Donna M. Veine

Crystal Structure of a Phosphoinositide Phosphatase, MTMR2: Insights into Myotubular Myopathy and Charcot-Marie-Tooth Syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.

Abstract 3890: The PhScN peptide as a highly potent inhibitor of basement membrane invasion in vitro and lung colonization in nude mice by metastatic human prostate cancer cells

Surgery and radiation can cure localized prostate cancer, but not metastatic disease. Our research has shown that activated Δ5α1 integrin fibronectin receptors mediate metastatic invasion by human prostate cancer cell lines, DU 145 and PC-3, as well as angiogenic invasion by human microvascular endothelial cells (hmvec). Thus, activated Δ5α1 receptors are key to metastatic progression. To inhibit metastatic invasion, we devised the Ac-PHSCN-NH2 peptide, PHSCN, licensed as ATN-161. Systemic ATN-161 monotherapy was well tolerated in Phase I trial, and halted metastatic progression for 4-14 months in 35% of patients; however, it was not potent enough for easy administration. Thus, there are some major difficulties with ATN-161 as a drug. Also, PHSCN can be degraded by endoproteinases, enzymes that cleave between its linked L-amino acids. Since tumors secrete abundant endoproteinases, we devised a potent, exo- and endoproteinase-resistant, Δ5α1αtargeted invasion inhibitor, the Ac-PhScN-NH2 peptide (PhScN). Because it contains mirror image D-isomers of His (h) and Cys (c) separating L-isomers of Pro, Ser, and Asn, is resistant to endoproteolytic degradation. Our results suggest that PhScN is 27,000 to 340,000-fold more potent than PHSCN at blocking Δ5α1-mediated, serum-induced, or serum-free PHSRN peptide-induced invasion in vitro by metastatic human DU 145 and PC-3 prostate cancer cells. Our results also indicate that a single pretreatment of suspended DU 145 and PC-3 cells with varying concentrations of the PhScN peptide prior to intravenous injection reduces lung colony formation in athymic, nude mice by 100,000 to 300,000-fold, relative to lung metastasis inhibition by the parental PHSCN peptide. Because Δ5α1 integrin also supports survival, we determined the effects of elevated PhScN concentrations on clonogenic DU 145 and PC-3 survival in vitro. We found that PhScN has no appreciable effect on clonogenic survival, suggesting that its potent anti-metastatic activity is due to invasion inhibition in lung microvasculature. Consistent with this hypothesis, our preliminary results suggest that PhScN pretreatment is also significantly more potent method for preventing extravasation into lung tissue than pretreatment with the parental PHSCN peptide. Since our published results show that the PHSCN polylysine dendrimer (PHSCNGGK-MAP) is 100-fold more potent than the PHSCN peptide at inhibiting DU 145 and PC-3 lung colonization, the results presented here suggest that D-amino acid substitution in the PHSCN sequence, thereby forming the PhScN peptide or dendrimer (PhScNGGK-MAP) may be an excellent strategy for the synthesis of highly potent Δ5α1-targeted inhibitors of metastatic invasion and lung colonization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3890. doi:1538-7445.AM2012-3890