Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.
The in vitro antiproliferative action of pineal indoles on several tumor cell lines including melanoma (B16), sarcoma (S180), macrophage-like cell line (PU5), fibroblasts (3T3), and choriocarcinoma (JAr) was examined by measuring the incorporation of 3H-thymidine by the tumor cells, and, in the case of melanoma cells, by also measuring the incorporation of 3H-leucine and 3H-uridine. Uptake of crystal violet was used to assess the viability of the tumor cells. The order of inhibitory potency of the indoles was found to be methoxytryptamine > melatonin, methoxytryptophol, hydroxytryptophol, and methoxyindoleacetic acid > serotonin and hydroxyindoleacetic acid. The possibility of an adverse effect of the indoles on the viability of normal cells was also investigated by employing a primary culture of rat hepatocytes. The release of glutamate-oxaloacetate transaminase by hepatocytes was not affected by the indoles, although the release of glutamate-pyruvate transaminase was increased to a small extent and the uptake of crystal violet was slightly inhibited.
Male C57 mice kept under a 14:10 (L:D) photoperiod received vehicle (VEH), melatonin (MEL) and methoxytryptamine (MTA) in the drinking water for 2 weeks. Splenocytes from MEL-treated mice showed an augmented mitogenic response to concanavalin A and lipopolysaccharide (LPS) while splenocytes from MTA-treated mice demonstrated an enhanced mitogenic response to LPS when compared to the VEH-treated control. Splenocytes from MEL-treated and MTA-treated mice also produced higher levels of gamma interferon and interleukin-2. Lymphokines prepared from splenocytes of MEL-treated mice stimulated peritoneal macrophages to produce more nitrite than those from splenocytes of MTA-treated and control mice, suggesting that MEL had a stronger stimulating effect on the lymphocytes than MTA. Understimulation of lymphokines from MEL-treated mice, peritoneal macrophages from MTA-treated mice produced a greater inhibition of the growth of murine mastocytoma P815 cells than that produced by macrophages from control and MEL-treated mice, suggesting that MTA was more potent than MEL in rendering the macrophages responsive to lymphokines. The results point to immunostimulatory actions of the pineal indoles MEL and MTA.
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