Negative regulation of Ire1 during the unfolded protein response

When cells undergo endoplasmic reticulum stress due to a build-up of unfolded or misfolded proteins, the cell must adapt to this stress and does so through the unfolded protein response (UPR). Ire1, a protein kinase endoribonuclease, is a protein found in the endoplasmic reticulum (ER) of Saccharomyces cerevisiae and plays a major role in the cell’s adaptive response to ER stress. Upon accumulation of unfolded proteins in the ER, Ire1 becomes active and splices HAC1 mRNA. After splicing the HAC1 mRNA is translated to produce the Hac1i protein, the Hac1i protein contains a bZIP transcription factor which leads to alleviation of ER stress by promoting inducing expression of UPR-associated genes. Previous work has shown that although phosphorylation is not essential to RNase activation, it still plays a critical role. Therefore, this study investigates previously identified phosphatases, Dcr2 and Ptc2, which were proposed to be negative regulators of Ire1. This study shows that out of the two investigated phosphatases, only Ptc2 was observed to negatively regulate the UPR. The mechanism of activation for the way in which the UPR was inactivated determined that interference of IRE1 clustering was affected by overexpression of either phosphatase, which suggests an alternative mechanism

Recent advances in signal integration mechanisms in the unfolded protein response [version 1; peer review: 2 approved]

Since its discovery more than 25 years ago, great progress has been made in our understanding of the unfolded protein response (UPR), a homeostatic mechanism that adjusts endoplasmic reticulum (ER) function to satisfy the physiological demands of the cell. However, if ER homeostasis is unattainable, the UPR switches to drive cell death to remove defective cells in an effort to protect the health of the organism. This functional dichotomy places the UPR at the crossroads of the adaptation versus apoptosis decision. Here, we focus on new developments in UPR signaling mechanisms, in the interconnectivity among the signaling pathways that make up the UPR in higher eukaryotes, and in the coordination between the UPR and other fundamental cellular processes

BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response

Computational modeling and experimentation in the unfolded protein response reveals a role for the ER-resident chaperone protein BiP in fine-tuning the system's response dynamics

The Unfolded Protein Response as a Guardian of the Secretory Pathway

The endoplasmic reticulum (ER) is the major site of membrane biogenesis in most eukary otic cells. As the entry point to the secretory pathway, it handles more than 10,000 different secretory and membrane proteins. The insertion of proteins into the membrane, their folding, and ER exit are affected by the lipid composition of the ER membrane and its collective membrane stiffness. The ER is also a hotspot of lipid biosynthesis including sterols, glycerophospholipids, ceramides and neural storage lipids. The unfolded protein response (UPR) bears an evolutionary conserved, dual sensitivity to both protein-folding imbalances in the ER lumen and aberrant compositions of the ER membrane, referred to as lipid bilayer stress (LBS). Through transcriptional and non-transcriptional mechanisms, the UPR upregulates the protein folding capacity of the ER and balances the production of proteins and lipids to maintain a functional secretory pathway. In this review, we discuss how UPR transducers sense unfolded proteins and LBS with a particular focus on their role as guardians of the secretory pathway

The integrated stress response directs cell fate decisions in response to perturbations in protein homeostasis

Indiana University-Purdue University Indianapolis (IUPUI)Disruptions of the endoplasmic reticulum (ER) cause perturbations in protein folding and result in a cellular condition known as ER stress. ER stress and the accumulation of unfolded protein activate the unfolded protein response (UPR) which is a cellular attempt to remedy the toxic accumulation of unfolded proteins. The UPR is implemented through three ER stress sensors PERK, ATF6, and IRE1. Phosphorylation of the α-subunit of eIF2 by PERK during ER stress represses protein synthesis and also induces preferential translation of ATF4, a transcriptional activator of stress response genes. Early UPR signaling involves translational and transcriptional changes in gene expression that is geared toward stress remedy. However, prolonged ER stress that is not alleviated can trigger apoptosis. This dual signaling nature of the UPR is proposed to mimic a 'binary switch' and the regulation of this switch is a key topic of this thesis. Adaptive gene expression aimed at balancing protein homeostasis encompasses the first phase of the UPR. In this study we show that the PERK/eIF2~P/ATF4 pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi where ATF6 is activated. Liver-specific depletion of PERK significantly lowers expression of survival genes, leading to reduced expression of protein chaperones. As a consequence, loss of PERK in the liver sensitizes cells to stress which ultimately leads to apoptosis. Despite important roles in survival, PERK signaling is often extended to the vii activation of other downstream transcription factors such as CHOP, a direct target of ATF4-mediated transcription. Accumulation of CHOP is a hallmark of the second phase in the binary switch model where CHOP is shown to be required for full activation of apoptosis. Here the transcription factor ATF5 is found to be induced by CHOP and that loss of ATF5 improves the survival of cells following changes in protein homeostasis. Taken together this study highlights the importance of UPR signaling in determining the balance between cell survival and cell death. A topic that is important for understanding the more complex pathological conditions of diseases such as diabetes, cancer, and neurodegeneration

The probable, possible, and novel functions of ERp29

The luminal endoplasmic reticulum (ER) protein of 29 kDa (ERp29) is a ubiquitously expressed cellular agent with multiple critical roles. ERp29 regulates the biosynthesis and trafficking of several transmembrane and secretory proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial sodium channel (ENaC), thyroglobulin, connexin 43 hemichannels, and proinsulin. ERp29 is hypothesized to promote ER t

Molecular characterization of the endoplasmic reticulum: Insights from proteomic studies

The endoplasmic reticulum (ER) is a multifunctional intracellular organelle responsible for the synthesis, processing and trafficking of a wide variety of proteins essential for cell growth and survival. Therefore, comprehensive characterization of the ER proteome is of great importance to the understanding of its functions and has been actively pursued in the past decade by scientists in the proteomics field. This review summarizes major proteomic studies published in the past decade that focused on the ER proteome. We evaluate the data sets obtained from two different organs, liver and pancreas each of which contains a primary cell type (hepatocyte and acinar cell) with specialized functions. We also discuss how the nature of the proteins uncovered is related to the methods of organelle purification, organelle purity and the techniques used for protein separation prior to MS. In addition, this review also puts emphasis on the biological insights gained from these studies regarding the molecular functions of the ER including protein synthesis and translocation, protein folding and quality control, ER-associated degradation and ER stress, ER export and membrane trafficking, calcium homeostasis and detoxification and drug metabolism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78298/1/4040_ftp.pd

Endoplasmic Reticulum Stress and Diabetic Cardiomyopathy

The endoplasmic reticulum (ER) is an organelle entrusted with lipid synthesis, calcium homeostasis, protein folding, and maturation. Perturbation of ER-associated functions results in an evolutionarily conserved cell stress response, the unfolded protein response (UPR) that is also called ER stress. ER stress is aimed initially at compensating for damage but can eventually trigger cell death if ER stress is excessive or prolonged. Now the ER stress has been associated with numerous diseases. For instance, our recent studies have demonstrated the important role of ER stress in diabetes-induced cardiac cell death. It is known that apoptosis has been considered to play a critical role in diabetic cardiomyopathy. Therefore, this paper will summarize the information from the literature and our own studies to focus on the pathological role of ER stress in the development of diabetic cardiomyopathy. Improved understanding of the molecular mechanisms underlying UPR activation and ER-initiated apoptosis in diabetic cardiomyopathy will provide us with new targets for drug discovery and therapeutic intervention

An investigation into the molecular mechanisms underlying retinitis pigmentosa 17 with the view to developing novel gene- based therapies

Includes bibliographical references.Retinitis pigmentosa (RP) is a highly heterogeneous form of inherited blindness that affects more than 1.3 million individuals worldwide. The RP17 form of the disease is caused by an arginine to tryptophan (R14W) mutation in the signal sequence of carbonic anhydrase IV (CAIV). In an effort to elucidate the molecular mechanisms underlying RP17, three cell types were transfected with the wild type (WT ) and the R14W mutant form of the protein. We show using immunocytochemistry that unlike transfected WT CAIV which is transported to the plasma membrane of transfected COS-7 and HT-1080 cells, R14W mutant CAIV is retained in the endoplasmic reticulum when transfected into the same cell type. Further analyses of these cells by western blotting reveal that whereas the WT CAIV is processed to its mature form in both these cell lines, significant levels of the R14W mutant protein remain in its immature form. Importantly, flow cytometry experiments demonstrate that compared to WT CAIV protein, expression of specifically the R14W CAIV results in an S and G2/M cell cycle block, followed by apoptosis. Interestingly, when the above experiments were repeated in the human embryonic kidney cell line, HEK-293, strikingly different results were obtained. These cells were unaffected by the expression of the R14W mutant CAIV and were able to process the mutant and WT protein equally effectively. These findings regarding cell type specificity were used as a basis to explore methods of therapy for RP17. In particular, allele-specific small hairpin RNA was used to silence expression of R14W mutant CAIV, and to rescue cells from undergoing cell cycle arrest and apoptosis. A study of specific chaperones involved in protein folding, as well as gene and protein expression studies (microarray and mass spectrometry analysis), were also carried out to determine which proteins that were expressed in HEK-293 cells play a part in the ability to fold, process and transport R14W mutant CAIV. The results of this study have important implications for our understanding of the RP17 phenotype, and in investigating gene and protein therapy for the prevention and treatment of RP17

Differential Transcriptome Analysis Reveals that Cache Valley PM2.5 Triggers the Unfolded Protein Response in Human Lung Cells

Worldwide, exposure to air pollution is a serious human health threat. Particulate matter (PM) air pollution is a mixture of suspended solid and/or liquid particles and particle size is determined by its aerodynamic diameter. Fine, or “respirable” particles, typically from vehicle emissions, manufacturing, power generation, agriculture, as well as secondary photochemical reactions, are classified as ≤2.5μm in diameter (PM2.5). Upon inhalation, PM2.5 particles can reach the lower, more sensitive regions of the lung, enter the bloodstream, and be distributed to other areas in the body. Large-scale epidemiology studies have shown that PM2.5 air pollution is associated with increases in all-cause mortality, cardiopulmonary and cardiovascular disease, stroke, cancer, and Alzheimer’s disease. The normally picturesque Cache Valley of Northern Utah frequently experiences some of the highest PM2.5 concentrations in the United States during inversion events in the winter months. Elevated wintertime PM2.5 concentrations in Cache Valley are primarily due to a combination of human activity and environmental factors. However, the exact mechanism(s) of Cache Valley PM2.5 (CVPM) toxicity, or how CVPM may affect the health of Cache Valley residents, are not fully understood. Previous studies from our laboratory showed that CVPM exposure in cultured human lung cells is associated with the inflammatory response, endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR), a well-known stress-response system in cells. The purpose of this study was to confirm our previous findings since our method for collecting local CVPM was updated to a more effective particle collection system. In the present study, next-generation RNA sequencing revealed that human lung cells exposed to CVPM had gene expression changes related to activation of the UPR. Disruptions in normal cell conditions, or homeostasis, were also observed. Identification of the UPR as an operative mechanism of PM2.5 toxicity will represent an important breakthrough in our understanding of pollutant toxicology because activation of the UPR has been linked to many serious diseases, such as diabetes, retinal degeneration, metabolic disease, and even cancer. My research is also significant because it will enable more accurate risk estimates of CVPM exposure and may help guide positive changes in government regulations to improve air quality