Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): A second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells.
Disintegrins are small peptides produced in viper venom that act as integrin antagonists. When bound to integrins, disintegrins induce altered cellular behaviors, such as apoptotic induction. Disintegrins with RGDDL or RGDDM motifs induce apoptosis of normal and cancer cells. We hypothesized that a second aspartate (D) carboxyl to the RGD is sufficient to induce apoptosis. Five recombinant mojastin D mutants were produced by site-directed mutagenesis (r-Moj-DA, r-Moj-DG, r-Moj-DL, r-Moj-DN, and r-Moj-DV). Stable αv integrin knockdown and shRNA scrambled control SK-Mel-28 cell lines were produced to test a second hypothesis: r-Moj-D_ peptides bind to αv integrin. Only r-Moj-DL, r-Moj-DM, and r-Moj-DN induced apoptosis of SK-Mel-28 cells (at 29.4%, 25.6%, and 36.2%, respectively). Apoptotic induction was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown (to 2%, 17%, and 2%, respectively), but not in SK-Mel-28 cells with a stable scrambled shRNA. All six r-Moj-D_ peptides inhibited cell proliferation; ranging from 49.56% (r-Moj-DN) to 75.6% (r-Moj-DA). Cell proliferation inhibition by r-Moj-D_ peptides was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown. All six r-Moj-D_ peptides inhibited SK-Mel-28 cell migration at high levels (69%-100%). As a consequence, rac-1 mRNA expression levels were significantly reduced as early as 1 h after treatment, suggesting that rac-1 is involved in the cell migration activity of SK-Mel-28.
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Carla J
CJ
Ramos
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Daniel A
DA
Gutierrez
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Ana S
AS
Aranda
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Melissa A
MA
Koshlaychuk
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
David A
DA
Carrillo
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Rafael
R
Medrano
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Terri D
TD
McBride
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Andrew
A
U
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Stephanie M
SM
Medina
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Melissa C
MC
Lombardo
National Natural Toxins Research Center, Texas A&M University, Kingsville, TX 78363, USA.
Sara E
SE
Lucena
National Natural Toxins Research Center, Texas A&M University, Kingsville, TX 78363, USA.
Elda E
EE
Sanchez
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA. Electronic address: julio.soto@sjsu.edu.
Julio G
JG
Soto
D020816:Amino Acid Motifs; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid / Q000737:chemistry; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D019483:Disintegrins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015195:Drug Design; D004794:Enzyme Repression / Q000187:drug effects; D006801:Humans; D039001:Integrin alpha Chains / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D039564:Integrin alphaV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D030162:Reptilian Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D014757:Viper Venoms / Q000737:chemistry; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D000970:Antineoplastic Agents; D019483:Disintegrins; D039001:Integrin alpha Chains; D039564:Integrin alphaV; D050505:Mutant Proteins; D010446:Peptide Fragments; C107953:RAC1 protein, human; D011994:Recombinant Proteins; D030162:Reptilian Proteins; D014757:Viper Venoms; C554078:mojastin protein, Crotalus scutulatus; D001224:Aspartic Acid; D020830:rac1 GTP-Binding Protein
Apoptosis induction; Cell migration; Cell proliferation; Melanoma cell line; Recombinant RGD disintegrin; rac-1 expression; αv knockdown; αvβ3 integrin