Chemoproteomic identification of CO2-dependent lysine carboxylation in proteins.

Carbon dioxide is an omnipresent gas that drives adaptive responses within organisms from all domains of life. The molecular mechanisms by which proteins serve as sensors of CO2 are, accordingly, of great interest. Because CO2 is electrophilic, one way it can modulate protein biochemistry is by carboxylation of the amine group of lysine residues. However, the resulting CO2-carboxylated lysines spontaneously decompose, giving off CO2, which makes studying this modification difficult. Here we describe a method to stably mimic CO2-carboxylated lysine residues in proteins. We leverage this method to develop a quantitative approach to identify CO2-carboxylated lysines of proteins and explore the lysine 'carboxylome' of the CO2-responsive cyanobacterium Synechocystis sp. We uncover one CO2-carboxylated lysine within the effector binding pocket of the metabolic signaling protein PII. CO2-carboxylatation of this lysine markedly lowers the affinity of PII for its regulatory effector ligand ATP, illuminating a negative molecular control mechanism mediated by CO2.

Bicyclic Picomolar OGA Inhibitors Enable Chemoproteomic Mapping of Its Endogenous Post-translational Modifications.

Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.

Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates.

The modification of proteins with O-linked N-acetylglucosamine ( O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.

A mechanism-based GlcNAc-inspired cyclophellitol inactivator of the peptidoglycan recycling enzyme NagZ reverses resistance to ß-lactams in Pseudomonas aeruginosa.

The development of a potent mechanism-based inactivator of NagZ, an enzyme critical to the production of inducible AmpC ß-lactamase in Gram-negative bacteria, is presented. This inactivator significantly reduces MIC values for important ß-lactams against a clinically relevant strain of Pseudomonas aeruginosa.

Pharmacological Inhibition of O-GlcNAcase Enhances Autophagy in Brain through an mTOR-Independent Pathway.

The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer's disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic.

Post-translational O-GlcNAcylation is essential for nuclear pore integrity and maintenance of the pore selectivity filter.

O-glycosylation of the nuclear pore complex (NPC) by O-linked N-acetylglucosamine (O-GlcNAc) is conserved within metazoans. Many nucleoporins (Nups) comprising the NPC are constitutively O-GlcNAcylated, but the functional role of this modification remains enigmatic. We show that loss of O-GlcNAc, induced by either inhibition of O-GlcNAc transferase (OGT) or deletion of the gene encoding OGT, leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups. Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation. The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells. These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter. The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.

HCN Channel C-Terminal Region Speeds Activation Rates Independently of Autoinhibition.

Hyperpolarization- and cyclic nucleotide-activated (HCN) channels contribute to rhythmic oscillations in excitable cells. They possess an intrinsic autoinhibition with a hyperpolarized V 1/2, which can be relieved by cAMP binding to the cyclic nucleotide binding (CNB) fold in the C-terminal region or by deletion of the CNB fold. We questioned whether V 1/2 shifts caused by altering the autoinhibitory CNB fold would be accompanied by parallel changes in activation rates. We used two-electrode voltage clamp on Xenopus oocytes to compare wildtype (WT) HCN2, a constitutively autoinhibited point mutant incapable of cAMP binding (HCN2 R591E), and derivatives with various C-terminal truncations. Activation V 1/2 and deactivation t 1/2 measurements confirmed that a truncated channel lacking the helix αC of the CNB fold (ΔαC) had autoinhibition comparable to HCN2 R591E; however, ΔαC activated approximately two-fold slower than HCN2 R591E over a 60-mV range of hyperpolarizations. A channel with a more drastic truncation deleting the entire CNB fold (ΔCNB) had similar V 1/2 values to HCN2 WT with endogenous cAMP bound, confirming autoinhibition relief, yet it surprisingly activated slower than the autoinhibited HCN2 R591E. Whereas CNB fold truncation slowed down voltage-dependent reaction steps, the voltage-independent closed-open equilibrium subject to autoinhibition in HCN2 was not rate-limiting. Chemically inhibiting formation of the endogenous lipid PIP2 hyperpolarized the V 1/2 of HCN2 WT but did not slow down activation to match ΔCNB rates. Our findings suggest a "quickening conformation" mechanism, requiring a full-length CNB that ensures fast rates for voltage-dependent steps during activation regardless of potentiation by cAMP or PIP2.

Cytoplasmic cAMP-sensing domain of hyperpolarization-activated cation (HCN) channels uses two structurally distinct mechanisms to regulate voltage gating.

Voltage gating of hyperpolarization-activated cation (HCN) channels is potentiated by direct binding of cAMP to a cytoplasmic cAMP-sensing domain (CSD). When unliganded, the CSD inhibits hyperpolarization-dependent opening of the HCN channel gate; cAMP binding relieves this autoinhibition so that opening becomes more favorable thermodynamically. This autoinhibition-relief mechanism is conserved with that of several other cyclic nucleotide receptors using the same ligand-binding fold. Besides its thermodynamic effect, cAMP also modulates the depolarization-dependent deactivation rate by kinetically trapping channels in an open state. Here we report studies of strong open-state trapping in an HCN channel showing that the well-established autoinhibition-relief model is insufficient. Whereas deletion of the CSD mimics the thermodynamic open-state stabilization usually associated with cAMP binding, CSD deletion removes rather than mimics the kinetic effect of strong open-state trapping. Substitution of different CSD sequences leads to variation of the degree of open-state trapping in the liganded channel but not in the unliganded channel. CSD-dependent open-state trapping is observed during a voltage-dependent deactivation pathway, specific to the secondary open state that is formed by mode shift after prolonged hyperpolarization activation. This hysteretic activation-deactivation cycle is preserved by CSD substitution, but the change in deactivation kinetics of the liganded channel resulting from CSD substitution is not correlated with the change in autoinhibition properties. Thus the liganded and the unliganded forms of the CSD respectively provide the structural determinants for open-state trapping and autoinhibition, such that two distinct mechanisms for cAMP regulation can operate in one receptor.

Sensitivity of HCN channel deactivation to cAMP is amplified by an S4 mutation combined with activation mode shift.

Hyperpolarisation-activation of HCN ion channels relies on the movement of a charged S4 transmembrane helix, preferentially stabilising the open conformation of the ion pore gate. The open state is additionally stabilised, (a) when cyclic AMP (cAMP) is bound to a cytoplasmic C-terminal domain or (b) when the "mode I" open state formed initially by gate opening undergoes a "mode shift" into a "mode II" open state with a new S4 conformation. We isolated a mutation (lysine 381 to glutamate) in S4 of mouse HCN4; patch-clamp of homomeric channels in excised inside-out membranes revealed a conditional phenotype. When cAMP-liganded K381E channels are previously activated by hyperpolarisation, tens of seconds are required for complete deactivation at a weakly depolarised potential; this "ultra-sustained activation" is not observed without cAMP. Whilst cAMP slows deactivation of wild-type channels, the K381E mutation amplifies this effect to enable extraordinary kinetic stabilisation of the open state. K381E channels retain S4-gate coupling, with strong voltage dependence of the rate-limiting step for deactivation of mode II channels near -40 mV. At these voltages, the mode I deactivation pathway shows a different rate-limiting step, lacking strong voltage or cAMP dependence. Ultra-sustained activation thus reflects stabilisation of the mode II open state by the K381E mutation in synergistic combination with cAMP binding. Thus, the voltage-sensing domain is subject to strong functional coupling not only to the pore domain but also to the cytoplasmic cAMP-sensing domain in a manner specific to the voltage sensor conformation.