Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER.

SUMMARY Accumulation of unfolded protein within the endoplasmic reticulum (ER) lumen attenuates mRNA translation through activation of the protein kinase PERK and subsequent phosphorylation of eukaryotic initiation factor 2 on Ser51 of the alpha subunit (eIF2α). Genetic disruption of the PERK/eIF2α pathway in humans and mice produces severe pancreatic beta cell deficiency and post-natal lethality. To elucidate the role of eIF2α phosphorylation in beta cells, we have rescued the lethality of homozygous eIF2α Ser51Ala mice by expression of a loxP-flanked wild-type eIF2α transgene. Beta cell-specific transgene deletion to prevent eIF2α phosphorylation caused a severe diabetic phenotype due to heightened, unregulated proinsulin translation, defective intracellular trafficking of secretory and plasma membrane proteins, increased oxidative damage, reduced expression of stress response and beta cell-specific genes, and apoptosis. However, glucose intolerance and beta cell death in these mice were attenuated by antioxidant treatment. We conclude that phosphorylation of eIF2α coordinately attenuates mRNA translation, prevents oxidative stress, and optimizes ER protein folding to support insulin production in the beta cell. These findings that show increased proinsulin synthesis causes oxidative stress leading to beta cell failure may reflect events in the beta cell loss associated with insulin resistance in type 2 diabetes.

• The D9D3 domains of VWF are sufficient to stabilize FVIII in vivo.• The prolongation of VWF D9D3 survival in vivo by Fc fusion elevates FVIII levels in the setting of VWF but not FVIII deficiency.Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D9D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf 2/2 mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D9D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t 1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf 2/2 mice. Although the VWF D9D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D9D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D9D3 and VWF D9D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D9D3/D9D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding. (Blood. 2014; 124(3):445-452)

• The VWF D9 domains are flexibly tethered entities projecting outside antiparallel dimers of the VWF D3 domain. • Extensive interactionsbetween the VWF D9 domain and primarily the FVIII C1 domain mediate VWF-FVIII association.Binding to the von Willebrand factor (VWF) D9D3 domains protects factor VIII (FVIII) from rapid clearance. We performed single-particle electron microscopy (EM) analysis of negatively stained specimens to examine the architecture of D9D3 alone and in complex with FVIII. The D9D3 dimer ([D9D3] 2 ) comprises 2 antiparallel D3 monomers with flexibly attached protrusions of D9. FVIII-VWF association is primarily established between the FVIII C1 domain and the VWF D9 domain, whereas weaker interactions appear to be mediated between both FVIII C domains and the VWF D3 core. IntroductionThe strong association of plasma factor VIII (FVIII) with circulating von Willebrand factor (VWF) secures FVIII from rapid clearance in the blood. The VWF-FVIII complex forms through a high-affinity interaction between the FVIII light chain and the VWF D9D3 domains. 1Mutations within VWF that abrogate or abolish this high-affinity binding lead to type 2N von Willebrand disease, a condition characterized by reduced plasma levels of FVIII. 2The tertiary structure of mature VWF, particularly at the N-terminal D9D3 domains, regulates the affinity for FVIII. VWF circulates as a multi-subunit protein comprising repeated domains that distinctly facilitate VWF packaging and hemostasis.3 The VWF propeptide (domains D1and D2) catalyzes the multimerization of VWF via intermolecular disulfide bonds at the D3 domain ( Figure 1A). 4 In the absence of propeptide-dependent posttranslational modifications to the D9D3 domains, VWF binds FVIII with reduced affinity.5 Cleavage of the propeptide by furin facilitates FVIII stabilization in the circulation. 6 We and others have previously reported that VWF fragments are sufficient to bind FVIII and that propeptide processing of these VWF fragments enhances the affinity for FVIII.7-9 Several of these VWF fragments were also sufficient to elevate FVIII levels in VWF-deficient mice. 7To further explore the association between VWF and FVIII, we used single-particle negative-stain electron microscopy (EM) to characterize the architecture of dimeric VWF D9D3 domains ([D9D3] 2 ) alone and in complex with FVIII. Study designProtein expression, purification, and analyses are detailed in supplemental Data available on the Blood Web site. The online version of this article contains a data supplement. Results and discussionThere is an Inside Blood Commentary on this article in this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734. , each monomer appears as an ovoid density along the dimer symmetry axis accompanied by a weaker elongated density, which we term the "handle," in the periphery. The dimensions of the handle are ;20Å ...

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73.