Browsing by Author "Morlacchi, Pietro"
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Item Nerolidol- terpene- and terpene deriviative synthesis(2012-05-08) McNeil, Caroline Virginia; Morlacchi, Pietro; Baevich, Alyssa; Matsuda, Seiichi P. T.; Rice University; United States Patent and Trademark OfficeAccording to one embodiment, the description relates to a method of nerolidol production. The method includes culturing a yeast strain lacking functional squalene synthase and overproducing HMG CoA reductase in synthetic medium lacking uracil and producing nerolidol. The pH of the medium may be adjusted to an acidic level to further increase nerolidol production. Other chemicals may also be produced by this method. The nerolidol or other chemicals may be removed from the yeast or medium or both. The medium may additionally contain a polyaromatic resin, which may adsorb nerolidol or other chemicals.Item Targeting the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 6 (STAT6) with Cell-Permeable, Phosphatase-Stable Phosphopeptide Mimics Potently Inhibits Tyr641 Phosphorylation and Transcriptional Activity(American Chemical Society, 2015) Mandal, Pijus K.; Morlacchi, Pietro; Knight, J. Morgan; Link, Todd M.; Lee, Gilbert R. IV; Nurieva, Roza; Singh, Divyendu; Dhanik, Ankur; Kavraki, Lydia; Corry, David B.; Ladbury, John E.; McMurray, John S.Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure–affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.Item Triterpene biosynthesis in plants: Oxidosqualene cyclization in Arabidopsis thaliana(2009) Morlacchi, Pietro; Matsuda, Seiichi P. T.The enzymatic conversion of oxidosqualene to cyclic triterpenes represents the key step in the biosynthesis of more than 20,000 triterpenoids. These compounds are widely distributed in nature among plants, animals, fungi, and some bacteria. Triterpenes serve as precursors of sterols, steroids, and numerous secondary metabolites of undefined function. Insights into triterpene biochemistry are emerging from studies of gene expression, metabolite profiles, and enzyme function in the reference plant Arabidopsis thaliana . This thesis describes the functional characterization by heterologous expression in Saccharomyces cerevisiae of four Arabidopsis oxidosqualene cyclases: PEN3 (At5g36150), LUP1 (At1g78970), LUP2 (At 1g78960), and LUP5 (At1g66960). PEN3, the last uncharacterized Arabidopsis cyclase makes predominantly tirucalla-7,24-dienol. The elucidation of the product profile of PEN3 illuminated alternative mechanistic routes to the formation of tetracyclic triterpene alcohols by dammarenyl-type oxidosqualene cyclases. LUP5, which had only been cursorily characterized, makes the same set of 6/6/6/5 tetracycles as that of PEN3. This represents a likely example of convergent evolution since these genes show rather low sequence identity. I found that LUP1 is a lupane-3β,20-diol (lupanediol) synthase not a lupeol synthase or a multifunctional enzyme. The fact that lupanediol is the major LUP1 product suggests that lupanediol is the biosynthetic precursor of trinorlupeol, a major nonsterol triterpenoid in Arabidopsis . The abundance of minor LUP1 products may reflect the difficulty for cyclases to make diols accurately. LUP2 is a truly multifunctional enzyme, making a mixture of β-amyrin and other cyclic triterpenes, any of which could have been better optimized by selective pressure to make a single product. I established that the product profile of LUP2 and LUP1 in previous characterizations was distorted by selective loss of triterpene products to the culture medium. These results underlined the importance of analyzing the medium to avoid misleading interpretation of in vivo product profiles. The characterization of PEN3 completes the first functional characterization of all triterpene synthases in a higher plant. These results together with more reliable product profiles of some LUP genes provide a better understanding of the catalytic properties of this class of enzymes.