Analysis of Serotonin <i>N</i>-Acetyltransferase Regulation in Vitro and in Live Cells Using Protein Semisynthesis

Szewczuk, Lawrence M.; Tarrant, Mary Katherine; Sample, Vedangi; Drury, William J.; Zhang, Jin; Cole, Philip A.

doi:10.1021/bi801189d

Cited by 22 publications

(24 citation statements)

References 29 publications

(80 reference statements)

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“…A classic example was reported by Cole and co-workers in 2003, in their study of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). [15] An unnatural protein named AANAT-Pma31 was first semi-synthetically produced with the use of NCL, by ligating the C-terminal fragment of AANAT with an N-terminal synthetic peptide containing 7 at position 31.…”

Section: Mimics Of Phosphoamino Acids (Paas)mentioning

confidence: 99%

Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

Tan

et al. 2011

Chemistry A European J

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Amongst different posttranslational events involved in cellular-signaling pathways, phosphorylation and dephosphorylation of proteins are the most prevalent. Aberrant regulations in the cellular phosphoproteome network are implicated in most major human diseases. Consequently, kinases and phosphatases are two of the most important groups of drug targets in medicinal research today. A major challenge in the understanding of protein phosphorylation and dephosphorylation is the sheer complexity of the phosphoproteome network and the lack of tools capable of studying protein phosphorylation and dephosphorylation as they occur in cells. We highlight herein various chemical biology tools that have emerged in the last decade for such studies. First, we discuss the use of small-molecule mimics of phosphoamino acids and their use in elucidating the function of protein phosphorylation and dephosphorylation. We also introduce recent advances in the field of activity-based protein profiling (ABPP) for proteome-wide detection of protein phosphorylation and dephosphorylation. We next discuss the key concepts in the design of peptide- and protein-based biosensors capable of real-time reporting of phosphorylation/dephosphorylation events. Finally, we highlight the application of peptide and small-molecule microarrays (SMMs), and their applications in high-throughput screening and discovery of new compounds related to phosphorylation/dephosphorylation.

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Section: Mimics Of Phosphoamino Acids (Paas)mentioning

confidence: 99%

Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

Tan

et al. 2011

Chemistry A European J

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“…Cole and co-workers have also used EPL in combination with FRET spectroscopy to study serotonin N-acetyltransferase [124]. Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT)] is a key circadian rhythm enzyme that drives the nocturnal production of melatonin in the pineal gland.…”

Section: Selected Applications Of Expressed Protein Ligationmentioning

confidence: 99%

Expressed protein ligation: a resourceful tool to study protein structure and function

Berrade

Camarero

2009

Cell. Mol. Life Sci.

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This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function.

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“…For example, we were the first to synthesize the CF 2 -phosphonate analogues of phosphoserine and phosphothreonine, 16 and these have proven to be very useful chemical mimics for the “constitutive phosphorylation” phenotype in studying kinase regulation in the cell. 20 …”

Section: Introductionmentioning

confidence: 99%

Combining a Clostridial Enzyme Exhibiting Unusual Active Site Plasticity with a Remarkably Facile Sigmatropic Rearrangement: Rapid, Stereocontrolled Entry into Densely Functionalized Fluorinated Phosphonates for Chemical Biology

et al. 2015

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Described is an efficient stereocontrolled route into valuable, densely functionalized fluorinated phosphonates that takes advantage of (i) a Clostridial enzyme to set the absolute stereochemistry and (ii) a new [3,3]-sigmatropic rearrangement of the thiono-Claisen variety that is among the fastest sigmatropic rearrangements yet reported. Here, a pronounced rate enhancement is achieved by distal fluorination. This rearrangement is completely stereoretentive, parlaying the enzymatically established β-C-O stereochemistry in the substrate into the δ-C-S stereochemistry in the product. The final products are of interest to chemical biology, with a platform for Zn-aminopeptidase A inhibitors being constructed here. The enzyme, Clostridium acetobutylicum (CaADH), recently expressed by our group, reduces a spectrum of γ,δ-unsaturated β-keto-α,α-difluorophosphonate esters (93–99% ee; 10 examples). The resultant β-hydroxy-α,α-difluorophosphonates possess the ‘L’-stereochemistry, opposite to that previously observed for the CaADH-reduction of ω-keto carboxylate esters (‘D’), indicating an unusual active site plasticity. For the thiono-Claisen rearrangement, a notable structure-reactivity relationship is observed. Measured rate constants vary by over three orders of magnitude, depending upon thiono-ester structure. Temperature-dependent kinetics reveal an unusually favorable entropy of activation (∆S‡ = 14.5 ± 0.6 e.u.). Most notably, a 400-fold rate enhancement is seen upon fluorination of the distal arene ring, arising from favorable enthalpic (∆∆H‡ = −2.3 kcal/mol) and entropic (∆∆S‡ = 4 e.u., i.e. 1.2 kcal/mol at RT) contributions. The unusual active site plasticity seen here is expected to drive structural biology studies on CaADH,, while the exceptionally facile sigmatropic rearrangement is expected to drive computational studies to elucidate its underlying entropic and enthalpic basis.

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

Cited by 22 publications

References 29 publications

Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

Current Chemical Biology Tools for Studying Protein Phosphorylation and Dephosphorylation

Expressed protein ligation: a resourceful tool to study protein structure and function

Combining a Clostridial Enzyme Exhibiting Unusual Active Site Plasticity with a Remarkably Facile Sigmatropic Rearrangement: Rapid, Stereocontrolled Entry into Densely Functionalized Fluorinated Phosphonates for Chemical Biology

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