Scratching below the Surface: Wound Healing and Alanine Mutagenesis Provide Unique Insights into Interactions between Eristostatin, Platelets and Melanoma Cells

Abstract

To study the molecular mechanism of the disintegrin eristostatin, cellular functional studies were performed using ten recombinant alanine mutants. ADP-induced platelet aggregation revealed critical contributions of seven residues within the ‘RGD loop’ (R24, R27, G28, N31) and C-terminus (W47, N48, G49) of this disintegrin. Using an in vitro scratch wound healing assay, four human melanoma cell lines yielded similar results when exposed to wildtype eristostatin. All eristostatin-treated cells healed less of the wounded area than control conditions. This phenomenon was reproduced when using fibronectin as the matrix. C8161 cells showed significant delay in wound closure with the N-terminal mutant P4A but not with R24A or G28A. Evidence from our laboratory and others suggests neither alpha IIb, alpha 4 nor alpha 5 integrins are directly involved in eristostatin’s interactions. Eristostatin did not affect the number of melanoma cells in culture after 24 h or the development of apoptosis. However, phosphorylation studies performed after these melanoma cells were exposed to eristostatin revealed changes in several tyrosine phosphorylated molecules.

Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition

“…Many studies have shown that the C-terminal tails of disintegrins are located in the proximity of the RGD loop, the integrin-binding loop, and that the C-terminal regions of disintegrins play synergistic roles in interacting with RGD-binding integrins [16, 18, 20, 21, 25]. For example, C-terminal W67 of flavoridin with an 48 ARGDFP motif is close to D55 [21], C-terminal Y67 of Rho with a 48 PRGDMP motif is close to D55 [25], and C-terminal W67 of trimestatin with a 48 ARGDNP motif is close to P53 [18].…”

“…In particular, eristostatin requires an ARGDW motif and an intact C-terminus (NPWNG) to interact with both platelets and melanoma cells [19]. Eristostatin and bitistatin contain an ARGDWN motif with different C-terminal tails, and eristostatin exhibits a higher affinity to resting platelets [20, 23]. However, the structural basis and mechanism underlying how integrins are recognized by the C-terminus and RGD loop of disintegrins are unclear.…”

Concomitant Activation of the JAK/STAT, PI3K/AKT, and ERK Signaling Is Involved in Leptin-Mediated Promotion of Invasion and Migration of Hepatocellular Carcinoma Cells

“…PCR generated 1,071-and 273-bp fragments of the Ob-Rb and Ob-Rt genes, respectively. To ensure that amplification of these genes was within the exponential range, different numbers of cycles (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) were run. Finally, 30 cycles of PCR amplification were chosen.…”

Isolation and characterization of four medium-size disintegrins from the venoms of Central American viperid snakes of the genera Atropoides, Bothrops, Cerrophidion and Crotalus

“…For example, interaction with integrin a v b 3 affects cell migration, with impact in angiogenesis, tumor metastasis, and atherosclerosis [42,43]. Various disintegrins have been shown to reduce experimental metastasis in melanomas [44,45], and others inhibit endothelial cell adhesion to the extracellular matrix [25]. Thus, the broad pharmacological scope of disintegrins underscores the need for a continuous exploration in search of novel disintegrins and the identification of their targets and pharmacological activities.…”