The Structure of a Conserved Piezo Channel Domain Reveals a Topologically Distinct β Sandwich Fold

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 β 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-tobe- 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

Débat avec les responsables scientifiques de l’axe 3

Valérie Carayol : Vous avez dit que « dans la mêlée du direct, nous participons plutôt que nous symbolisons » et que « l’induction se vit au présent ». Hier, avec Wolfgang Settekorn qui nous a parlé de métaphorisations mutuelles avec des exemples visuels et avec Philippe Breton qui nous a parlé d’amalgame, on avait déjà esquissé un rapprochement entre l’induction et les dynamiques spatiales, pas obligatoirement une dynamique temporelle. Est-ce que vous pourriez préciser cette idée du direct, ..

Innovative Internships in Protein Biomanufacturing

In this video, published by InnovATEBIO, Aron Kamajaya describes the development of a training program for protein biomanufacturing at Los Angeles Pierce College. Kamajaya explores the biotechnology program's coursework, internship locations and duties, student production of Taq polymerase, various internship projects, and student response data on internships. The video recording runs 46:28 minutes in length

Structural Study of Piezo Channel, a Unique Family of Eukaryotic Mechanosensitive Channel

Piezo is a unique family of eukaryotic mechanosensitive (MS) channel. With over 2500 amino acids per subunit, intact Piezo channel is one of the largest ion channels known to date. Two versions of Piezo can be found in vertebrates, namely PIEZO1 and PIEZO2. PIEZO1 appears to play roles in processes which control physiological homeostasis, whereas PIEZO2 assumes roles in mechanical somatosensation. A number of mutations mapped onto PIEZO1 or PIEZO2 are found in several hereditary human diseases, such as Dehydrated Hereditary Stomatocytosis, Gordon syndrome, and Distal Arthrogryposis. Although biochemical and functional studies provided many insightful findings, structural study of Piezo was very minimal. Herein, I described the structural investigation of Piezo channel. In the first study, we isolated a conserved soluble domain of Piezo (C-terminal loop 2, CTL2) from the C. elegans homolog, and provided the first molecular glimpse into this enigmatic MS channel. Subsequently, I described challenges that are associated with the expression and protein preparation of the full length Piezo channel. Recently, the full length mouse PIEZO1 structure solved by single particle cryo-EM revealed trimeric arrangement of the intact channel. CTL2 domain forms an extracellular cap which makes up the central core in this Piezo model. Lastly, we isolated a stable C-terminal fragment of Piezo. This fragment corresponds to the entire central core of Piezo channel and a few upstream transmembrane helices. This fragment can be localized to the plasma membrane. Further investigation is needed to look at the functionality of this fragment

Dual Credit HS DNA Sequencing and Internships in Protein Biomanufacturing

This resource, published by InnovATEBIO, features two presentations: one that explains the Dual Credit High School-Community College DNA Sequencing and Genomics Project at Austin Community College, followed by one on the development of a training program for protein biomanufacturing at Los Angeles Pierce College. The DNA sequencing project provides hands-on work experience in student-led research using "industry-level DNA sequencing technology." The Protein Biomanufacturing project places interns in a student-led Contract Manufacturing organization to create Taq polymerase. Both presentations describe the internship activities in-detail along with outcomes for participating students. This video runs 56:29 minutes in length

Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals

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

Cell cycle-dependent adaptor complex for ClpXP-mediated proteolysis directly integrates phosphorylation and second messenger signals

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 K(m) 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

Microscopy Using Fluorescent Drug Biosensors for "Inside-Out Pharmacology"

Neuropharmacology offers many tools for connecting molecular to acute behavioral phenomena but has few tools to explain effects of chronic drugs. In the biophysically based “inside-out” approach to neuropharmacology, drugs bind to their nascent targets within the endoplasmic reticulum (ER) (i.e. pharmacological chaperoning). Now we visualize, quantify, and time the first steps: drugs entering the cell and entering organelles. Our fluorescent biosensors are built on an OpuBC-GFP fusion scaffold. OpuBC is a bacterial periplasmic binding protein with two key features: a cation-π box, favorable for binding amines well represented in drugs, and a binding-induced “Venus fly-trap” conformational change. The suitably mutated OpuBC domain is connected, at the hinge regions, to a circularly permuted “superfolder” GFP (cpGFP). Aided by structural information, we use directed evolution to create a family of drug-sensing biosensors meeting the criterion of ΔF/F0 > 1 at 1 μM drug. Our proof of principle study concerns nicotine entering the ER, as measured by an “intensity-based nicotine-sensing fluorescent reporter” (iNicSnFR). Previous data demonstrated that exposure to nicotine causes changes in number and stoichiometry of nicotinic receptors by chaperoning within the ER; however nicotine itself entering the ER had not yet been measured. In live cell imaging of an iNicSnFR targeted to the ER, we found that nicotine enters the ER within 10 s of application at concentrations experienced by a cigarette smoker. Moreover, we found that varenicline, a smoking cessation drug, enters the ER almost as rapidly as nicotine, helping to explain varenicline's biochemical and behavioral effects. We are currently developing other “iDrugSnFRs” for antidepressants, antipsychotics, and opioids. These tools to study subcellular pharmacokinetics will help to clarify chronic effects of several families of neural drugs. Support: DA037161, GM123582, NARSAD, California TRDRP, and HHMI

Microscopy Using Fluorescent Drug Biosensors for "Inside-Out Pharmacology"

Neuropharmacology offers many tools for connecting molecular to acute behavioral phenomena but has few tools to explain effects of chronic drugs. In the biophysically based “inside-out” approach to neuropharmacology, drugs bind to their nascent targets within the endoplasmic reticulum (ER) (i.e. pharmacological chaperoning). Now we visualize, quantify, and time the first steps: drugs entering the cell and entering organelles. Our fluorescent biosensors are built on an OpuBC-GFP fusion scaffold. OpuBC is a bacterial periplasmic binding protein with two key features: a cation-π box, favorable for binding amines well represented in drugs, and a binding-induced “Venus fly-trap” conformational change. The suitably mutated OpuBC domain is connected, at the hinge regions, to a circularly permuted “superfolder” GFP (cpGFP). Aided by structural information, we use directed evolution to create a family of drug-sensing biosensors meeting the criterion of ΔF/F0 > 1 at 1 μM drug. Our proof of principle study concerns nicotine entering the ER, as measured by an “intensity-based nicotine-sensing fluorescent reporter” (iNicSnFR). Previous data demonstrated that exposure to nicotine causes changes in number and stoichiometry of nicotinic receptors by chaperoning within the ER; however nicotine itself entering the ER had not yet been measured. In live cell imaging of an iNicSnFR targeted to the ER, we found that nicotine enters the ER within 10 s of application at concentrations experienced by a cigarette smoker. Moreover, we found that varenicline, a smoking cessation drug, enters the ER almost as rapidly as nicotine, helping to explain varenicline's biochemical and behavioral effects. We are currently developing other “iDrugSnFRs” for antidepressants, antipsychotics, and opioids. These tools to study subcellular pharmacokinetics will help to clarify chronic effects of several families of neural drugs. Support: DA037161, GM123582, NARSAD, California TRDRP, and HHMI

Biosensors Show the Pharmacokinetics of S-Ketamine in the Endoplasmic Reticulum.

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 drugs 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