Structural insights into the mechanism of nucleotide regulation of pancreatic KATP channel

ATP-sensitive potassium channels (KATP) are metabolic sensors that convert the intracellular ATP/ADP ratio to the excitability of cells. They are involved in many physiological processes and implicated in several human diseases. Here we present the cryo-EM structures of the pancreatic KATP channel in both the closed state and the pre-open state, resolved in the same sample. The nucleotides bind at the inhibitory sites of the Kir6.2 channel in the closed state but not in the pre-open state. Structural comparisons reveal the mechanism for ATP inhibition and Mg-ADP activation, two fundamental properties of KATP channels. Moreover, the structure also uncovers the activation mechanism of diazoxide-type KATP openers.

HSP-90/kinase complexes are stabilized by the large PPIase FKB-6

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

A nucleotide-sensing endonuclease from the Gabija bacterial defense system

The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation.

A compendium of G-protein–coupled receptors and cyclic nucleotide regulation of adipose tissue metabolism and energy expenditure

With the ever-increasing burden of obesity and Type 2 diabetes, it is generally acknowledged that there remains a need for developing new therapeutics. One potential mechanism to combat obesity is to raise energy expenditure via increasing the amount of uncoupled respiration from the mitochondria-rich brown and beige adipocytes. With the recent appreciation of thermogenic adipocytes in humans, much effort is being made to elucidate the signaling pathways that regulate the browning of adipose tissue. In this review, we focus on the ligand–receptor signaling pathways that influence the cyclic nucleotides, cAMP and cGMP, in adipocytes. We chose to focus on G-protein–coupled receptor (GPCR), guanylyl cyclase and phosphodiesterase regulation of adipocytes because they are the targets of a large proportion of all currently available therapeutics. Furthermore, there is a large overlap in their signaling pathways, as signaling events that raise cAMP or cGMP generally increase adipocyte lipolysis and cause changes that are commonly referred to as browning: increasing mitochondrial biogenesis, uncoupling protein 1 (UCP1) expression and respiration.

Metabolic Energy Sensing by Mammalian CLC Anion/Proton Exchangers

Mammalian CLC anion/proton exchangers control the pH and [Cl-] of the endolysosomal system, one of the major cellular nutrient uptake pathways. We explored the regulation of the vesicular transporters ClC-3, ClC-4, and ClC-5 by the adenylic system components ATP, ADP, and AMP. Using heterologous expression and whole-cell electrophysiology, we demonstrated that cytosolic ATP and ADP but not AMP and Mg2+-free ADP enhance CLC ion transport via binding to the protein C-terminal CBS domains. Biophysical investigations revealed that the effects depend on the delivery of intracellular protons into the CLC transport machinery and result from modified voltage-dependence and altered probability that CLC proteins undergo silent non-transporting cycles. Our findings demonstrate that the CLC CBS domains are able to serve as energy sensors by detecting changes in the cytosolic ATP/ADP/AMP equilibrium. The adenine nucleotide regulation of vesicular Cl-/H+ exchange creates a link between the activity of the endolysosomal system and the cellular metabolic state.