Chemoproteomic identification of CO-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 CO are, accordingly, of great interest. Because CO is electrophilic, one way it can modulate protein biochemistry is by carboxylation of the amine group of lysine residues.

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 -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.

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.

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

The development of a potent mechanism-based inactivator of NagZ, an enzyme critical to the production of inducible AmpC β-lactamase in Gram-negative bacteria, is presented. This inactivator significantly reduces MIC values for important β-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.

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.

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.

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.

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.