Factors affect the structure of plexin-B1 and plexin-C1

Buck Lab

Abstract:Plexins are semaphorin receptors that play essential roles in many biological processes. Plexin signaling relies on a semaphorin-induced dimerization mechanism and is regulated by small GTPases of the Rho family. In addition, the binding of small Rho GTPases with the Rho GTPase Binding Domain (RBD) of plexin is necessary to the function of plexin. There are a couple of cysteine in RBDs, and cysteine can form intermolecular disulfide bonds. It is believed that disulfide bonds can modulate the structure and the function of RBDs. Understanding what factors can affect the formation of disulfide bonds can help us decipher the structure and the function of plexin. In the research, plexin-B1 and plexin-C1 proteins, different pH values buffers, an oxidizing reagent (_H2O2) and two reducing agents (β-ME, or DTT), are used to study the structure of plexin. SDS-PAGE is used to determine the molecular weight of the protein. ¹H- ¹⁵N HSQC is utilized to investigate the chemical shift perturbation, helping understand the structure of plexin. The results show that the protein prefers to form a dimer, trimer, or oligomer in some specific conditions. When the dimer forms, protein-protein interaction happens between the dimer interfaces. The effect leads to some missing peaks in NMR spectra. This analysis of interaction provides an approach to help understand the structure and the function of plexin.

Pharmacological activation of unfolded protein response promotes proteostasis of  epilepsy-associated GABAA receptors 

Mu Lab

Protein homeostasis between GABA_A receptor folding, trafficking and degradation is essential to  ensure normal physiological functions. Mutations in GABA_A receptors lead to numerous neurological disorders, including genetic epilepsy, and many patients suffering from which are  resistant to current drug treatments. Therefore, developing novel therapeutic strategies to target  defective GABA_A receptors is critical to effectively treat genetic epilepsy. In this study, we used  HEK293T cells that exogenously express disease-associated variants in the β2 subunit of  GABA_A receptor (i.e., I246T) with wild type α1 and γ2 subunits. Cell surface biotinylation assay  demonstrated that these β2 variants result in significantly reduced surface expression of the β2  protein compared to the wild type, mainly by decreasing folding and surface trafficking of the  mutant receptors. Automated patch clamping experiments further showed that these β2 mutants  have significantly diminished current compared to the WT receptor. Additionally, we searched  for small molecules that rescue the functional surface expression of the pathogenic receptors.  We found that GABA_A receptors-specific pharmacological chaperones restored the surface  expression of these β2 mutants. Western blot analysis further showed that pharmacologically  activating the unfolded protein response enhanced the total and surface protein levels of β2  variants. Overall, our results demonstrated that these compounds, while having distinct  mechanisms of action, hold great therapeutic potentials to treat genetic epilepsy by targeting the  disease-associated GABA_A receptor variants. 

Investigating the effect of GABRA1 frameshift mutations on GABAAR function

Mu Lab

Abstract:Frameshift mutations in the GABRA1 gene of the gamma-aminobutyric acid type A receptor  (GABA_AR) have been commonly linked to epilepsy disorders, particularly childhood absence  epilepsy and epileptic encephalopathy. However, due to its rarity and de novo nature, there is little  literature found about the disease mechanisms that underlie these mutations. Because these  mutations have substantial structural effects on the GABA_AR α1 subunit through an introduction  of a premature stop codon for translation, they have been mostly regarded as pathogenic despite  their heterozygosity. We hypothesize that the frameshift mutant α1 subunit presents as a severely  misfolded protein with trafficking defects causing accumulation in the endoplasmic reticulum  (ER) and defective GABA_AR function. Focusing on four particular frameshift mutations: K401,  S326, V290, and F272, protein overexpression and trafficking of the GABAAR α1 subunit were examined in a transiently transfected HEK293T cell model. Total and surface protein expression  of the mutants differed from the wild-type (WT) indicating defects in trafficking. Additionally,  automated patch clamp recordings revealed functionally defective GABA_AR with frameshift  mutations in GABRA1. Investigation of these mutations’ effect on assembly, trafficking, and  function will provide critical insights necessary to understanding the mechanisms of the epilepsy linked disorders as well as reveal potential therapeutic targets and pathways that could advance  current treatment research. 

PR-B Proteolytic Cleavage as a Mechanism for Functional Progesterone Withdrawal

Mesiano Lab

Progesterone (P4) is the most important hormone for the maintenance of pregnancy, and its  withdrawal, whether functional or systemic, initiates parturition. Targeting factors that lead to  inflammatory events within Progesterone Receptor (PR) pathways may help keep a fetus at risk  for preterm birth (PTB) in the uterus longer, increasing chances of survival and decreasing  potential negative consequences. However, gaps in our understanding of these highly complex  progesterone signaling pathways remain.  Canonically, once liganded, the full length PR (PR-B), is known to have anti-inflammatory  actions by interfering with AP-1’s transcriptional activity. This is a major pro-gestational drive  during the quiescent period of pregnancy. However, the anti-inflammatory activity associated  with PR-B decreases drastically as pregnancy nears its culmination—the mechanism of which is  currently unknown.   We postulate that when an “Inflammatory Threshold” is met during pregnancy, PR-B’s activity  may decline due to proteolytic cleavage. This cleavage likely occurs near the 164th residue of  PR-B, directly distal to the B-Upstream Segment (BUS) that is unique to PR-B. We hypothesize  that this proteolytic cleavage, for all intents and purposes, deactivates PR-B’s anti-inflammatory  action, allowing for uninhibited pro-inflammatory signaling and pro-labor mechanisms.   This research addresses a major physiological knowledge-gap, i.e.: the mechanism by which  pregnancy is both maintained through quiescence and concluded through parturition. The goal of  this study is to determine the mechanism of PR-B cleavage and its association with the onset of  labor. This will allow us to understand the function of the progesterone receptors in the human  myometrium, and provide specific, targetable mechanisms for potential preterm birth therapies. Ultimately, this research will contribute to the development of effective PR-based therapies to  prevent PTB.  These aims will be achieved using several innovative model systems including genetically  modified human myometrial hTERT cell lines, frozen/fixed and explant cultures of gravid  human myometrium, mouse models of inflammation-induced PTB and physiological pregnancy  phenotypes, and optimized assays using validated reagents to measure 1) relative abundance of  specific mRNAs; 2) abundance of specific proteins; 3) physical interaction pathways between  PRs and AP-1 subunits; 4) specific sub-cellular localizations of PR-B; and 5) development of  model systems in human myometrial cell lines and transgenic mice, within which PRs are  mutated to observe functionality. 

Glycolytic Dysregulation in Amyotrophic Lateral Sclerosis

Qi Lab

Abstract:Amyotrophic Lateral Sclerosis is a late-onset neurodegenerative disease characterized by upper and lower motor neuron loss [1]. Clinically, ALS patients have documented an increased resting energy expenditure, defined as hypermetabolism, creating an imbalance between energy supply and demand [2]. Since the brain consumes about 20% of the body’s total energy to maintain regulatory functions [3], a drop in energy supply could be fatal. The primary source of this energy, glucose, is broken down through glycolysis and oxidative phosphorylation. Alterations in glycolytic output via lactate production and transcription levels of rate limiting glycolytic enzymes have been found in ALS patients and patient derived IPSC motor neurons, respectively [4-6]. Preliminary data from our lab corroborates previous data with increased lactate production in post mortem ALS patient spinal cord samples. Despite the evidence showing a dysregulation of glycolysis in ALS, the role of glycolysis in motor neurodegeneration and the impact of glycolysis impairment on the development of ALS are unknown. In this study, I will be investigating the role of glycolysis and rate-limiting enzymes functions in ALS. Based on preliminary data, a significant increase in the mRNA level of Hexokinase 1 (HK1), the first enzyme of the glycolytic pathway, in ALS patient postmortem spinal cord samples. Either an increase or decrease in HK1 level has been shown to associate with cell death and neuroinflammation [7, 8]. However, whether the increase in HK1 and lactate are contributing factors of motor neurodegeneration in ALS or the consequences of disease progression remains to be determined.

Glutamate is the primary excitatory neurotransmitter in the brain and is required for proper brain function

Tajima Lab

Abstract:Glutamate is the primary excitatory neurotransmitter in the brain and is required for proper brain function. Excitatory transmission affects the sympathetic and parasympathetic nervous system thereby directly modulating cardiovascular activity. Kainate receptors (KARs) are a type of glutamate receptor that are involved in the genesis of epilepsy thereby contributing to an increase in heart rate. KARs are expressed in the brain and therefore have the added effect of contributing to processes associated with memory and learning. Neuropilin-and-tolloid- like (NETO) proteins are single-transmembrane proteins that will interact with KARs and affect their desensitization and deactivation rates. NETO proteins also play role in localizing glutamate receptors to the synapse. However, little is known about the structure of KARs in complex with NETO proteins. Understanding this structure will help us understand how the amino acid residues play key roles in modulating KAR activity. With this understanding, we will be able to develop therapeutics that are aimed to treat various mental disorders including epilepsy and schizophrenia as well as potentially target tachycardia. There are two types of NETO proteins; NETO1 and NETO2, of which, NETO1 is less understood. Structural studies using cryogenic electron microscopy (CryoEM) have elucidated the structure of NETO2 but not NETO1 and have shown how NETO2 forms a complex with GluK2. Therefore, the structure of NETO1 has yet to be determined as well as the structure of it bound with GluK1. The overall hypothesis of this proposal is that NETO1 is structurally different from NETO2 thereby increasing the rate of desensitization and deactivation of the GluK1 homomer. We will address this hypothesis and achieve the goals that are necessary to prove or disprove this hypothesis by purifying NETO1 and the GluK1 homomer and subsequently using CryoEM to determine the structure of the complex. We will plan on doing the same procedure with NETO2 and GluK1 to compare the relevant interactions that take place. This will help us determine the amino acids in the GluK1 homomer and NETO1 protein that are important in modulating the desensitization and deactivation rates of these glutamate receptors. The findings of this proposal will provide a complete molecular basis for excitatory neurotransmission.

Heart failure (HF) represents one of the largest global health concerns of the modern world

Stelzer Lab

Abstract:Heart failure (HF) represents one of the largest global health concerns of the modern world. Unfortunately, current HF therapies do little to improve prognosis or quality of life. Given that health experts forecast that HF's prevalence and economic burden will only continue to rise in the coming decade, effective HF therapies are in high demand. Despite extensive research and testing of several potential therapeutic strategies, none have proven clinically viable. A recent approach to treating HF uses small molecules to activate myosin motors within the heart to improve force-generating capabilities. Omecamtiv mecarbil (OM) represents the first of such molecules to pass clinical trials and obtain FDA approval. While many had high hopes for OM, recent large-scale clinical trials reveal its therapeutic limitations, suggesting that OM may not be the silver bullet HF needs. Furthermore, the lack of in vivo mechanistic studies dissecting how OM interacts with the complex pathophysiology of HF may underscore our misunderstanding of OM’s clinical behavior. Since drug companies continue to pursue small molecule myosin activators as an HF therapy, this talk outlines how rodent models of HF can improve our preclinical understanding of myosin activation in the failing heart and better assess its therapeutic potential.

Progesterone Receptor Pathways in Preterm Birth

Chakravarti Lab

Abstract:Progesterone is the most important hormone for the maintenance of pregnancy, and its withdrawal, whether functional or systemic, initiates parturition. There are two main progesterone receptors (PRs) found in human myometrium, PR-A and PR-B. Previous research has indicated that PR-B has an anti-inflammatory function and favors pregnancy, while PR-A has an inflammatory function and favors parturition. However, gaps in our understanding of these highly complex progesterone signaling pathways remain. Due the potency of progesterone and the consequences of its withdrawal during pregnancy, understanding these pathways is integral to the prevention and mitigation of preterm birth. Targeting factors that lead to inflammatory events within PR pathways may help keep a fetus at risk for preterm birth in the uterus longer, increasing chances of survival and decreasing potential negative consequences. Emerging research shows that the phosphorylated conformation of PR-A may be responsible for the inflammatory action associated with it, while the non-phosphorylated and inactivated (unliganded) states of PR-A may have other undetermined actions. The goal of this study is to determine if there are indeed three separate PR-A pathways; the phosphorylated state (with canonical inflammatory effects), the non-phosphorylated state (with anti-inflammatory effects comparable to PR-B), and the inactive state (in which PR-B actions dominate). Preliminary data generated using immortalized cell lines, western blots, PCR, and Elisa assays support this hypothesis, demonstrating the anti-inflammatory effects of PR-B alone and the modulated effects with phospho-PR-A.