Publications

2020
Z. A. Wang and P. A. Cole. 2020. “The Chemical Biology of Reversible Lysine Post-translational Modifications.” Cell Chem Biol.Abstract
Lysine (Lys) residues in proteins undergo a wide range of reversible post-translational modifications (PTMs), which can regulate enzyme activities, chromatin structure, protein-protein interactions, protein stability, and cellular localization. Here we discuss the "writers," "erasers," and "readers" of some of the common protein Lys PTMs and summarize examples of their major biological impacts. We also review chemical biology approaches, from small-molecule probes to protein chemistry technologies, that have helped to delineate Lys PTM functions and show promise for a diverse set of biomedical applications.
C. D. Morrison-Smith, T. M. Knox, I. Filic, K. M. Soroko, B. K. Eschle, M. K. Wilkens, P. C. Gokhale, F. Giles, A. Griffin, B. Brown, G. I. Shapiro, B. E. Zucconi, P. A. Cole, M. E. Lemieux, and C. A. French. 2020. “Combined Targeting of the BRD4-NUT-p300 Axis in NUT Midline Carcinoma by Dual Selective Bromodomain Inhibitor, NEO2734.” Mol. Cancer Ther., 19, 7, Pp. 1406–1414.Abstract
NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET-selective bromodomain inhibitors have demonstrated on-target activity in patients with NMC, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces cooperative depletion of MYC and synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain-selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or "standard" chemotherapy. Our findings provide a strong rationale for clinical study of NEO2734 in patients with NMC. Moreover, the synergistic inhibition of NMC growth by CBP/p300 and BET bromodomain inhibition lays the groundwork for greater mechanistic understanding of the interplay between p300 and BRD4-NUT that drives this cancer.
Z. A. Wang, C. J. Millard, C. L. Lin, J. E. Gurnett, M. Wu, K. Lee, L. Fairall, J. W. Schwabe, and P. A. Cole. 2020. “Diverse nucleosome Site-Selectivity among histone deacetylase complexes.” Elife, 9.Abstract
Histone acetylation regulates chromatin structure and gene expression and is removed by histone deacetylases (HDACs). HDACs are commonly found in various protein complexes to confer distinct cellular functions, but how the multi-subunit complexes influence deacetylase activities and site-selectivities in chromatin is poorly understood. Previously we reported the results of studies on the HDAC1 containing CoREST complex and acetylated nucleosome substrates which revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al, 2018). Here we analyze the enzymatic properties of five class I HDAC complexes: CoREST, NuRD, Sin3B, MiDAC and SMRT with site-specific acetylated nucleosome substrates. Our results demonstrate that these HDAC complexes show a wide variety of deacetylase rates in a site-selective manner. A Gly13 in the histone H3 tail is responsible for a sharp reduction in deacetylase activity of the CoREST complex for H3K14ac. These studies provide a framework for connecting enzymatic and biological functions of specific HDAC complexes.
Wang L. Han R. Belfer U. Kalin J. Cole P. A. Hancock W. Xiong, Y. 2020. “Essential Role of the CoREST Complex in Foxp3+ T-Regulatory Cell Functions.” J. Clin. Invest., 130, Pp. 1830-1842.
Dagil L. Fairall L. Robertson N. Wu M. Ragan T. J. Savva G. C. Morone N. Kunze M. B. A. Jamieson A. G Song, Y. 2020. “Functional and Structural Coupling Between LSD1 and HDAC1 in the CoREST Corepressor Complex.” Cell Reports, 30, Pp. 2699-2711.
S. A. Miller, R. A. Policastro, S. S. Savant, S. Sriramkumar, N. Ding, X. Lu, H. P. Mohammad, S. Cao, J. H. Kalin, P. A. Cole, G. E. Zentner, and H. M. O'Hagan. 2020. “Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer.” Mol. Cancer Res., 18, 2, Pp. 264–277.Abstract
Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.
Z. A. Wang and P. A. Cole. 2020. “Methods and Applications of Expressed Protein Ligation.” Methods Mol. Biol., 2133, Pp. 1–13.Abstract
Expressed protein ligation is a method of protein semisynthesis and typically involves the reaction of recombinant protein C-terminal thioesters with N-cysteine containing synthetic peptides in a chemoselective ligation. The recombinant protein C-terminal thioesters are produced by exploiting the action of nature's inteins which are protein modules that catalyze protein splicing. This chapter discusses the basic principles of expressed protein ligation and recent advances and applications in this protein semisynthesis field. Comparative strengths and weaknesses of the method and future challenges are highlighted.
J. W. Hsu, M. Bai, K. Li, J. S. Yang, N. Chu, P. A. Cole, M. J. Eck, J. Li, and V. W. Hsu. 2020. “The protein kinase Akt acts as a coat adaptor in endocytic recycling.” Nat. Cell Biol., 22, 8, Pp. 927–933.Abstract
Coat proteins have a central role in vesicular transport by binding to cargoes for their sorting into intracellular pathways. Cargo recognition is mediated by components of the coat complex known as adaptor proteins1-3. We previously showed that Arf-GAP with coil-coil, ANK repeat and PH domain-containing protein 1 (ACAP1) functions as an adaptor for a clathrin coat complex that has a function in endocytic recycling4-6. Here, we show that the protein kinase Akt acts as a co-adaptor in this complex, and is needed in conjunction with ACAP1 to bind to cargo proteins to promote their recycling. In addition to advancing the understanding of endocytic recycling, we uncover a fundamentally different function in which a kinase acts, as Akt in this case is an effector rather than a regulator in a cellular event.
H. Jiang, G. D. D'Agostino, P. A. Cole, and D. R. Dempsey. 2020. “Selective protein N-terminal labeling with N-hydroxysuccinimide esters.” Meth. Enzymol., 639, Pp. 333–353.Abstract
In order to gain detailed insight into the biochemical behavior of proteins, researchers have developed chemical tools to incorporate new functionality into proteins beyond the canonical 20 amino acids. Important considerations regarding effective chemical modification of proteins include chemoselectivity, near stoichiometric labeling, and reaction conditions that maintain protein stability. Taking these factors into account, we discuss an N-terminal labeling strategy that employs a simple two-step "one-pot" method using N-hydroxysuccinimide (NHS) esters. The first step converts a R-NHS ester into a more chemoselective R-thioester. The second step reacts the in situ generated R-thioester with a protein that harbors an N-terminal cysteine to generate a new amide bond. This labeling reaction is selective for the N-terminus with high stoichiometry. Herein, we provide a detailed description of this method and further highlight its utility with a large protein (>100kDa) and labeling with a commonly used cyanine dye.
N. Chu, T. Viennet, H. Bae, A. Salguero, A. Boeszoermenyi, H. Arthanari, and P. A. Cole. 2020. “The Structural Determinants of PH Domain-Mediated Regulation of Akt Revealed by Segmental Labeling.” Elife, 9.Abstract
Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.
Mattevi A. Cole, P. A. 2020. “Editorial Overview: Biological catalysis at the cross-roads of signaling and metabolism.” Curr. Opinion in Struct. Biol., 59, Pp. iii-iv.
2019
Jamie N Anastas, Barry M Zee, Jay H Kalin, Mirhee Kim, Robyn Guo, Sanda Alexandrescu, Mario Andres Blanco, Stefanie Giera, Shawn M Gillespie, Jayanta Das, Muzhou Wu, Sarah Nocco, Dennis M Bonal, Quang-De Nguyen, Mario L Suva, Bradley E Bernstein, Rhoda Alani, Todd R Golub, Philip A Cole, Mariella G Filbin, and Yang Shi. 10/2019. “Re-programing Chromatin with a Bifunctional LSD1/HDAC Inhibitor Induces Therapeutic Differentiation in DIP.” Cancer Cell. Publisher's VersionAbstract
H3K27M mutations resulting in epigenetic dysfunction are frequently observed in diffuse intrinsic pontine glioma (DIPGs), an incurable pediatric cancer. We conduct a CRISPR screen revealing that knockout of KDM1A encoding lysine-specific demethylase 1 (LSD1) sensitizes DIPG cells to histone deacetylase (HDAC) inhibitors. Consistently, Corin, a bifunctional inhibitor of HDACs and LSD1, potently inhibits DIPG growth in vitro and in xenografts. Mechanistically, Corin increases H3K27me3 levels suppressed by H3K27M histones, and simultaneously increases HDAC-targeted H3K27ac and LSD1-targeted H3K4me1 at differentiation-associated genes. Corin treatment induces cell death, cell-cycle arrest, and a cellular differentiation phenotype and drives transcriptional changes correlating with increased survival time in DIPG patients. These data suggest a strategy for treating DIPG by simultaneously inhibiting LSD1 and HDACs.
Hanjie Jiang, Stefani N. Thomas, Zan Chen, Claire Y. Chiang, and Philip A. Cole. 10/2019. “Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases.” J Biol Chem. Publisher's VersionAbstract
NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities are important for controlling cellular protein homeostasis and their dysregulation may lead to cancer and other diseases. Previous work has implicated non-catalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1 related ubiquitin ligases.
Philip A Cole, Nam Chu, Antonieta L Salguero, and Hwan Bae. 2019. “AKTivation mechanisms.” Curr Opin Struct Biol, 59, Pp. 47-53.Abstract
Akt1-3 (Akt) are a subset of the AGC protein Ser/Thr kinase family and play important roles in cell growth, metabolic regulation, cancer, and other diseases. We describe some of the roles of Akt in cell signaling and the biochemical and structural mechanisms of the regulation of Akt catalysis by the phospholipid PIP3 and by phosphorylation. Recent findings highlight a diverse set of strategies to control Akt catalytic activity to ensure its normal biological functions.
Samuel Henager, Stephanie Henriquez, Daniel Dempsey, and Philip A Cole. 2019. “Analysis of Site-specific Phosphorylation of PTEN using Enzyme-Catalyzed Expressed Protein Ligation.” Chembiochem.Abstract
The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C-terminus. Prior enzymatic characterization of the four mono-phosphorylated (1p) PTENs using classical expressed protein ligation (EPL) was complicated by the inclusion of a non-native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here we apply subtiligase-mediated EPL to create wt 1p-PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with Tyr379's role in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p-PTENs, which are poorly recognized by an anti-phospho-PTEN Ab, three of the four wt 1p-PTENs are recognized by a commonly used anti-phospho-PTEN Ab.
Beth E Zucconi, Jessica L Makofske, David J Meyers, Yousang Hwang, Mingxuan Wu, Mitzi I Kuroda, and Philip A Cole. 2019. “Combination Targeting of the Bromodomain and Acetyltransferase Active Site of p300/CBP.” Biochemistry, 58, 16, Pp. 2133-2143.Abstract
p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation. We find that the combination confers a dramatic reduction in p300 chromatin occupancy compared to the individual effects of blocking either domain alone. Accompanying this loss of p300 on chromatin, combination treatment leads to the reduction of specific mRNAs including androgen-dependent and pro-oncogenic prostate genes such as KLK3 (PSA) and c-Myc. Consistent with p300 directly affecting gene expression, mRNAs that are significantly reduced by combination treatment also exhibit a strong reduction in p300 chromatin occupancy at their gene promoters. The relatively few mRNAs that are up-regulated upon combination treatment show no correlation with p300 occupancy. These studies provide support for the pharmacologic advantage of concurrent targeting of two domains within one key epigenetic modification enzyme.
Hanjie Jiang, Stefani N Thomas, Zan Chen, Claire Y Chiang, and Philip A Cole. 2019. “Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases.” J Biol Chem.Abstract
NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities are important for controlling cellular protein homeostasis and their dysregulation may lead to cancer and other diseases. Previous work has implicated non-catalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1 related ubiquitin ligases.
Yujie Liu, Chunrong Bao, Liqing Wang, Rongxiang Han, Ulf H Beier, Tatiana Akimova, Philip A Cole, Sharon YR Dent, and Wayne W Hancock. 2019. “Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells.” Cancers (Basel), 11, 4.Abstract
Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acute allograft rejection. PCAF deletion also enhanced anti-tumor immunity in immunocompetent mice. Dual deletion of GCN5 and PCAF led to decreased Treg stability and numbers in peripheral lymphoid tissues, and mice succumbed to severe autoimmunity by 3-4 weeks of life. These data indicate that HATs of the GNAT family have contributions to Treg function that cannot be replaced by the functions of previously characterized Treg HATs (CBP, p300, and Tip60), and may be useful targets in immuno-oncology.
Sam Van de Velde, Ezra Wiater, Melissa Tran, Yousang Hwang, Philip A Cole, and Marc Montminy. 2019. “CREB Promotes Beta Cell Gene Expression by Targeting Its Coactivators to Tissue-Specific Enhancers.” Mol Cell Biol, 39, 17.Abstract
CREB mediates effects of cyclic AMP on cellular gene expression. Ubiquitous CREB target genes are induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell-specific genes by targeting CBP/p300 to promoter-distal enhancer regions. Subsequent increases in histone acetylation facilitate recruitment of the coactivators CRTC2 and BRD4, leading to release of RNA polymerase II over the target gene body. Indeed, CREB-induced hyperacetylation of chromatin over superenhancers promoted beta cell-restricted gene expression, which is sensitive to inhibitors of CBP/p300 and BRD4 activity. Neurod1 appears critical in establishing nucleosome-free regions for recruitment of CREB to beta cell-specific enhancers. Deletion of a CREB-Neurod1-bound enhancer within the superenhancer disrupted the expression of both genes and decreased beta cell function. Our results demonstrate how cross talk between signal-dependent and lineage-determining factors promotes the expression of cell-type-specific gene programs in response to extracellular cues.
P. A. Cole and A. Mattevi. 2019. “Editorial overview: Biological catalysis at the cross-roads of signaling and metabolism.” Curr. Opin. Struct. Biol., 59, Pp. iii-v.

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