Novel peptide inhibitors of Leishmania gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein.
The zinc metalloprotease gp63 (leishmanolysin; promastigote surface protease) is expressed at high density at the surface of Leishmania promastigotes. Efficient non-toxic inhibitors of gp63 do not exist, and its precise role in parasite physiology remains unknown. MARCKS (myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in various cells, including macrophages. We reported previously that MRP is an excellent substrate for gp63. A major cleavage site was identified within the MRP effector domain (ED), a highly basic 24-amino-acid sequence, and the synthetic ED peptide (MRP(ED)) was shown to inhibit MRP hydrolysis. In the present study, MRP cleavage was used as an assay to measure the capacity of various MRP or MARCKS ED peptides to block gp63 activity. On a molar basis, MRP(ED) inhibited gp63 to a greater extent than two previously described gp63 inhibitors, o -phenanthroline and benzyloxycarbonyl-Tyr-Leu-NHOH. MARCKS(ED) analogues containing modifications in the gp63 consensus cleavage site showed significant differences in inhibitory capacity. As phosphorylation of ED serine residues prevented gp63-mediated MRP degradation, we synthesized a pseudophosphorylated peptide in which serine residues were substituted by aspartate (3DMRP(ED)). 3DMRP(ED) was a highly effective inhibitor of both soluble and parasite-associated gp63. Finally, MRP ED peptides were synthesized together with an N-terminal HIV-1 Tat transduction domain (TD) to obtain cell-permeant peptide constructs. Such peptides retained gp63 inhibitory activity and efficiently entered both macrophages and parasites in a Tat TD-dependent manner. These studies may provide the basis for developing potent cell-permeant inhibitors of gp63.
Institute of Biochemistry, University of Lausanne, Chemin de Boveresses, 1066 Epalinges, Switzerland. Sally.Corradin-Betz@ib.unil.ch
Sally
S
Corradin
Adriana
A
Ransijn
Giampietro
G
Corradin
Jacques
J
Bouvier
Maria Belen
MB
Delgado
Jimena
J
Fernandez-Carneado
Jeremy C
JC
Mottram
Guy
G
Vergères
Jacques
J
Mauël
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D006868:Hydrolysis; D018320:Leishmania major / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013379:Substrate Specificity
D008565:Membrane Proteins; D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania