Remarkably, it has been 40 years since the isolation of the 2 genes involved in hemophilia A (HA) and hemophilia B (HB), encoding clotting factor (F) VIII (FVIII) and FIX, respectively. Over the ...years, these advances led to the development of purified recombinant protein factors that are free of contaminating viruses from human pooled plasma for hemophilia treatments, reducing the morbidity and mortality previously associated with human plasma-derived clotting factors. These discoveries also paved the way for modified factors that have increased plasma half-lives. Importantly, more recent advances have led to the development and Food and Drug Administration approval of a hepatocyte-targeted, adeno-associated viral vector-mediated gene transfer approach for HA and HB. However, major concerns regarding the durability and safety of HA gene therapy remain to be resolved. Compared with FIX, FVIII is a much larger protein that is prone to misfolding and aggregation in the endoplasmic reticulum and is poorly secreted by the mammalian cells. Due to the constraint of the packaging capacity of adeno-associated viral vector, B-domain deleted FVIII rather than the full-length protein is used for HA gene therapy. Like full-length FVIII, B-domain deleted FVIII misfolds and is inefficiently secreted. Its expression in hepatocytes activates the cellular unfolded protein response, which is deleterious for hepatocyte function and survival and has the potential to drive hepatocellular carcinoma. This review is focused on our current understanding of factors limiting FVIII secretion and the potential pathophysiological consequences upon expression in hepatocytes.
The endoplasmic reticulum (ER) imports ATP and uses energy from ATP hydrolysis for protein folding and trafficking. However, little is known about how this vital ATP transport occurs across the ER ...membrane. Here, using three commonly used cell lines (CHO, INS1 and HeLa), we report that ATP enters the ER lumen through a cytosolic Ca2+-antagonized mechanism, or CaATiER (Ca2+-Antagonized Transport into ER). Significantly, we show that mitochondria supply ATP to the ER and a SERCA-dependent Ca2+ gradient across the ER membrane is necessary for ATP transport into the ER, through SLC35B1/AXER. We propose that under physiological conditions, increases in cytosolic Ca2+ inhibit ATP import into the ER lumen to limit ER ATP consumption. Furthermore, the ATP level in the ER is readily depleted by oxidative phosphorylation (OxPhos) inhibitors and that ER protein misfolding increases ATP uptake from mitochondria into the ER. These findings suggest that ATP usage in the ER may increase mitochondrial OxPhos while decreasing glycolysis, i.e. an ‘anti-Warburg’ effect.
RATIONALE:ER stress causes accumulation of misfolded proteins in the ER, activating the transcription factor, ATF6, which induces ER stress response genes. Myocardial ischemia induces the ER stress ...response; however, neither the function of this response nor whether it is mediated by ATF6 is known.
OBJECTIVE:Here, we examined the effects of blocking the ATF6-mediated ER stress response on ischemia/reperfusion (I/R) in cardiac myocytes and mouse hearts.
METHODS AND RESULTS:Knockdown of ATF6 in cardiac myocytes subjected to I/R increased ROS and necrotic cell death, which were mitigated by ATF6 overexpression. Under non-stressed conditions, WT and ATF6 knockout (KO) mouse hearts were similar. However, compared to WT, ATF6 KO hearts showed increased damage and decreased function upon I/R. Mechanistically, gene array analysis showed that ATF6, which is known to induce genes encoding ER proteins that augment ER protein-folding, induced numerous oxidative stress response genes not previously known to be ATF6-inducible. Many of the proteins encoded by the ATF6-induced oxidative stress genes identified here reside outside the ER, including catalase, which is known to decrease damaging ROS in the heart. Catalase was induced by the canonical ER stressor, tunicamycin, and by I/R in cardiac myocytes from WT but not in cardiac myocytes from ATF6 KO mice. ER stress response elements were identified in the catalase gene and were shown to bind ATF6 in cardiac myocytes, which increased catalase promoter activity. Overexpression of catalase, in vivo, restored ATF6 KO mouse heart function to WT levels in a mouse model of I/R, as did AAV9-mediated ATF6 overexpression.
CONCLUSIONS:ATF6 serves as a previously unappreciated link between the ER stress and oxidative stress gene programs, supporting a novel mechanism by which ATF6 decreases myocardial I/R damage.
Cells respond to the accumulation of unfolded proteins by activating signal transduction cascades that improve protein folding. One example of such a cascade is the unfolded protein response (UPR), ...which senses protein folding stress in the endoplasmic reticulum (ER) and leads to improvement in the protein folding and processing capacity of the organelle. A central paradox of the UPR, and indeed of all such stress pathways, is that the response is designed to facilitate both adaptation to stress and apoptosis, depending upon the nature and severity of the stressor. Understanding how the UPR can allow for adaptation, instead of apoptosis, is of tremendous physiological importance. Recent advances have improved our understanding of ER stress and the vertebrate UPR, which suggest possible mechanisms by which cells adapt to chronic stress.
IRE1α, the most conserved transducer of the unfolded protein response, plays critical roles in many biological processes and cell fate decisions. Reporting in Science, Upton et al. (2012) broadened ...our understanding of IRE1α as a cell-death executioner, showing that upon ER stress, IRE1α degrades microRNAs to promote translation of caspase-2.
Retinitis Pigmentosa (RP) is a blinding disease that arises from loss of rods and subsequently cones. The P23H rhodopsin knock-in (P23H-KI) mouse develops retinal degeneration that mirrors RP ...phenotype in patients carrying the orthologous variant. Previously, we found that the P23H rhodopsin protein was degraded in P23H-KI retinas, and the Unfolded Protein Response (UPR) promoted P23H rhodopsin degradation in heterologous cells in vitro. Here, we investigated the role of a UPR regulator gene, activating transcription factor 6 (Atf6), in rhodopsin protein homeostasis in heterozygous P23H rhodopsin (Rho
) mice. Significantly increased rhodopsin protein levels were found in Atf6
Rho
retinas compared to Atf6
Rho
retinas at early ages (~ P12), while rhodopsin mRNA levels were not different. The IRE1 pathway of the UPR was hyper-activated in young Atf6
Rho
retinas, and photoreceptor layer thickness was unchanged at this early age in Rho
mice lacking Atf6. By contrast, older Atf6
Rho
mice developed significantly increased retinal degeneration in comparison to Atf6
Rho
mice in all retinal layers, accompanied by reduced rhodopsin protein levels. Our findings demonstrate that Atf6 is required for efficient clearance of rhodopsin protein in rod photoreceptors expressing P23H rhodopsin, and that loss of Atf6 ultimately accelerates retinal degeneration in P23H-KI mice.
cAMP responsive element‐binding protein, hepatocyte specific (CREBH), is a liver‐specific transcription factor localized in the endoplasmic reticulum (ER) membrane. Our previous work demonstrated ...that CREBH is activated by ER stress or inflammatory stimuli to induce an acute‐phase hepatic inflammation. Here, we demonstrate that CREBH is a key metabolic regulator of hepatic lipogenesis, fatty acid (FA) oxidation, and lipolysis under metabolic stress. Saturated FA, insulin signals, or an atherogenic high‐fat diet can induce CREBH activation in the liver. Under the normal chow diet, CrebH knockout mice display a modest decrease in hepatic lipid contents, but an increase in plasma triglycerides (TGs). After having been fed an atherogenic high‐fat (AHF) diet, massive accumulation of hepatic lipid metabolites and significant increase in plasma TG levels were observed in the CrebH knockout mice. Along with the hypertriglyceridemia phenotype, the CrebH null mice displayed significantly reduced body‐weight gain, diminished abdominal fat, and increased nonalcoholic steatohepatitis activities under the AHF diet. Gene‐expression analysis and chromatin‐immunoprecipitation assay indicated that CREBH is required to activate the expression of the genes encoding functions involved in de novo lipogenesis, TG and cholesterol biosynthesis, FA elongation and oxidation, lipolysis, and lipid transport. Supporting the role of CREBH in lipogenesis and lipolysis, forced expression of an activated form of CREBH protein in the liver significantly increases accumulation of hepatic lipids, but reduces plasma TG levels in mice. Conclusion: All together, our study shows that CREBH plays a key role in maintaining lipid homeostasis by regulating the expression of the genes involved in hepatic lipogenesis, FA oxidation, and lipolysis under metabolic stress. The identification of CREBH as a stress‐inducible metabolic regulator has important implications in the understanding and treatment of metabolic disease. (Hepatology 2012)
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, ...there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome.
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