ali, De Planque, Huynh, Manolios, & Separovic, 2002;Amon et al, 2006Amon et al, , 2008Bender, Ali, Amon, Diefenbach, & Manolios, 2004;Collier et al, 2006;Gerber, Quintana, Bloch, Cohen, & Shai, 2005;Gollner et al, 2000;Huynh, Ffrench, Boadle, & Manolios, 2003;Kurosaka, Bolte, et al, 2007;Manolios, Huynh, & Collier, 2002;Sigalov, 2007bSigalov, , 2008aWang, Djordjevic, Bender, et al, 2002;Wang, Djordjevic, Kurosaka, et al, 2002).…”

Exogenous Agents that Target Transmembrane Domains of Proteins

“…Conjugation of the CP at the carboxyl terminus with palmitic acid through a tris(hydroxymethyl)aminomethane (Tris) linkage resulted in greater inhibition of T-cell interleukin-2 (IL-2) production in vitro and in vivo than did just the peptide alone. [50] A similar enhancement was also observed with sugar (1-aminoglucosesuccinate) conjugations, [51] which implies potential applications of these CP peptides in drug delivery. When examined for effects in vivo, the CP reduced inflammation in three T-cell-mediated disease models including adjuvantinduced arthritis, experimental allergic neuritis, and cyclophosphamide-induced diabetes in NOD/Lt(F) mice.…”

Novel Mechanistic Insights into Viral Modulation of Immune Receptor Signaling

“…As shown, TCR CP might be a proper treatment for human T cell-mediated dermatoses substituting for corticosteroids [17,18]. However, despite extensive studies [2,[17][18][19][20][21][22][23][24][25][26][27][28], the mode of action of this clinically relevant peptide was not explained until 2004 when the SCHOOL model was first introduced [6].…”