Structure of the hypothetical protein Ton 1535 from Thermococcus onnurineus NA1 reveals unique structural properties by a left‐handed helical turn in normal α‐solenoid protein

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α‐helices in a helix‐turn‐helix motif as a repeating unit, Ton1535 consists of right‐handed coiled N‐ and C‐terminal regions that are stacked together using helix bundles containing a left‐handed helical turn. One left‐handed helical turn in the right‐handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α‐helices on the convex surfaces of the N‐terminal region to the extended surface of the concave groove of the C‐terminal region and vice versa. Proteins 2014; 82:1072–1078. © 2013 Wiley Periodicals, Inc.     

HDAC‐inhibitor (S)‐8 disrupts HDAC6‐PP1 complex prompting A375 melanoma cell growth arrest and apoptosis

Histone deacetylase inhibitors (HDACi) are agents capable of inducing growth arrest and apoptosis in different tumour cell types. Previously, we reported a series of novel HDACi obtained by hybridizing SAHA or oxamflatin with 1,4‐benzodiazepines. Some of these hybrids proved effective against haematological and solid cancer cells and, above all, compound (S)‐8 has emerged for its activities in various biological systems. Here, we describe the effectiveness of (S)‐8 against highly metastatic human A375 melanoma cells by using normal PIG1 melanocytes as control. (S)‐8 prompted: acetylation of histones H3/H4 and α‐tubulin; G₀/G₁ and G₂/M cell cycle arrest by rising p21 and hypophos‐phorylated RB levels; apoptosis involving the cleavage of PARP and caspase 9, BAD protein augmentation and cytochrome c release; decrease in cell motility, invasiveness and pro‐angiogenic potential as shown by results of wound‐healing assay, down‐regulation of MMP‐2 and VEGF‐A/VEGF‐R2, besides TIMP‐1/TIMP‐2 up‐regulation; and also intracellular accumulation of melanin and neutral lipids. The pan‐caspase inhibitor Z‐VAD‐fmk, but not the antioxidant N‐acetyl‐cysteine, contrasted these events. Mechanistically, (S)‐8 allows the disruption of cytoplasmic HDAC6‐protein phosphatase 1 (PP1) complex in A375 cells thus releasing the active PP1 that dephosphorylates AKT and blocks its downstream pro‐survival signalling. This view is consistent with results obtained by: inhibiting PP1 with Calyculin A; using PPP1R2‐transfected cells with impaired PP1 activity; monitoring drug‐induced HDAC6‐PP1 complex re‐shuffling; and, abrogating HDAC6 expression with specific siRNA. Altogether, (S)‐8 proved very effective against melanoma A375 cells, but not normal melanocytes, and safe to normal mice thus offering attractive clinical prospects for treating this aggressive malignancy.