Enzymatic reactions in teleocidin B biosynthesis

The teleocidin B family members are terpene indole compounds isolated from Streptomyces bacteria, and they strongly activate protein kinase C (PKC). Their unique structures have attracted many researchers in the natural product chemistry and pharmacology fields, and numerous isolation and bioactivity studies have been conducted. The accumulated information has facilitated the identification of the enzymatic reactions in teleocidin biosynthesis, and new developments in structural biology have strongly aided efforts to clarify the finer points of these reactions. This review describes the recent biochemical and structural biological studies to reveal their reaction mechanisms, with a primary focus on the terpene cyclization triggered by the C-N bond formation by P450 oxygenase (TleB), the prenyltransferase (TleC), and the methyltransferase (TleD). This new knowledge will benefit future engineering studies to create unnatural PKC activators.

GPCR signaling inhibits mTORC1 via PKA phosphorylation of Raptor

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth, metabolism, and autophagy. Extensive research has focused on pathways that activate mTORC1 like growth factors and amino acids; however, much less is known about signaling cues that directly inhibit mTORC1 activity. Here, we report that G-protein coupled receptors (GPCRs) paired to Gαs proteins increase cyclic adenosine 3’5’ monophosphate (cAMP) to activate protein kinase A (PKA) and inhibit mTORC1. Mechanistically, PKA phosphorylates the mTORC1 component Raptor on Ser 791, leading to decreased mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, mTORC1 activity is partially rescued even after PKA activation. Gαs-coupled GPCRs stimulation leads to inhibition of mTORC1 in multiple cell lines and mouse tissues. Our results uncover a signaling pathway that directly inhibits mTORC1, and suggest that GPCRs paired to Gαs proteins may be potential therapeutic targets for human diseases with hyperactivated mTORC1.

Using RNA Editing and ADARs to Increase Cell Sensitivity to Interferon Chemotherapies

Background and Hypothesis: During viral infection, viral double stranded RNAs (dsRNAs) are bound by and activate pattern recognition receptors (PRRs). Activated PRRs signal several downstream cellular events, including activating transcription of interferon (IFN). IFN-β can bind to cellular receptors and increase transcription of PRRs, interferon stimulated genes (ISGs), activate Protein Kinase R (PKR) and trigger apoptosis. The ability of IFN to trigger apoptosis makes it a potential chemotherapeutic. However, IFN therapy has had mixed efficacy in inducing tumor cell apoptosis. One mechanism for this failure may be intrinsic mechanisms that prevent dsRNA from binding to and activating PRRs. ADAR1 binds to dsRNA and catalyzes deamination of adenosine to inosine, a process known as RNA editing. Normally, ADAR1 inhibits cellular dsRNAs from initiating the IFN response. Recent studies have shown ADAR1 is overexpressed in several cancers and contributes to oncogenic phenotypes, suggesting inhibition of ADAR1 may increase sensitivity to IFN-β. The Hundley lab recently identified an inhibitor of ADAR1 called ADAR3. ADAR3 has no deaminase activity and is highly expressed in glioblastoma (GBM) tumors. We hypothesize that ADAR3 expressing GBM cells should exhibit a greater antiproliferative effect in response to IFN-b treatment compared to control. Experimental Design or Project Methods: An MTT assay was conducted in which U87 ADAR3 +/- cell lines were exposed to IFN-β after 24 hours. Cell viability was measured for 3 days post-treatment. Results: U87 ADAR3 + cells are more sensitive to IFN-β treatment (n=2 biological replicates, p-value= 0.04). Conclusion and Potential Impact: The results suggest a mechanism to enhance IFN therapy for patients with ADAR3 expressing GBM.

Protein kinase Cδ protects against bile acid apoptosis by suppressing proapoptotic JNK and BIM pathways in human and rat hepatocytes

Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.