Bioengineering of coagulation factor VIII for improved secretion

Miao, Hongzhi; Sirachainan, Nongnuch; Palmer, L. E.; Kucab, Phillip; Cunningham, Michael; Kaufman, Randal J.; Pipe, Steven W.

doi:10.1182/blood-2003-10-3591

Cited by 191 publications

(208 citation statements)

References 73 publications

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“…In contrast, a smaller BDD molecule (226/N6, which retains an additional 226 aa with 6 N-linked glycosylation sites from the B domain) is secreted Ϸ10-fold more efficiently than either wtFVIII or BDD, presumably because of increased engagement with the lectin chaperone machinery of the cell (Fig. 1A) (20). wtFVIII, BDD, and 226/N6 each contain eight disulfide bonds so that proper oxidative folding of each of these molecules requires the transfer of 16 electrons from cysteine residues within FVIII to molecular oxygen to form hydrogen peroxide.…”

Section: Compared With Wtfviii and B Domain Deletion 226/n6 Is Morementioning

confidence: 99%

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Malhotra¹,

Miao

Zhang³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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564

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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.factor VIII ͉ oxidative stress ͉ unfolded protein response A lthough endoplasmic reticulum (ER) stress and oxidative stress are closely linked events, the molecular pathways that couple these processes are poorly understood (1). Reactive oxygen species (ROS) originate during oxygen-using cellular metabolic processes, such as oxidative phosphorylation within mitochondria. The ER provides a unique oxidizing environment for protein folding and disulfide bond formation before transit to the Golgi compartment. During disulfide bond formation ROS are formed as a product of electron transport from thiol groups in proteins through protein disulfide isomerase (PDI) and ER oxidoreductase 1 (ERO1) to reduce molecular oxygen to form the oxidant hydrogen peroxide. It has been suggested that oxidation of cysteine residues during disulfide bond formation in the ER may significantly contribute to oxidative stress (2, 3). The unfolded protein response (UPR) is an adaptive signaling pathway designed to prevent the accumulation of misfolded proteins in the ER lumen. Studies also suggest the UPR is designed to minimize the stress of oxidative protein folding (2). The UPR is signaled through the protein kinases inositolrequiring protein 1␣ and PKR-related ER kinase and the activating transcription factor 6␣ (4, 5). Chronic unresolved accumulation of unfolded proteins in the ER leads to apoptosis. To elucidate the relationship between unfolded protein accumulation in the ER lumen, oxidative stress, and apoptosis, we have analyzed the secretion of coagulation factor VIII (FVIII), a large glycoprotein that is deficient in the X chromosome-linked bleeding disorder hemophilia A. As FVIII is prone to misfolding in the ER lumen, FVIII expression provides a unique approach to manipulate the ER stress response that does not rely on pharmacological intervention, where any connection between ER stress and ROS would be difficult to dissect.FVIII is comprised of three domains in the ord...

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Section: Compared With Wtfviii and B Domain Deletion 226/n6 Is Morementioning

confidence: 99%

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Malhotra¹,

Miao

Zhang³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

605

564

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“…Plasmid DNA samples (100 mg) were diluted in 2.5 ml lactated Ringer buffer and infused over 10 s into the tail-vein as previously described (Miao et al, 2004). Retro-orbital blood collection was performed at 24 h after injection for measurement of hFVIII activity and antigen in the plasma (Miao et al, 2004).…”

Section: Hydrodynamic Tail-vein Injection and Quantification Of Hfviimentioning

confidence: 99%

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

et al. 2011

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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.

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“…12 Pipe et al demonstrated that restoration of several N-linked glycosylation sites from the deleted B domain of a B domain deleted (BDD) human (h)-FVIII construct facilitated increased interaction with mannose-binding lectin protein-1 (LMAN1), resulting in more efficient carbohydrate-facilitated transport from the endoplasmic reticulum to the Golgi apparatus. 13 Nathwani et al combined a similar approach with codon optimization and demonstrated significant improvement in FVIII production from liver-directed AAV gene transfer systems. 14 In addition, we have shown that BDD porcine FVIII (p-FVIII) is expressed at 10-to 100-fold greater levels than BDD-h-FVIII in vitro.…”

Section: Origins and Limitations Of Fviii And Fix Biosynthesismentioning

confidence: 99%

Replacing bad (F)actors: hemophilia

Doering

Spencer

2014

Hematology

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Hemophilia A and B are bleeding disorders that result from functional deficiencies in specific circulating blood clotting factors termed factor VIII (FVIII) and factor IX (FIX), respectively, and collectively display an incidence of 1 in 4000 male births. Stem cell transplantation therapies hold the promise of providing a cure for hemophilia, but currently available transplantable stem cell products do not confer endogenous FIX or FVIII biosynthesis. Learning Objective• To gain an understanding of the key issues surrounding the implementation of stem cell therapies for hemophilia

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Bioengineering of coagulation factor VIII for improved secretion

Cited by 191 publications

References 73 publications

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

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

Replacing bad (F)actors: hemophilia

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