Yun Luo - US grants

? DESCRIPTION (provided by applicant): Angelman Syndrome is rare but severe neurodevelopmental disorder characterized by intellectual disability, motor impairment, and happy demeanors. Genetic cause of the disease is the deletion/abnormal expression of maternal chromosome 15q11-q13, which includes the UBE3A gene that codes for the E6-associated protein (E6-AP). A mouse model (AS mice) of the human disease has been generated by deletion of maternal Ube3a; these mice exhibit impaired long-term potentiation (LTP) of synaptic transmission in hippocampus, learning of various hippocampus-dependent tasks, and motor functions. Preliminary results indicated that abnormal calpain-2 activity might contribute to synaptic and cognitive impairment in AS mice, as treatment with a calpain-2 inhibitor restored normal LTP in slices prepared from AS mice. The rationale for the preliminary study was based on our recent discovery that calpain-2 activation during LTP consolidation functions as a molecular brake that limits the magnitude of long-term potentiation in hippocampus. A major component of the molecular brake is calpain-2-mediated degradation of the tumor suppressor PTEN, and AS mice have enhanced calpain-2 expression and decreased PTEN levels in hippocampus. These results suggest that calpain-2 might be a good target to restore normal learning in the Angelman Syndrome. While we have identified the dipeptide ketoamide, Z-Leu-Abu-CONH-CH2-C6H3(3,5-(OMe)2) as a relatively selective calpain-2 inhibitor at low concentrations, at higher concentrations it also inhibits calpain-1, which is necessary for LTP induction. As PTEN is selectively cleaved by calpain-2 and not calpain-1, we posit that identifying PTEN properties underlying its selectivity for calpain-2 will provide criticl information to develop calpain-2 selective inhibitors, which will not inhibit calpain-1. The proposed studies are directed at using detailed molecular dynamics simulation to identify critical features in PTEN that account for calpain-2 selectivity. We will then use a multi-level virtual screening method to design new and selective calpain- 2 inhibitors. These inhibitors will be tested first on in vitro assays for calpain-1 and -2 and for interactions with other cysteine proteases and then for their effects on LTP and in learning in the AS mice. Successful design and testing of a selective calpain-2 inhibitor will then lead to a potential U01 submission for using such inhibitors not only for AS treatment but also for other indications with learning and memory impairment.

Abstract Piezo1 and Piezo2 are mammalian cation-selective mechanosensitive ion channels homologs which open their pore in response to various mechanical stimuli. Mechanotransduction signaling through Piezo channels plays a central role in a bewildering variety of important physiological processes including red blood cell osmotic homeostasis, somatic and visceral mechanosensation, proprioception, blood pressure regulation and development and differentiation of many tissues and organ systems. Several human diseases including xerocytosis and lymphedema have been directly linked to genetic mutations in Piezo channels and many studies further indicate a role of Piezo-mediated signaling in allodynia and hyperalgesia and a possible role of Piezo channels in sleep apnea. The development of drugs capable of selectively activating or inhibiting Piezo channels represent a promising therapeutic opportunity for the treatment of some of these Piezo-related pathologies. To date, Yoda1, a synthetic small molecule agonist capable of selectively activating Piezo1 with micromolar affinity, represents the best small molecule candidate to expand the pharmacome of Piezo channels. Unfortunately, the fundamental mechanisms by which Piezo channel sense mechanical forces and activates in the presence of Yoda1 are still unknown. In this proposal we will address these two unsolved questions using a multidisciplinary approach combining molecular dynamic (MD) stimulations and experimental assays. In our first aim, we will identify rapid, force-induced structural rearrangements in Piezo1 by simulating the channel molecule in a membrane under tension. On another hand, using force-clamp fluorimetry, we will probe local conformational changes using spectroscopic measurements. This will be done by inserting conformational probes into strategic positions of the channel expressed in cells while protein function is being monitored in real-time. This combination of computations and experiments will allow us to capture structural dynamic information that happens in a temporal window spanning several orders of magnitude, from microsecond to minutes. In our second Aim, we will identify how Yoda1 interacts with and activates Piezo1. We have already identified a Yoda1 binding site using a combination of predictive MD simulations and experimental validations. We will characterize structural changes, changes in transition free energy, and modifications of allosteric residue-residue interactions that happen upon Yoda1 binding. This aim will shed light on the mechanism of chemical activation of a Piezo channel and will be invaluable to develop pharmacological agents with clinical value.

Abstract The ability of cells to rapidly sense and respond to mechanical stimuli is vital for all living beings. In vertebrates, this task is mainly achieved by mechanosensitive ion channels PIEZO1 and PIEZO2. Indeed, a growing number of studies have outlined the central role played by these channels to numerous biological processes including sensory physiology, osmotic homeostasis, and organ development. Not surprisingly, abnormal PIEZO channel activity is associated with many clinical conditions such as lymphedema, anemia, arthrogryposis, cancer, inflammation, and pain. These proteins are formed by three long polypeptide chains (subunits) assembled around a central ion conduction pathway (pore). The first specific Aim of our awarded project consists of identifying and characterizing specific conformational rearrangements taking place in these subunits as the channel opens (gates) its pore upon mechanical stimulation. Recent studies from our team and others suggest that the gating motion of one subunit may cooperatively influence that of the others. The purpose of the requested administrative supplement is to test this hypothesis. To this aim, we intend to purchase a Total Internal Reflection Fluorescence (TIRF) upgrade for our epifluroescence microscope. TIRF measurements will enable single-molecule fluorescence measurements of PIEZO1 channels in which each subunit is genetically-tagged with a shear-stress sensitive fluorescence probe. Our new epifluorescence data indicate that these probes emit large fluorescence signals that correlate with pore opening when channels are stimulated by fluid shear stress. We anticipate that the flow-mediated gating motion of each subunit will be accompanied by a discrete, jump-like increase of pixel brightness in TIRF images. If this gating motion is coupled, these discrete jumps are predicted to be temporally correlated. If not, these fluorescence jumps are predicted to occur independently. If successful, these experiments will deepen our fundamental understanding of how these essential ion channels open their pore in response to a physiological stimulus.