Jyoti D. Malhotra

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.

Tenascin-R is a functional modulator of sodium channel beta subunits.

Voltage-gated sodium channels isolated from mammalian brain are composed of α, β1, and β2 subunits. The α subunit forms the ion conducting pore of the channel, whereas the β1 and β2 subunits modulate channel function, as well as channel plasma membrane expression levels. β1 and β2 each contain a single, extracellular Ig-like domain with structural similarity to the neural cell adhesion molecule (CAM), myelin Po. β2 contains strong amino acid homology to the third Ig domain and to the juxtamembrane region of F3/contactin. Many CAMs of the Ig superfamily have been shown to interact with extracellular matrix molecules. We hypothesized that β2 may interact with tenascin-R (TN-R), an extracellular matrix molecule that is secreted by oligodendrocytes during myelination and that binds F3-contactin. We show here that cells expressing sodium channel β1 or β2 subunits are functionally modulated by TN-R. Transfected cells stably expressing β1 or β2 subunits initially recognized and then were repelled from TN-R substrates. The cysteine-rich amino-terminal domain of TN-R expressed as a recombinant peptide, termed EGF-L, appears to be responsible for the repellent effect on β subunit-expressing cells. The epidermal growth factor-like repeats and fibronectin-like repeats 6–8 are most effective in the initial adhesion of β subunit-expressing cells. Application of EGF-L to αIIAβ1β2 channels expressed in Xenopus oocytes potentiated expressed sodium currents without significantly altering current time course or the voltage dependence of current activation or inactivation. Thus, sodium channel β subunits appear to function as CAMs, and TN-R may be an important regulator of sodium channel localization and function in neurons.

The unfolded protein response transducer IRE1α prevents ER stress-induced hepatic steatosis

The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol‐requiring enzyme 1 α (IRE1α), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1α‐mediated signalling in the liver, we generated hepatocyte‐specific Ire1α‐null mice by utilizing an albumin promoter‐controlled Cre recombinase‐mediated deletion. Deletion of Ire1α caused defective induction of genes encoding functions in ER‐to‐Golgi protein transport, oxidative protein folding, and ER‐associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro‐apoptotic UPR trans‐activators. We show that IRE1α is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte‐specific Ire1α deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1α represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer‐binding protein (C/EBP) β, C/EBPδ, peroxisome proliferator‐activated receptor γ (PPARγ), and enzymes involved in triglyceride biosynthesis. IRE1α was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1α in preventing intracellular lipid accumulation, mice with hepatocyte‐specific deletion of Ire1α developed severe hepatic steatosis after treatment with an ER stress‐inducing anti‐cancer drug Bortezomib, upon expression of a misfolding‐prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1α as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

Reduced sodium channel density, altered voltage dependence of inactivation, and increased susceptibility to seizures in mice lacking sodium channel β2-subunits

Sodium channel β-subunits modulate channel gating, assembly, and cell surface expression in heterologous cell systems. We generated β2−/− mice to investigate the role of β2 in control of sodium channel density, localization, and function in neurons in vivo. Measurements of [3H]saxitoxin (STX) binding showed a significant reduction in the level of plasma membrane sodium channels in β2−/− neurons. The loss of β2 resulted in negative shifts in the voltage dependence of inactivation as well as significant decreases in sodium current density in acutely dissociated hippocampal neurons. The integral of the compound action potential in optic nerve was significantly reduced, and the threshold for action potential generation was increased, indicating a reduction in the level of functional plasma membrane sodium channels. In contrast, the conduction velocity, the number and size of axons in the optic nerve, and the specific localization of Nav1.6 channels in the nodes of Ranvier were unchanged. β2−/− mice displayed increased susceptibility to seizures, as indicated by reduced latency and threshold for pilocarpine-induced seizures, but seemed normal in other neurological tests. Our observations show that β2-subunits play an important role in the regulation of sodium channel density and function in neurons in vivo and are required for normal action potential generation and control of excitability.

ER Stress and Its Functional Link to Mitochondria: Role in Cell Survival and Death

The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.

The intracellular segment of the sodium channel β1 subunit is required for its efficient association with the channel α subunit

Sodium channels consist of a pore‐forming α subunit and auxiliary β1 and β2 subunits. The subunit β1 alters the kinetics and voltage‐dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N‐terminal extracellular domain of β1, but does not require the intracellular C‐terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain NaV1.2a sodium channel α subunits expressed in Chinese hamster lung cells by a mutant β1 construct with 34 amino acids deleted from the C‐terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co‐immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant β1 with α, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10 000 times more mutant β1 RNA was required to achieve the level of functional modulation observed with injection of full‐length β1. Together, these findings suggest that the C‐terminal cytoplasmic domain of β1 is an important determinant of β1 binding to the sodium channel α subunit in both mammalian cells and Xenopus oocytes.

Detection of oxidative damage in response to protein misfolding in the endoplasmic reticulum.

Disulfide bond formation in the endoplasmic reticulum (ER) requires the sequential transfer of electrons from thiol residues to protein disulfide isomerase and ER oxidase 1, with the final reduction of molecular oxygen to form hydrogen peroxide. Conditions that perturb correct protein folding lead to accumulation of misfolded proteins in the ER lumen, which induce ER stress and oxidative stress. Oxidative damage of cellular macromolecules is a common marker of aging and various pathological conditions including diabetes, cancer, and neurodegenerative disease. As accumulating evidence suggests a tight connection between the ER stress and oxidative stress, analysis of appropriate markers becomes particularly important. Here, we describe methods to analyze markers of oxidative damage associated with ER stress.

ER and Oxidative Stress

Publisher Summary The endoplasmic reticulum (ER) is a membranous network extending throughout the cytoplasm of the eukaryotic cell and is contiguous with the nuclear envelope. The ER is the site of synthesis of sterols, lipids, core-asparagine linked oligosaccharides, and membrane and secreted proteins biosynthesis. It has evolved as a protein folding machine and a major intracellular signaling organelle and provides a unique environment for protein folding, assembly, and disulfide bond formation prior to transit to the Golgi compartment. The unfolded protein response (UPR) evolved as a complex homeostatic mechanism to balance the load of newly synthesized proteins with the capacity for chaperone assisted protein folding in the lumen of the ER. UPR plays an important role in numerous disease states, including diabetes mellitus, atherosclerosis, neoplasia, and neurodegenerative diseases. The development of Type 2 diabetes is associated with a combination of insulin resistance in fat, muscle, and liver and a failure of pancreatic beta cells to adequately compensate by increased insulin production. There is also evidence that oxidative damage is associated with development of the diabetic state. Antioxidants have been reported to preserve glucose stimulated insulin secretion, prevent apoptosis, and expand beta cell mass, without significantly affecting cell proliferation. Neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Oxidative stress and protein misfolding play critical roles in the pathogenesis of these neurodegenerative diseases.Publisher Summary The endoplasmic reticulum (ER) is a membranous network extending throughout the cytoplasm of the eukaryotic cell and is contiguous with the nuclear envelope. The ER is the site of synthesis of sterols, lipids, core-asparagine linked oligosaccharides, and membrane and secreted proteins biosynthesis. It has evolved as a protein folding machine and a major intracellular signaling organelle and provides a unique environment for protein folding, assembly, and disulfide bond formation prior to transit to the Golgi compartment. The unfolded protein response (UPR) evolved as a complex homeostatic mechanism to balance the load of newly synthesized proteins with the capacity for chaperone assisted protein folding in the lumen of the ER. UPR plays an important role in numerous disease states, including diabetes mellitus, atherosclerosis, neoplasia, and neurodegenerative diseases. The development of Type 2 diabetes is associated with a combination of insulin resistance in fat, muscle, and liver and a failure of pancreatic beta cells to adequately compensate by increased insulin production. There is also evidence that oxidative damage is associated with development of the diabetic state. Antioxidants have been reported to preserve glucose stimulated insulin secretion, prevent apoptosis, and expand beta cell mass, without significantly affecting cell proliferation. Neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Oxidative stress and protein misfolding play critical roles in the pathogenesis of these neurodegenerative diseases.

Chapter 276 – ER and Oxidative Stress: Implications in Disease

Publisher Summary The endoplasmic reticulum (ER) is a membranous network extending throughout the cytoplasm of the eukaryotic cell and is contiguous with the nuclear envelope. The ER is the site of synthesis of sterols, lipids, core-asparagine linked oligosaccharides, and membrane and secreted proteins biosynthesis. It has evolved as a protein folding machine and a major intracellular signaling organelle and provides a unique environment for protein folding, assembly, and disulfide bond formation prior to transit to the Golgi compartment. The unfolded protein response (UPR) evolved as a complex homeostatic mechanism to balance the load of newly synthesized proteins with the capacity for chaperone assisted protein folding in the lumen of the ER. UPR plays an important role in numerous disease states, including diabetes mellitus, atherosclerosis, neoplasia, and neurodegenerative diseases. The development of Type 2 diabetes is associated with a combination of insulin resistance in fat, muscle, and liver and a failure of pancreatic beta cells to adequately compensate by increased insulin production. There is also evidence that oxidative damage is associated with development of the diabetic state. Antioxidants have been reported to preserve glucose stimulated insulin secretion, prevent apoptosis, and expand beta cell mass, without significantly affecting cell proliferation. Neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Oxidative stress and protein misfolding play critical roles in the pathogenesis of these neurodegenerative diseases.Publisher Summary The endoplasmic reticulum (ER) is a membranous network extending throughout the cytoplasm of the eukaryotic cell and is contiguous with the nuclear envelope. The ER is the site of synthesis of sterols, lipids, core-asparagine linked oligosaccharides, and membrane and secreted proteins biosynthesis. It has evolved as a protein folding machine and a major intracellular signaling organelle and provides a unique environment for protein folding, assembly, and disulfide bond formation prior to transit to the Golgi compartment. The unfolded protein response (UPR) evolved as a complex homeostatic mechanism to balance the load of newly synthesized proteins with the capacity for chaperone assisted protein folding in the lumen of the ER. UPR plays an important role in numerous disease states, including diabetes mellitus, atherosclerosis, neoplasia, and neurodegenerative diseases. The development of Type 2 diabetes is associated with a combination of insulin resistance in fat, muscle, and liver and a failure of pancreatic beta cells to adequately compensate by increased insulin production. There is also evidence that oxidative damage is associated with development of the diabetic state. Antioxidants have been reported to preserve glucose stimulated insulin secretion, prevent apoptosis, and expand beta cell mass, without significantly affecting cell proliferation. Neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Oxidative stress and protein misfolding play critical roles in the pathogenesis of these neurodegenerative diseases.

ER and Oxidative Stress: Implications in Disease

Publisher Summary The endoplasmic reticulum (ER) is a membranous network extending throughout the cytoplasm of the eukaryotic cell and is contiguous with the nuclear envelope. The ER is the site of synthesis of sterols, lipids, core-asparagine linked oligosaccharides, and membrane and secreted proteins biosynthesis. It has evolved as a protein folding machine and a major intracellular signaling organelle and provides a unique environment for protein folding, assembly, and disulfide bond formation prior to transit to the Golgi compartment. The unfolded protein response (UPR) evolved as a complex homeostatic mechanism to balance the load of newly synthesized proteins with the capacity for chaperone assisted protein folding in the lumen of the ER. UPR plays an important role in numerous disease states, including diabetes mellitus, atherosclerosis, neoplasia, and neurodegenerative diseases. The development of Type 2 diabetes is associated with a combination of insulin resistance in fat, muscle, and liver and a failure of pancreatic beta cells to adequately compensate by increased insulin production. There is also evidence that oxidative damage is associated with development of the diabetic state. Antioxidants have been reported to preserve glucose stimulated insulin secretion, prevent apoptosis, and expand beta cell mass, without significantly affecting cell proliferation. Neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Oxidative stress and protein misfolding play critical roles in the pathogenesis of these neurodegenerative diseases.Publisher Summary The endoplasmic reticulum (ER) is a membranous network extending throughout the cytoplasm of the eukaryotic cell and is contiguous with the nuclear envelope. The ER is the site of synthesis of sterols, lipids, core-asparagine linked oligosaccharides, and membrane and secreted proteins biosynthesis. It has evolved as a protein folding machine and a major intracellular signaling organelle and provides a unique environment for protein folding, assembly, and disulfide bond formation prior to transit to the Golgi compartment. The unfolded protein response (UPR) evolved as a complex homeostatic mechanism to balance the load of newly synthesized proteins with the capacity for chaperone assisted protein folding in the lumen of the ER. UPR plays an important role in numerous disease states, including diabetes mellitus, atherosclerosis, neoplasia, and neurodegenerative diseases. The development of Type 2 diabetes is associated with a combination of insulin resistance in fat, muscle, and liver and a failure of pancreatic beta cells to adequately compensate by increased insulin production. There is also evidence that oxidative damage is associated with development of the diabetic state. Antioxidants have been reported to preserve glucose stimulated insulin secretion, prevent apoptosis, and expand beta cell mass, without significantly affecting cell proliferation. Neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Oxidative stress and protein misfolding play critical roles in the pathogenesis of these neurodegenerative diseases.