Instructor, Molecular & Cellular Physiology
Honors & Awards
NIH Pathway to Independence Award (K99/R00), National Institutes of Health, NIGMS (2022-07-01 - Present)
Damon Runyon Cancer Research Foundation Postdoctoral Fellowship, Damon Runyon Cancer Research Foundation (2018-01-01 - 2022-06-30)
- Viewpoint on the Second Transatlantic GPCR Symposium for Early Career Investigators ACS PHARMACOLOGY & TRANSLATIONAL SCIENCE 2022
Membrane phosphoinositides regulate GPCR-beta-arrestin complex assembly and dynamics.
Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with beta-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and invitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in beta-arrestin recruitment and GPCR-beta-arrestin complexdynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for beta-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of beta-arrestin and stabilize GPCR-beta-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-beta-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for beta-arrestin recruitment, this provides a mechanism for beta-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.
View details for DOI 10.1016/j.cell.2022.10.018
View details for PubMedID 36368322
Membrane Phosphoinositides Stabilize GPCR-arrestin Complexes and Provide Temporal Control of Complex Assembly and Dynamics.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2022; 36 Suppl 1
Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for gating signaling. Once internalized some GPCRs remain stably associated with arrestin, while others interact transiently; this difference affects signaling and recycling behaviors of these GPCRs. Using cell-based and in vitro biophysical assays we examined the role of membrane phosphoinositides (PIPs) in arrestin recruitment and GPCR-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one which requires PIP-binding for arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of arrestin and stabilize GPCR-arrestin complexes by promoting a core-engaged state of the complex. As allosteric modulators of GPCR-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. The dependance on membrane PIPs provides a mechanism for arrestin release from transiently associated GPCRs, allowing their rapid recycling, while explaining how stably associated GPCRs are able to engage G proteins at endosomes.
View details for DOI 10.1096/fasebj.2022.36.S1.R6320
View details for PubMedID 35553909
Protein Substrates Engage the Lumen of O-GlcNAc Transferase's Tetratricopeptide Repeat Domain in Different Ways.
Glycosylation of nuclear and cytoplasmic proteins is an essential post-translational modification in mammals. O-GlcNAc transferase (OGT), the sole enzyme responsible for this modification, glycosylates more than 1000 unique nuclear and cytoplasmic substrates. How OGT selects its substrates is a fundamental question that must be answered to understand OGT's unusual biology. OGT contains a long tetratricopeptide repeat (TPR) domain that has been implicated in substrate selection, but there is almost no information about how changes to this domain affect glycosylation of individual substrates. By profiling O-GlcNAc in cell extracts and probing glycosylation of purified substrates, we show here that ladders of asparagines and aspartates that extend the full length of OGT's TPR lumen control substrate glycosylation. Different substrates are sensitive to changes in different regions of OGT's TPR lumen. We also found that substrates with glycosylation sites close to the C-terminus bypass lumenal binding. Our findings demonstrate that substrates can engage OGT in a variety of different ways for glycosylation.
View details for DOI 10.1021/acs.biochem.0c00981
View details for PubMedID 33709700
Structure of the neurotensin receptor 1 in complex with β-arrestin 1.
Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization, and affecting various downstream signalling pathways1,2. Although there is a wealth of structural information delineating the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-EM structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human β-arrestin 1 (βarr1ΔCT). We found that phosphorylation of NTSR1 was critical for obtaining a stable complex with βarr1ΔCT, and identified phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observed a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared to a structure of rhodopsin-arrestin-1, our structure displays an approximately 85° rotation of arrestin relative to the receptor. These findings highlight both conserved aspects but also the plasticity of arrestin-receptor interactions.
View details for DOI 10.1038/s41586-020-1953-1
View details for PubMedID 31945771
Structure-Based Evolution of Low Nanomolar O-GlcNAc Transferase Inhibitors.
Journal of the American Chemical Society
Reversible glycosylation of nuclear and cytoplasmic proteins is an important regulatory mechanism across metazoans. One enzyme, O-linked N-acetylglucosamine transferase (OGT), is responsible for all nucleocytoplasmic glycosylation and there is a well-known need for potent, cell-permeable inhibitors to interrogate OGT function. Here we report the structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity. In addition to disclosing useful OGT inhibitors, the structures we report provide insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.
View details for PubMedID 30285435
Aspartate Glycosylation Triggers Isomerization to Isoaspartate
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2017; 139 (9): 3332-3335
O-Linked β-N-acetylglucosamine transferase (OGT) is an essential human enzyme that glycosylates numerous nuclear and cytoplasmic proteins on serine and threonine. It also cleaves Host cell factor 1 (HCF-1) by a mechanism in which the first step involves glycosylation on glutamate. Replacing glutamate with aspartate in an HCF-1 proteolytic repeat was shown to prevent peptide backbone cleavage, but whether aspartate glycosylation occurred was not examined. We report here that OGT glycosylates aspartate much faster than it glycosylates glutamate in an otherwise identical model peptide substrate; moreover, once formed, the glycosyl aspartate reacts further to form a succinimide intermediate that hydrolyzes to produce the corresponding isoaspartyl peptide. Aspartate-to-isoaspartate isomerization in proteins occurs in cells but was previously thought to be exclusively non-enzymatic. Our findings suggest it may also be enzyme-catalyzed. In addition to OGT, enzymes that may catalyze aspartate to isoaspartate isomerization include PARPs, enzymes known to ribosylate aspartate residues in the process of poly(ADP-ribosyl)ation.
View details for DOI 10.1021/jacs.6b12866
View details for Web of Science ID 000396185700007
View details for PubMedID 28207246
View details for PubMedCentralID PMC5431074
How the glycosyltransferase OGT catalyzes amide bond cleavage
NATURE CHEMICAL BIOLOGY
2016; 12 (11): 899-?
The essential human enzyme O-linked β-N-acetylglucosamine transferase (OGT), known for modulating the functions of nuclear and cytoplasmic proteins through serine and threonine glycosylation, was unexpectedly implicated in the proteolytic maturation of the cell cycle regulator host cell factor-1 (HCF-1). Here we show that HCF-1 cleavage occurs via glycosylation of a glutamate side chain followed by on-enzyme formation of an internal pyroglutamate, which undergoes spontaneous backbone hydrolysis.
View details for DOI 10.1038/NCHEMBIO.2173
View details for Web of Science ID 000386798800007
View details for PubMedID 27618188
View details for PubMedCentralID PMC5172607
Development and Characterization of Potent Cyclic Acyldepsipeptide Analogues with Increased Antimicrobial Activity
JOURNAL OF MEDICINAL CHEMISTRY
2016; 59 (2): 624-646
The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms of action. Analogues of the A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class of antimicrobial agents that act through dysregulation of caseinolytic protease (ClpP). Previous studies have shown that ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there are currently few studies examining Gram-negative bacteria. In this study, the synthesis and biological evaluation of 14 novel ADEPs against a variety of pathogenic Gram-negative and Gram-positive organisms is outlined. Optimization of the macrocyclic core residues and N-acyl side chain culminated in the development of 26, which shows potent activity against the Gram-negative species Neisseria meningitidis and Neisseria gonorrheae and improved activity against the Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis in comparison with known analogues. In addition, the co-crystal structure of an ADEP-ClpP complex derived from N. meningitidis was solved.
View details for DOI 10.1021/acs.jmedchem.5b01451
View details for Web of Science ID 000369115700010
View details for PubMedID 26818454
A Small Molecule That Inhibits OGT Activity in Cells
ACS CHEMICAL BIOLOGY
2015; 10 (6): 1392-1397
O-GlcNAc transferase (OGT) is an essential mammalian enzyme that regulates numerous cellular processes through the attachment of O-linked N-acetylglucosamine (O-GlcNAc) residues to nuclear and cytoplasmic proteins. Its targets include kinases, phosphatases, transcription factors, histones, and many other intracellular proteins. The biology of O-GlcNAc modification is still not well understood, and cell-permeable inhibitors of OGT are needed both as research tools and for validating OGT as a therapeutic target. Here, we report a small molecule OGT inhibitor, OSMI-1, developed from a high-throughput screening hit. It is cell-permeable and inhibits protein O-GlcNAcylation in several mammalian cell lines without qualitatively altering cell surface N- or O-linked glycans. The development of this molecule validates high-throughput screening approaches for the discovery of glycosyltransferase inhibitors, and further optimization of this scaffold may lead to yet more potent OGT inhibitors useful for studying OGT in animal models.
View details for DOI 10.1021/acschembio.5b00004
View details for Web of Science ID 000356845400005
View details for PubMedID 25751766
View details for PubMedCentralID PMC4475500
The Making of a Sweet Modification: Structure and Function of O-GlcNAc Transferase
JOURNAL OF BIOLOGICAL CHEMISTRY
2014; 289 (50): 34424-34432
O-GlcNAc transferase is an essential mammalian enzyme responsible for transferring a single GlcNAc moiety from UDP-GlcNAc to specific serine/threonine residues of hundreds of nuclear and cytoplasmic proteins. This modification is dynamic and has been implicated in numerous signaling pathways. An unexpected second function for O-GlcNAc transferase as a protease involved in cleaving the epigenetic regulator HCF-1 has also been reported. Recent structural and biochemical studies that provide insight into the mechanism of glycosylation and HCF-1 cleavage will be described, with outstanding questions highlighted.
View details for DOI 10.1074/jbc.R114.604405
View details for Web of Science ID 000346260800002
View details for PubMedID 25336649
View details for PubMedCentralID PMC4263849
Organoboron-Based Allylation Approach to the Total Synthesis of the Medium-Ring Dilactone (+)-Antimycin A(1b)
JOURNAL OF ORGANIC CHEMISTRY
2014; 79 (16): 7415-7424
The stereoselective synthesis of (+)-antimycin A1b has been accomplished in 12 linear steps and 18% overall yield from (-)-ethyl lactate. A robust, scalable, and highly diastereoselective montmorillonite K10-promoted allylation reaction between an α-silyloxy aldehyde and a substituted potassium allyltrifluoroborate salt provides a general approach to the core stereochemical triad of the antimycin A family. The requisite (Z)-substituted potassium allyltrifluoroborate salt was synthesized using a syn-selective hydroboration/protodeboration of an alkynylboronate ester, followed by a Matteson homologation reaction. The total synthesis leverages an MNBA (Shiina's reagent)-mediated macrolactonization to generate the 9-membered dilactone ring and a late-stage PyBOP-mediated amide coupling employing an unprotected 3-formamidosalicylic acid fragment, thereby shortening the longest linear sequence and, perhaps most notably, generating the antimycin A C7-C8-C9 stereotriad in a single step using a single chiral pool-derived stereocenter.
View details for DOI 10.1021/jo501134d
View details for Web of Science ID 000340517600016
View details for PubMedID 25019929
HCF-1 Is Cleaved in the Active Site of O-GlcNAc Transferase
2013; 342 (6163): 1235-1239
Host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the carboxyl-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above uridine diphosphate-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.
View details for DOI 10.1126/science.1243990
View details for Web of Science ID 000327857900048
View details for PubMedID 24311690
View details for PubMedCentralID PMC3930058
- Palladium beta-diiminate chemistry: Reactivity towards monodentate ligands and arylboronic acids INORGANICA CHIMICA ACTA 2012; 380: 308-321
Indium-Promoted Chemo- and Diastereoselective Allylation of alpha,beta-Epoxy Ketones with Potassium Allyltrifluoroborate
2010; 12 (23): 5490-5493
A practical method for the chemo- and diastereoselective allylation of α,β-epoxy ketones has been developed by using the convenient air and moisture stable reagent potassium allyltrifluoroborate. Indium metal was found to promote addition in stoichiometric or catalytic amounts, to afford α,β-epoxyhomoallylic tertiary alcohols in high yields and diastereoselectivities, without competing ring-scission of the epoxide.
View details for DOI 10.1021/ol1023757
View details for Web of Science ID 000284555600034
View details for PubMedID 21070014
Novel dinuclear and trinuclear palladium beta-diiminate complexes containing amido-chloro double-bridges
We report the formation of an unexpected trinuclear palladium beta-diiminate complex from the decomposition of [Pd(Ph(2)nacnac)(Cl)(4-H(2)NC(6)H(4)-(t)Bu)] (nacnac = beta-diiminate derived from acetylacetone), the proposed reaction pathway, and the synthesis of the first dinuclear palladium complex with an amido-chloro double-bridge.
View details for DOI 10.1039/b804792h
View details for Web of Science ID 000256832300004
View details for PubMedID 18560659