12 research outputs found
A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome
Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.Peer reviewe
Depletion of Endoplasmic Reticulum Calcium Triggers the Loss of ER Resident Proteins
The endoplasmic reticulum (ER) contains proteins that carry out the diverse functions of the ER including calcium storage, protein folding, modification, and trafficking, lipid metabolism, and drug detoxification. When soluble ER resident proteins with an ER retention sequence (ERS) depart from the ER they interact with KDEL receptors in the Golgi membrane and are retrogradely transported to the ER lumen via the KDEL receptor retrieval pathway. ER calcium depletion disrupts this process resulting in the mass departure of ERS-containing proteins into the extracellular space. Such a loss of ER resident proteins has two potential consequences to an affected cell. First, there is a loss of proteins involved in the aforementioned critical ER functions. Second, the relocation of such proteins and their associated functions outside of the cell may cause changes in the extracellular environment. This dissertation describes the identification and characterization of a phenomenon whereby ER resident protein secretion is triggered by pathophysiological ER calcium depletion. By exploiting the enzymatic activity of one of the identified ERS-containing proteins we developed an assay to monitor changes in the ER proteome. We also developed a high-throughput screen that identified drugs that could prevent the release of ER resident proteins following ER calcium depletion and showed that several of these compounds have therapeutic potential in models of ER stress and ischemia. Taken together, the work described in my dissertation identifies a novel molecular mechanism of cellular dysfunction for which I have identified both endogenous biomarkers and possible therapeutics
Caffeine and MDMA (Ecstasy) Exacerbate ER Stress Triggered by Hyperthermia
Drugs of abuse can cause local and systemic hyperthermia, a known trigger of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Another trigger of ER stress and UPR is ER calcium depletion, which causes ER exodosis, the secretion of ER-resident proteins. In rodent models, club drugs such as 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’) can create hyperthermic conditions in the brain and cause toxicity that is affected by the environmental temperature and the presence of other drugs, such as caffeine. In human studies, MDMA stimulated an acute, dose-dependent increase in core body temperature, but an examination of caffeine and MDMA in combination remains a topic for clinical research. Here we examine the secretion of ER-resident proteins and activation of the UPR under combined exposure to MDMA and caffeine in a cellular model of hyperthermia. We show that hyperthermia triggers the secretion of normally ER-resident proteins, and that this aberrant protein secretion is potentiated by the presence of MDMA, caffeine, or a combination of the two drugs. Hyperthermia activates the UPR but the addition of MDMA or caffeine does not alter the canonical UPR gene expression despite the drug effects on ER exodosis of UPR-related proteins. One exception was increased BiP/GRP78 mRNA levels in MDMA-treated cells exposed to hyperthermia. These findings suggest that club drug use under hyperthermic conditions exacerbates disruption of ER proteostasis, contributing to cellular toxicity
KDEL Receptors Are Differentially Regulated to Maintain the ER Proteome under Calcium Deficiency
Summary: Retention of critical endoplasmic reticulum (ER) luminal proteins needed to carry out diverse functions (e.g., protein synthesis and folding, lipid metabolism) is mediated through a carboxy-terminal ER retention sequence (ERS) and its interaction with KDEL receptors. Here, we demonstrate that depleting ER calcium causes mass departure of ERS-containing proteins from cells by overwhelming KDEL receptors. In addition, we provide evidence that KDELR2 and KDELR3, but not KDELR1, are unfolded protein response (UPR) genes upregulated as an adaptive response to counteract the loss of ERS-containing proteins, suggesting previously unknown isoform-specific functions of the KDEL receptors. Overall, our findings establish that decreases in ER calcium change the composition of the ER luminal proteome and secretome, which can impact cellular functions and cell viability. The redistribution of the ER proteome from inside the cell to the outside has implications for dissecting the complex relationship of ER homeostasis with diverse disease pathologies. : Trychta et al. identify an exodus of resident proteins from the endoplasmic reticulum (ER) in response to the loss of luminal ER calcium. They show that the cell works to maintain the ER proteome under calcium depletion by upregulating KDEL receptors, which act as unfolded protein response genes. Keywords: endoplasmic reticulum, calcium, KDEL receptor, ER retention sequence, XBP1, UPR, unfolded protein response, MAN
Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a <em>Gaussia</em> Luciferase SERCaMP
Dual luciferase assay can be used to examine ER-calcium-dependent (GLuc-SERCaMP) and constitutive (secNLuc) secretion.
<p>(A) Schematic of AAV-constructs used to express secNLuc and GLuc-SERCaMP. Orange represents hMANF signal peptide, pink is hMANF C-terminal peptide, and purple is SV40 polyA tail. (B, C) Constitutive secretion and ER calcium-dependent secretion can be monitored in rat primary cortical neurons using the dual GLuc and secNLuc reporter approach. Neurons were transduced with AAV-secNLuc, AAV-GLuc-SERCaMP, or a combination of both viruses. Secreted levels of the reporter proteins were assessed after 8 hrs of Tg (0–300 nM) treatment using 5 μL of culture medium and 100 μL of the indicated substrate (mean ± SEM, n = 12). (D, E) Blood was collected from rats expressing AAV-secNLuc, AAV-GLuc-SERCaMP, or a combination of both, prior to and following 1 mg/kg Tg exposure. Plasma was assayed for GLuc-SERCaMP or secNLuc using CTZ plus NLuc inhibitor or FMZ, respectively. Raw luminescence values are shown (mean ± SEM, n = 4 per group, 2-way ANOVA, * p<0.05, Dunnett’s multiple comparison test vs. day 14; # p<0.05 Tukey’s multiple comparison test between viral groups). (F) Design of viral construct expressing both secNLuc and GLuc-SERCaMP from a single transgene using a self-cleaving 2A peptide. (G, H) SH-SY5Y cells transfected with pAAV-EF1α-secNLuc-2A-GLuc- SERCaMP were treated with 300 nM Tg for 8 hrs. (G) Medium was collected and assayed for secNLuc using FMZ (mean ± SD, n = 6; *p<0.01, two-tailed t-test). (H) Medium was collected and assayed for GLuc-SERCaMP activity using 8 μM CTZ + 5 μM NLuc inhibitor (mean ± SD, n = 6; *p<0.01, two-tailed t- test).</p
NLuc inhibitor blocks NLuc activity using CTZ as a substrate
<p>(A) Schematic showing expected activities of NLuc and GLuc with CTZ and FMZ substrates. (B) Chemical structure of the NLuc inhibitor. (C) The NLuc Inhibitor blocks the enzymatic activity of secNLuc, but not GLuc, towards CTZ. The inhibitor was added to 8 μM CTZ substrate and light emission was measured (mean ± SD, n = 3).</p
UPRE-induced secNLuc can be used to monitor ER stress.
<p>(A) Schematic of AAV constructs used to express UPRE-dependent and control (minimal promoter, minP) secNLuc reporters. Tg treatment (100 nM) increases (B) 5X-UPRE-secNLuc activity (culture medium, 24 hrs, mean ± SD, n = 9) and endogenous BiP protein levels in the cell (cell lysates, 24 hrs, mean ± SD, n = 4). (C) Representative blot of BiP/actin as function of Tg treatment used for graph in (B). (D) Expression levels of UPR-responsive genes BiP, ERdj4, and ASNS (mRNA analysis, 8 hrs, mean ± SD n = 9) **p<0.01, ****p<0.0001, 1-way ANOVA, Dunnett’s multiple comparison test vehicle vs Tg). (E) TMP induces 5X-UPRE-secNLuc but has minimal effect on MinP-secNLuc (mean ± SD, n = 6 wells/transfection/TMP treatment ****p<0.0001, 2-way ANOVA). (F) Doxycycline-inducible XBP1 activates 5X-UPRE-secNLuc in HEK<sup>DAX</sup> cells (mean ± SEM, n = 4 wells/transfection/doxycycline treatment ****p<0.0001, 2-way ANOVA, Sidak’s multiple comparison test).</p
Longitudinal monitoring of <i>Gaussia</i> and Nano luciferase activities to concurrently assess ER calcium homeostasis and ER stress <i>in vivo</i>
<div><p>The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism and calcium storage. The ability to longitudinally monitor ER homeostasis in the same organism would offer insight into progressive molecular and cellular adaptations to physiologic or pathologic states, but has been challenging. We recently described the creation of a <i>Gaussia</i> luciferase (GLuc)-based secreted ER calcium-modulated protein (SERCaMP or GLuc-SERCaMP) to longitudinally monitor ER calcium homeostasis. Here we describe a complementary tool to measure the unfolded protein response (UPR), utilizing a UPRE-driven secreted Nano luciferase (UPRE-secNLuc) to examine the activating transcription factor-6 (ATF6) and inositol-requiring enzyme 1 (IRE1) pathways of the UPR. We observed an upregulation of endogenous ATF6- and XBP1-regulated genes following pharmacologically-induced ER stress that was consistent with responsiveness of the UPRE sensor. Both GLuc and NLuc-based reporters have favorable properties for <i>in vivo</i> studies, however, they are not easily used in combination due to overlapping substrate activities. We describe a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. This study is the first to describe the <i>in vivo</i> measurement of UPRE activation by sampling blood, using an approach that allows concurrent interrogation of two components of ER homeostasis.</p></div