Dissecting OGT's TPR domain to identify determinants of cellular function.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification.

Viewpoint on the Second Transatlantic GPCR Symposium for Early Career Investigators.

The second Transatlantic Early Career Investigator (ECI) G Protein-Coupled Receptor (GPCR) Symposium was an online scientific meeting geared at young GPCR investigators, with the primary goal of expanding opportunities for sharing research and networking among trainees in North America and Europe. Here, we discuss the format of our meeting, its impact, and the challenges and opportunities facing meetings like it in the future.

Membrane phosphoinositides regulate GPCR-ß-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 ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-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 ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.

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.

Structure of the neurotensin receptor 1 in complex with ß-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 detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human ß-arrestin 1 (ßarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with ßarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.

Structure-Based Evolution of Low Nanomolar O-GlcNAc Transferase Inhibitors.

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.

Aspartate Glycosylation Triggers Isomerization to Isoaspartate.

O-Linked ß-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.

How the glycosyltransferase OGT catalyzes amide bond cleavage.

The essential human enzyme O-linked ß-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.

Development and Characterization of Potent Cyclic Acyldepsipeptide Analogues with Increased Antimicrobial Activity.

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.

A small molecule that inhibits OGT activity in cells.

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.

The making of a sweet modification: structure and function of O-GlcNAc transferase.

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.

Organoboron-based allylation approach to the total synthesis of the medium-ring dilactone (+)-antimycin A(1b).

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.

HCF-1 is cleaved in the active site of O-GlcNAc transferase.

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.

Indium-promoted chemo- and diastereoselective allylation of α,ß-epoxy ketones with potassium allyltrifluoroborate.

A practical method for the chemo- and diastereoselective allylation of α,ß-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,ß-epoxyhomoallylic tertiary alcohols in high yields and diastereoselectivities, without competing ring-scission of the epoxide.

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.