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1.
Front Cell Neurosci ; 13: 499, 2019.
Article in English | MEDLINE | ID: mdl-31798415

ABSTRACT

The target for the "rapid" (<24 h) antidepressant effects of S-ketamine is unknown, vitiating programs to rationally develop more effective rapid antidepressants. To describe a drug's target, one must first understand the compartments entered by the drug, at all levels-the organ, the cell, and the organelle. We have, therefore, developed molecular tools to measure the subcellular, organellar pharmacokinetics of S-ketamine. The tools are genetically encoded intensity-based S-ketamine-sensing fluorescent reporters, iSKetSnFR1 and iSKetSnFR2. In solution, these biosensors respond to S-ketamine with a sensitivity, S-slope = delta(F/F0)/(delta[S-ketamine]) of 0.23 and 1.9/µM, respectively. The iSKetSnFR2 construct allows measurements at <0.3 µM S-ketamine. The iSKetSnFR1 and iSKetSnFR2 biosensors display >100-fold selectivity over other ligands tested, including R-ketamine. We targeted each of the sensors to either the plasma membrane (PM) or the endoplasmic reticulum (ER). Measurements on these biosensors expressed in Neuro2a cells and in human dopaminergic neurons differentiated from induced pluripotent stem cells (iPSCs) show that S-ketamine enters the ER within a few seconds after appearing in the external solution near the PM, then leaves as rapidly after S-ketamine is removed from the extracellular solution. In cells, S-slopes for the ER and PM-targeted sensors differ by <2-fold, indicating that the ER [S-ketamine] is less than 2-fold different from the extracellular [S-ketamine]. Organelles represent potential compartments for the engagement of S-ketamine with its antidepressant target, and potential S-ketamine targets include organellar ion channels, receptors, and transporters.

2.
Structure ; 22(10): 1520-7, 2014 Oct 07.
Article in English | MEDLINE | ID: mdl-25242456

ABSTRACT

Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct ß sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels.


Subject(s)
Caenorhabditis elegans Proteins/chemistry , Ion Channels/chemistry , Protein Folding , Acid-Base Imbalance/genetics , Amino Acid Sequence , Anemia, Hemolytic, Congenital/genetics , Caenorhabditis elegans Proteins/metabolism , Conserved Sequence , Crystallography, X-Ray , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Erythrocytes, Abnormal , Humans , Ion Channels/genetics , Ion Channels/metabolism , Metabolism, Inborn Errors/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Structure, Tertiary
3.
Proc Natl Acad Sci U S A ; 111(39): 14229-34, 2014 Sep 30.
Article in English | MEDLINE | ID: mdl-25197043

ABSTRACT

The cell-division cycle of Caulobacter crescentus depends on periodic activation and deactivation of the essential response regulator CtrA. Although CtrA is critical for transcription during some parts of the cell cycle, its activity must be eliminated before chromosome replication because CtrA also blocks the initiation of DNA replication. CtrA activity is down-regulated both by dephosphorylation and by proteolysis, mediated by the ubiquitous ATP-dependent protease ClpXP. Here we demonstrate that proteins needed for rapid CtrA proteolysis in vivo form a phosphorylation-dependent and cyclic diguanylate (cdG)-dependent adaptor complex that accelerates CtrA degradation in vitro by ClpXP. The adaptor complex includes CpdR, a single-domain response regulator; PopA, a cdG-binding protein; and RcdA, a protein whose activity cannot be predicted. When CpdR is unphosphorylated and when PopA is bound to cdG, they work together with RcdA in an all-or-none manner to reduce the Km of CtrA proteolysis 10-fold. We further identified a set of amino acids in the receiver domain of CtrA that modulate its adaptor-mediated degradation in vitro and in vivo. Complex formation between PopA and CtrA depends on these amino acids, which reside on alpha-helix 1 of the CtrA receiver domain, and on cdG binding by PopA. These results reveal that each accessory factor plays an essential biochemical role in the regulated proteolysis of CtrA and demonstrate, to our knowledge, the first example of a multiprotein, cdG-dependent proteolytic adaptor.


Subject(s)
Bacterial Proteins/metabolism , Caulobacter crescentus/cytology , Caulobacter crescentus/metabolism , DNA-Binding Proteins/metabolism , Endopeptidase Clp/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Caulobacter crescentus/genetics , Cell Cycle/physiology , Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Endopeptidase Clp/chemistry , Endopeptidase Clp/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Structure, Tertiary , Proteolysis , Second Messenger Systems , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/genetics
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