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Novel peptide inhibitors of Leishmania gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein.

ABSTRACT: 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.

SUBMITTER: Corradin S

PROVIDER: S-EPMC1222923 | biostudies-other | 2002 Nov

REPOSITORIES: biostudies-other

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Project description:The myristoylated alanine-rich C-kinase substrate (MARCKS) protein has been at the crossroads of multiple signaling pathways that govern several critical operations in normal and malignant cellular physiology. Functioning as a target of protein kinase C, MARCKS shuttles between the phosphorylated cytosolic form and the unphosphorylated plasma membrane-bound states whilst regulating several molecular partners including, but not limited to calmodulin, actin, phosphatidylinositol-4,5-bisphosphate, and phosphoinositide-3-kinase. As a result of these interactions, MARCKS directly or indirectly modulates a host of cellular functions, primarily including cytoskeletal reorganization, membrane trafficking, cell secretion, inflammatory response, cell migration, and mitosis. Recent evidence indicates that dysregulated expression of MARCKS is associated with the development and progression of hematological cancers. While it is understood that MARCKS impacts the overall carcinogenesis as well as plays a part in determining the disease outcome in blood cancers, we are still at an early stage of interpreting the pathophysiological roles of MARCKS in neoplastic disease. The situation is further complicated by contradictory reports regarding the role of phosphorylated versus an unphosphorylated form of MARCKS as an oncogene versus tumor suppressor in blood cancers. In this review, we will investigate the current body of knowledge and evolving concepts of the physical properties, molecular network, functional attributes, and the likely pathogenic roles of MARCKS in hematological malignancies. Key emphasis will also be laid upon understanding the novel mechanisms by which MARCKS determines the overall disease prognosis by playing a vital role in the induction of therapeutic resistance. Additionally, we will highlight the importance of MARCKS as a valuable therapeutic target in blood cancers and will discuss the potential of existing strategies available to tackle MARCKS-driven blood cancers.

Project description:The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to and is a reversible source of PIP2 at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP2, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH2-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.

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Novel peptide inhibitors of Leishmania gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein.

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