Analysis of Serotonin N -Acetyltransferase Regulation in Vitro and in Live Cells Using Protein Semisynthesis †

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry (Impact Factor: 3.02). 10/2008; 47(39):10407-19. DOI: 10.1021/bi801189d
Source: PubMed


Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal. Prior studies have suggested that its light and diurnal regulation involves phosphorylation on key AANAT Ser and Thr residues which results in 14-3-3zeta recruitment and changes in catalytic activity and protein stability. Here we use protein semisynthesis by expressed protein ligation to systematically explore the effects of single and dual phosphorylation of AANAT on acetyltransferase activity and relative affinity for 14-3-3zeta. AANAT Thr31 phosphorylation on its own can enhance catalytic efficiency up to 7-fold through an interaction with 14-3-3zeta that lowers the substrate K m. This augmented catalytic profile is largely abolished by double phosphorylation at Thr31 and Ser205. A possible basis for this difference is the dual anchoring of doubly phosphorylated AANAT via one 14-3-3zeta heterodimer. We have developed a novel solution phase assay for accurate K D measurements of 14-3-3zeta-AANAT interaction using 14-3-3zeta fluorescently labeled with rhodamine by expressed protein ligation. We have also generated a doubly fluorescently labeled AANAT which can be used to assess the stability of this protein in a live cell, real-time assay by fluorescence resonance energy transfer measured by microscopic imaging. These studies offer new insights into the molecular basis of melatonin regulation and 14-3-3zeta interaction.

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    • "The protein was hypothesized to be shielded from proteosomal proteolysis by association to 14-3-3 z through pSer-205. The Cole group synthesized the octapeptide containing a difluoromethyl phosphoserine mimetic and used their EPL technology to look for an interaction between pSer-205 AANAT and its 14-3-3 z binding partner (Szewczuk et al., 2008). Through this construct they were able to demonstrate that phosphorylation at this site results in binding to 14- 3-3 z and enhances cellular stability of AANAT protein. "
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    ABSTRACT: Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues. To create small molecules that inhibit such interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. For this study, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The prodrug is cytotoxic at low micromolar concentrations when applied to cancer cells and induces caspase activation resulting in apoptosis. The prodrug reverses the 14-3-3-mediated inhibition of FOXO3a resulting from its phosphorylation by Akt1 in a concentration-dependent manner that correlates well with its ability to inhibit cell growth. This methodology can be applied to target a variety of proteins containing phosphoserine and other phosphoamino acid binding domains.
    Chemistry & biology 06/2012; 19(6):764-71. DOI:10.1016/j.chembiol.2012.05.011 · 6.65 Impact Factor
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    • "Subsequent studies showed that this effect persists in live cells, expanding the domain in which such " teflon phosphates " may be deployed to ask fundamental questions in chemical biology (Szewczuk et al., 2008). "
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    ABSTRACT: This overview focuses on the (alpha,alpha-difluoromethylene)phosphonate mimic of phosphoserine (pCF(2)Ser) and its application to the study of kinase-mediated signal transduction-pathways of great interest to drug development. The most versatile modes of access to these chemical biological tools are discussed, organized by method of PCF(2)-C bond formation. The pCF(2)-Ser mimic may be site-specifically incorporated into peptides (SPPS) and proteins (expressed protein ligation). This isopolar, dianionic pSer mimic results in a "constitutive phosphorylation" phenotype and is seen to support native protein-protein interactions that depend on serine phosphorylation. Signal transduction pathways studied with this chemical biological approach include the regulation of p53 tumor suppressor protein activity and of melatonin production. Given these successes, the future is bright for the use of such "teflon phospho-amino acid mimics" to map kinase-based signaling pathways.
    Chemistry & biology 09/2009; 16(9):928-36. DOI:10.1016/j.chembiol.2009.08.008 · 6.65 Impact Factor
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    ABSTRACT: Die Untersuchung biologischer Fragestellungen mit chemischen Methoden wird meist als “Chemische Biologie” bezeichnet und setzt voraus, dass biologisch relevante Makromoleküle, wie Peptide und Proteine, chemisch zugänglich sind. Auf der Festphasensynthese von Peptiden aufbauend wurden viele chemoselektive Ligations- und Modifikationstechniken zur Verknüpfung synthetischer Peptide oder funktionaler Einheiten zu größeren synthetischen, biologisch relevanten Makromolekülen entwickelt. Dieser Aufsatz fasst die aktuellen Entwicklungen auf dem Gebiet der chemoselektiven Ligations- und Modifikationsstrategien zusammen und illustriert ihre Anwendbarkeit an Beispielen aus der chemischen Totalsynthese von Proteinen bis hin zur Semisynthese natürlicher modifizierter Proteine.
    Angewandte Chemie 12/2008; 120(52):10182-10228. DOI:10.1002/ange.200801313
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