Hongzhi Miao

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.

UPR Pathways Combine to Prevent Hepatic Steatosis Caused by ER Stress-Mediated Suppression of Transcriptional Master Regulators

The unfolded protein response (UPR) is linked to metabolic dysfunction, yet it is not known how endoplasmic reticulum (ER) disruption might influence metabolic pathways. Using a multilayered genetic approach, we find that mice with genetic ablations of either ER stress-sensing pathways (ATF6alpha, eIF2alpha, IRE1alpha) or of ER quality control (p58(IPK)) share a common dysregulated response to ER stress that includes the development of hepatic microvesicular steatosis. Rescue of ER protein processing capacity by the combined action of UPR pathways during stress prevents the suppression of a subset of metabolic transcription factors that regulate lipid homeostasis. This suppression occurs in part by unresolved ER stress perpetuating expression of the transcriptional repressor CHOP. As a consequence, metabolic gene expression networks are directly responsive to ER homeostasis. These results reveal an unanticipated direct link between ER homeostasis and the transcriptional regulation of metabolism, and suggest mechanisms by which ER stress might underlie fatty liver disease.

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.

A membrane-interactive surface on the factor VIII C1 domain cooperates with the C2 domain for cofactor function

Factor VIII binds to phosphatidylserine (PS)-containing membranes through its tandem, lectin-homology, C1 and C2 domains. However, the details of C1 domain membrane binding have not been delineated. We prepared 4 factor VIII C1 mutations localized to a hypothesized membrane-interactive surface (Arg2090Ala/Gln2091Ala, Lys2092Ala/Phe2093Ala, Gln2042Ala/Tyr2043Ala, and Arg2159Ala). Membrane binding and cofactor activity were measured using membranes with 15% PS, mimicking platelets stimulated by thrombin plus collagen, and 4% PS, mimicking platelets stimulated by thrombin. All mutants had at least 10-fold reduced affinities for membranes of 4% PS, and 3 mutants also had decreased apparent affinity for factor X. Monoclonal antibodies against the C2 domain produced different relative impairment of mutants compared with wild-type factor VIII. Monoclonal antibody ESH4 decreased the V(max) for all mutants but only the apparent membrane affinity for wild-type factor VIII. Monoclonal antibody BO2C11 decreased the V(max) of wild-type factor VIII by 90% but decreased the activity of 3 mutants more than 98%. These results identify a membrane-binding face of the factor VIII C1 domain, indicate an influence of the C1 domain on factor VIII binding to factor X, and indicate that cooperation between the C1 and C2 domains is necessary for full activity of the factor Xase complex.

C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis.

Significance Acute myeloid leukemia (AML) is a highly heterogeneous form of cancer that results from the uncontrolled proliferation of primitive immune cells. Homeobox A9 (HOXA9) is an evolutionarily conserved transcription factor that is overexpressed in a large percentage of AML cases and is associated with a poor prognosis. Here, we show that CCAAT/enhancer binding protein alpha (C/EBPα), a transcription factor involved in immune cell development that is commonly mutated in AML, is a critical collaborator required for HOXA9-mediated leukemic transformation. We also establish that the cell cycle regulator cyclin-dependent kinase inhibitors Cdkn2a/b are corepressed by the Hoxa9–C/EBPα complex. These findings suggest a novel functional interaction between two leukemic transcription factors, HOXA9 and C/EBPα, that is altered in a large percentage of AML cases. Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity

Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FV(MTTS/Y)). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FV(MTTS/Y), had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FV(MTTS/Y) showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.

The same site on the integrase-binding domain of lens epithelium-derived growth factor is a therapeutic target for MLL leukemia and HIV.

Lens epithelium-derived growth factor (LEDGF) is a chromatin-associated protein implicated in leukemia and HIV type 1 infection. LEDGF associates with mixed-lineage leukemia (MLL) fusion proteins and menin and is required for leukemic transformation. To better understand the molecular mechanism underlying the LEDGF integrase-binding domain (IBD) interaction with MLL fusion proteins in leukemia, we determined the solution structure of the MLL-IBD complex. We found a novel MLL motif, integrase domain binding motif 2 (IBM2), which binds to a well-defined site on IBD. Point mutations within IBM2 abolished leukemogenic transformation by MLL-AF9, validating that this newly identified motif is essential for the oncogenic activity of MLL fusion proteins. Interestingly, the IBM2 binding site on IBD overlaps with the binding site for the HIV integrase (IN), and IN was capable of efficiently sequestering IBD from the menin-MLL complex. A short IBM2 peptide binds to IBD directly and inhibits both the IBD-MLL/menin and IBD-IN interactions. Our findings show that the same site on IBD is involved in binding to MLL and HIV-IN, revealing an attractive approach to simultaneously target LEDGF in leukemia and HIV.

Property Focused Structure-Based Optimization of Small Molecule Inhibitors of the Protein-Protein Interaction between Menin and Mixed Lineage Leukemia (MLL).

Development of potent small molecule inhibitors of protein-protein interactions with optimized druglike properties represents a challenging task in lead optimization process. Here, we report synthesis and structure-based optimization of new thienopyrimidine class of compounds, which block the protein-protein interaction between menin and MLL fusion proteins that plays an important role in acute leukemias with MLL translocations. We performed simultaneous optimization of both activity and druglike properties through systematic exploration of substituents introduced to the indole ring of lead compound 1 (MI-136) to identify compounds suitable for in vivo studies in mice. This work resulted in the identification of compound 27 (MI-538), which showed significantly increased activity, selectivity, polarity, and pharmacokinetic profile over 1 and demonstrated a pronounced effect in a mouse model of MLL leukemia. This study, which reports detailed structure-activity and structure-property relationships for the menin-MLL inhibitors, demonstrates challenges in optimizing inhibitors of protein-protein interactions for potential therapeutic applications.

Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

Summary.  Background: Current manufacturing methods for recombinant human factor VIII (rFVIII) within mammalian cell cultures are inefficient, hampering the production of sufficient amounts for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary glands of mice, rabbits, sheep, and pigs. Unfortunately, it is secreted into milk with low specific activity, owing in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives: To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods: Transgenic mice were made with a mammary‐specific FVIII gene (226/N6) bioengineered for efficient expression and stability, encoding a protein containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand factor (VWF). 226/N6 was evaluated by ELISA, SDS‐PAGE, western blot, and one‐stage and two‐stage clotting assays. The hemostatic activity of immunoaffinity‐enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions: With or without coexpression of VWF, 226/N6 was secreted into milk as a biologically active single‐chain molecule that retained high specific activity, similar to therapeutic‐grade FVIII. 226/N6 had > 450‐fold higher IU mL−1 than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma‐derived VWF as therapeutic‐grade rFVIII, and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof‐of‐principle for the study of expression of 226/N6 and perhaps other single‐chain bioengineered rFVIIIs in the milk of transgenic livestock.

Menin-MLL inhibitors block oncogenic transformation by MLL fusion proteins in a fusion partner independent manner

Menin-MLL inhibitors block oncogenic transformation by MLL-fusion proteins in a fusion partner-independent manner