Search

BACKGROUND We have recently reported on a recombinant von Willebrand factor (VWF) D'D3 albumin fusion protein (rD'D3-FP) developed to extend the half-life of coagulation factor VIII (FVIII) for the treatment of hemophilia A. Based on predictive modelling presented in this study, we hypothesized that modifying rD'D3-FP to improve FVIII interaction would reduce exchange with endogenous VWF and provide additional FVIII half-life benefit. OBJECTIVES The aim of this study was to identify novel rD'D3-FP variants with enhanced therapeutic efficacy in extending FVIII half-life. METHODS Through both directed mutagenesis and random mutagenesis using a novel mammalian display platform, we identified novel rD'D3-FP variants with increased affinity for FVIII (rVIII-SingleChain) under both neutral and acidic conditions and assessed their ability to extend FVIII half-life in vitro and in vivo. RESULTS In rat preclinical studies, rD'D3-FP variants with increased affinity for FVIII displayed enhanced potency, with reduced dose levels required to achieve equivalent rVIII-SingleChain half-life extension. In cell-based imaging studies in vitro, we also demonstrated reduced dissociation of rVIII-SingleChain from the rD'D3-FP variants within acidic endosomes and more efficient co-recycling of the rD'D3-FP/rVIII-SingleChain complex via the FcRn recycling system. CONCLUSIONS In summary, at potential clinical doses, the rD'D3-FP variants provide marked benefits with respect to dose levels and half-life extension of co-administered FVIII, supporting their development for use in the treatment of hemophilia A.

Mimivirus (Acanthamoeba polyphaga mimivirus) was the first giant DNA virus identified in an amoeba species. Its genome contains at least 979 genes. One of these, L276, encodes a nucleotide translocator with similarities to mitochondrial metabolite carriers, provisionally named viral mitochondrial carrier 1 (VMC1). In this study, we investigated the intracellular distribution of VMC1 upon expression in HeLa cells and in the yeast Saccharomyces cerevisiae. We found that VMC1 is specifically targeted to mitochondria and to the inner mitochondrial membrane. Newly synthesized VMC1 binds to the mitochondrial outer-membrane protein Tom70 and translocates through the import channel formed by the β-barrel protein Tom40. Derivatization of the four cysteine residues inside Tom40 by N-ethylmaleimide caused a delay in translocation but not a complete occlusion. Cell viability was not reduced by VMC1. Neither the mitochondrial membrane potential nor the intracellular production of reactive oxygen species was affected. Similar to endogenous metabolite carriers, mimivirus-encoded VMC1 appears to act as a specific translocator in the mitochondrial inner membrane. Due to its permeability for deoxyribonucleotides, VMC1 confers to the mitochondria an opportunity to contribute nucleotides for the replication of the large DNA genome of the virus.

Pex8p of Saccharomyces cerevisiae is located at the inner surface of the peroxisomal membrane and is essential for the assembly of the importomer, a multi-protein complex of the peroxisomal protein import machinery. By means of the yeast two-hybrid system as well as in vitro binding studies, we demonstrate that Pex8p is capable to interact with the PTS2-receptor Pex7p and the docking complex component Pex13p with its C-terminal SH3-domain providing the binding site. Analysis of the importomer composition of pex-mutants revealed that both PTS-receptors as well as the auxiliary proteins Pex18p and Pex21p are neither required for Pex8p association with the importomer nor for its function as an organizer of the peroxisomal protein import machinery.

The gamma-subunit is required for the assembly of ATP synthases and plays a crucial role in their catalytic activity. We stepwise shortened the N-terminus and the C-terminus of the gamma-subunit in the mitochondrial ATP synthase of yeast and investigated the relevance of these segments in the assembly of the enzyme and in the growth of the cells. We found that a deletion of 9 residues at the N-terminus or 20 residues at the C-terminus still allowed efficient import of the subunit into mitochondria; however, the assembly of both monomeric and dimeric holoenzymes was partially impaired. gamma-Subunits lacking 13 N-terminal residues or 30 C-terminal residues were not assembled. Yeast strains expressing either of the truncated gamma-subunits did not grow on non-fermentable carbon sources, indicating that non-assembled parts of the ATP synthase accumulated and impaired essential mitochondrial functions.

SummaryThe cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.

During budding of yeast cells peroxisomes are distributed over mother cell and bud, a process that involves the myosin motor protein Myo2p and the peroxisomal membrane protein Inp2p. Here, we show that Pex19p, a peroxin implicated in targeting and complex formation of peroxisomal membrane proteins, also plays a role in peroxisome partitioning. Binding studies revealed that Pex19p interacts with the cargo-binding domain of Myo2p. We identified mutations in Myo2p that specifically reduced binding to Pex19p, but not to Inp2p. The interaction between Myo2p and Pex19p was also reduced by a mutation that blocked Pex19p farnesylation. Microscopy revealed that the Pex19p-Myo2p interaction is important for peroxisome inheritance, because mutations that affect this interaction hamper peroxisome inheritance in vivo. Together these data suggest that both Inp2p and Pex19p are required for proper association of peroxisomes to Myo2p.