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Andrew J. Andrews, PhD

Andrew J. Andrews, PhD
About

Associate Professor

Research Program

Lab Overview

The goal of the Andrews lab is to elucidate the mechanisms of decision making in cells. Most of the time a single cell acts as the perfect CEO, using multitudes of data to make decisions, manage resources, and grow. Cells make decisions using a deep learning network, by having an input layer (biological signals), one or more hidden or interconnected layers, and finally, biological output. Our goal is to understand how these networks function when making correct and incorrect decisions. To do this, we need to map the interconnectedness of these hidden networks and mathematically model their relationships. To accomplish this goal, we combined classical enzymology with modern proteomics. This approach allows for the study of complex multi-substrate/product systems that can’t be understood using classical approaches.

Education and Training

Educational Background

  • PhD, Biological Chemistry, University of Michigan, Ann Arbor, MI, 2006
  • BS/MS Microbiology, NCSU, Raleigh, NC, 2000
Research Profile

Research Program

Research Interests

  • Histone chaperone mediated acetylation (Rtt109-Vps75)
  • Specificity and selectivity of p300 and CBP KATs
  • How acetyl-CoA concentration can alter selectivity of histone acetylation
  • How chromatin dynamics influence histone acetylation

Lab Description

There are >100 lysine residues within the nucleosome and greater than 90% have been shown to have post-translational modifications (PTMs) (Figure 1). If you considered PTMs as a chemical indexing system and each PTM creates a unique nucleosome you could make >1018 distinct nucleosomes. Not all of these modifications are observed and coordinated mechanisms of crosstalk suggest the potential that they may make up a learning network.

Mass spectrometry coupled enzyme kinetics in multiproduct systems (Figure 2). We were the first group to use validated quantitative and targeted mass spectrometry (UPLC-QqQ) to study lysine acetyltransferases (KATs) under steady state conditions, allowing us to monitor one substrate turning over into multiple products or acetylation states. This was a technical challenge due to the 100’s of samples that have to be quantitated for this analysis (typically 300 samples for one steady-state curve). We also needed to develop a new theoretical framework for the analysis of one substrate to multiple product turnover.

Specificity and selectivity of p300 and CBP are different and regulated by acetyl-CoA concentration.  These two KATs share ~90% identity in their functional domains but are often correlated with different diseases and developmental responses. Thus, understanding their unique, site-specific activity has provided insight into how the function of the two proteins differ. The findings of our initial study were highlighted in F1000 for its importance in identifying unique differences in these two homologous enzymes. From this starting point we were then able to link acetyl-CoA concentration with the residue preferences of these enzymes (Figure 2). We proposed these enzymes potentially function through a monomeric cooperative model (manifesting as hysteresis), which predicts that at certain concentrations competitive inhibitors could activate the enzyme, resulting in a paradoxical activity. We were able to demonstrate this effect both in vitro and in vivo.

Histone chaperones can recognize histone PTMs, and direct crosstalk (Figure 3). Histone chaperones both interact with KATs and assemble and disassemble nucleosomes, strongly suggesting that they could regulate which histone residue could be modified. Rtt109 is a yeast KAT which is an active site structural homolog of human p300 and CBP and forms a complex with the histone chaperone Vps75 (Nap1 family). In vivo, Rtt109 functions with a second histone chaperone Asf1, to facilitate the acetylation of H3K56, which is critical for DNA damage in yeast. Early biochemical data was able to show that Asf1 could increase the activity of Rtt109-Vps75 but did not result in a change in selectivity. One of the major goals was to determine the mechanism of this change in selectivity observed in vivo. To test our hypothesis that pre-existing acetylation influences the selectivity of Rtt109-Vps75 we made a library of singly acetylated histones using a strain of E. coli containing an orthogonal Nε-acetyllysyl-tRNA synthetase/tRNACUA pair. Using this library with Rtt109-Vps75 under steady-state conditions in the presence and absence of Asf1 we found that only the combination of Asf1 and H3K14ac/H4 could completely change the selectivity of Rtt109-Vps75. The ability of Asf1 to alter the selectivity of Rtt109-Vps75 only in the presence of H3K14ac, suggest that Asf1 was not only a histone chaperone but an acetyl reader. We identified an acidic patch on the backside of Asf1, which we propose ‘reads’ the acetylation state of the H3 tail (Figure 3) and from these data we proposed a new model that reconciles previously conflicting genetic and biochemical data.

The regulation of hydroxylated estrogens in cancer. The oxidative metabolite of estrogen, 4-hydroxyestrogen, could be oncogenic, while 2-hydroxyestrogen is suggested to be anti-proliferative. Both of these products could be further broken down by the P450 enzymes CYP1A1 and 1B1 to produce semiquinones and/or quinones causing DNA damage. Preventing the formation of quinones or semiquinones is the enzyme catechol-O-methyltransferase (COMT) can convert the catechol estrogens to methoxyestrogens. To begin to understand the role of these enzymes in oncogenesis we recently developed a UPLC-QqQ based assay for estrogens and their derivatives. Our assay improves on the sensitivity and speed of existing assays allowing for both in vivo (human urine and cell culture) and in vitro quantitation (Figure 4).

Figure 1: Lysine modifications sites within a nucleosome.
Figure 2: Histone acetylation assay. Top left: Chromatographs of various time points of acetylated and non-acetylated K9K14 peptide (UPLC-QqQ). Top right: normalized intensity for the initial 10% acetylation for K9. Middle: steady-state acetylation as a function of acetyl-CoA for either H3 alone (left) or H3/H4 (right). Bottom: Structure of H3/H4 tetramer, with the H3 in blue and the sites of acetylation in red and the H4 in red with the sites of acetylation in blue, highest observed enzyme dependence (CBP or p300) for each residue under either limiting acetyl-CoA or tetramer is shown.
Figure 3: Acidic patch on the opposite face of the histone binding region of Asf1 is responsible for the recognition of H3K14ac. Left) Electronic potential surface for Asf1 (PDB: 2io5), calculated using PyMOL plugin APBS electrostatics and default settings. Middle) Heat map of free energy difference of catalytic proficiency as a function of Asf1 mutations using H3/H4 and H3K14ac/H4 as a substrate. The specificity of E105A is the most similar to wild-type Asf1 in the absence of H3K14ac but differs in the presence of H3K14ac. Top right) kcat values for Rtt109-Vps75 using wt, E105A, and E105R Asf1 interacting with various acetylation states of H3 (H3KXac/H4). Bottom right) Binding of H3K14ac/H4 and H3/H4 to Asf1 or Asf1(E105A).
Figure 4: Profile of estrogens secreted into the media by cultured lung adenocarcinoma (A549 cells), determined by UPLC-MS-MS.
Lab Staff

Joy M. Cote, PhD

Postdoctoral Associate

Room: P3117
215-728-3558

Daniel D. Krzizike, PhD

Postdoctoral Associate

Room: P3117
215-728-3558

Ryan A. Henry, PhD

Visiting Scientist / Past Lab Member

Darlene Curran

Administrative Assistant

Room: P3048
215-214-1643
Publications

Selected Publications

Cote J.M., Kuo Y.M., Henry R.A., Scherman H., Krzizike D.D., Andrews A.J., Two factor authentication: Asf1 mediates crosstalk between h3 k14 and k56 acetylation. Nucleic Acids Res. 47(14): 7380-7391, 2019. PMC6698667. 11.147

Sidoli S., Kori Y., Lopes M., Yuan Z.F., Kim H.J., Kulej K., Janssen K.A., Agosto L.M., Cunha J.P.C., Andrews A.J., Garcia B.A., One  minute analysis of 200 histone posttranslational modifications by direct injection mass spectrometry. Genome Res. 29(6): 978-987, 2019. PMC6581051. 9.944

Gordon R.E., Zhang L., Peri S., Kuo Y.M., Du F., Egleston B.L., Ng J.M.Y., Andrews A.J., Astsaturov I., Curran T.,Yang Z.J., Statins synergize with hedgehog pathway inhibitors for treatment of medulloblastoma. Clin Cancer Res. 24(6): 1375-1388, 2018. PMC5856627. 8.911

Lee HO, Wang L, Kuo YM, Andrews AJ, Gupta S, Kruger WD. S-adenosylhomocysteine hydrolase over-expression does not alter S-adenosylmethionine or S-adenosylhomocysteine levels in CBS deficient mice. Molecular Genetics and Metabolism Reports, 15:15-21, 2018. ScienceDirect.com

Anthony S.A., Burrell A.L., Johnson M.C., Duong-Ly K.C., Kuo Y.M., Simonet J.C., Michener P., Andrews A., Kollman J.M.,Peterson J.R., Reconstituted impdh polymers accommodate both catalytically active and inactive conformations. Mol Biol Cell. 28(20): 2600-8, 2017. PMC5620369. 14.797

Stepanova D.S., Semenova G., Kuo Y.M., Andrews A.J., Ammoun S., Hanemann C.O., Chernoff J., An essential role for the tumor suppressor merlin in regulating fatty acid synthesis. Cancer Res. 77(18): 5026-5038, 2017. PMC5600854. 8.378

Kuo, Y.M., Henry, R.A., Huang, L., Chen, X., Stargell, L.A., Andrews, A.J. Utilizing targeted mass spectrometry to demonstrate Asf1-dependent increases in residue specificity for Rtt109-Vps75 mediated histone acetylation. PLoS One 10(3), 2015    PubMed

Henry, R.A., Kuo, Y.M., Bhattacharjee, V., Yen, T.J., Andrews, A.J.  Changing the selectivity of p300 by acetyl-CoA modulation of histone acetylation.  ACS Chem. Biol.  10(1): 146-156, 2015 PubMed

Kuo, Y.M., Henry, R.A., Andrews, A.J.  A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation.  Methods.  70(2-3), 127-133, 2014. PubMed

Haery, L., Lugo-Picó, J.G., Henry, R.A., Andrews, A.J., Gilmore, T.D.  Histone acetyltransferase-deficient p300 mutants in diffuse large B cell lymphoma have altered transcriptional regulatory activities and are required for optimal cell growth.  Mol. Cancer 13:29, 2014. PMC3930761  PubMed

Kuo, Y.M., Andrews, A.J.  Correction: Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.  PLoS One. 8(10), 2013. PMC3806868  PubMed

Henry, R.A., Kuo, Y.M., Andrews, A.J.  Differences in specificity and selectivity between CBP and p300 acetylation of histone H3 and H3/H4.  Biochemistry 52(34):5746-59, 2013. PMC3756530  PubMed

Kuo, Y.M., Andrews, A.J.  Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.  PLoS One 8:e54896, 2013.  PMC3578832  PubMed

Andrews, A.J., Luger, K.  Nucleosome structure(s) and stability: Variations on a theme.  Annu. Rev. Biophys. 40:99-117, 2011. Review  PubMed

Böhm, V., Hieb, A.R., Andrews, A.J., Gansen, A., Rocker, A., Tóth, K., Luger, K., Langowski, J.  Nucleosome accessibility governed by the dimer/tetramer interface.  Nucleic Acids Res. 39(8):3093-3102, 2011. PMC3082900  PubMed

Andrews, A.J., Chen, X., Zevin, A., Stargell, L.A., Luger, K.  The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions.  Mol. Cell 37:834-842, 2010.  PMC2880918  PubMed

Koutmou, K.S., Casiano-Negroni, A., Getz, M.M., Pazicni, S., Andrews, A.J., Penner-Hahn, J.E., Al-Hashimi, H.M., Fierke, C.A.  NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P.  Proc. Natl. Acad. Sci. USA 107:2479-2484, 2010.  PMC2823894   PubMed

Geiss, B.J., Thompson, A.A., Andrews, A., Sons, R.L., Gari, H.H., Keenan, S.M., Peersen, O.B.  Analysis of flavivirus NS5 methyltransferase cap binding.  J. Mol. Biol. 385(5):1643-1654, 2009.  PMC2680092  PubMed

Andrews, A., Downing, G., Brown, K., Park Y., Luger, K.  A thermodynamic model for Nap1-histone interactions.  J. Biol. Chem. 283(47):32412-32418, 2008. PMC2583301   PubMed

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