Abstract Background Heterotopic ossification (HO) frequently causes complications following orthopaedic and trauma surgery and may drastically reduce the postoperative outcome due to pain and joint ...contracture. Current therapeutic options include NSAID's and local radiation. However, both options of prevention show disadvantages such as delayed fracture healing and impaired ossification as well as other side effects. Our goal was to investigate a novel approach in the prevention of heterotopic ossification by pharmacologically interfering with the molecular signalling pathways involved in this process. Hypoxia leads to numerous effects on a cellular level, one of which is the activation of the transcriptional complex hypoxia-inducible factor (HIF). Among several other actions, the HIF1-α signalling pathway in turn regulates angiogenesis through induction of the expression of vascular endothelial growth factor (VEGF). We hypothesised that by pharmacologically interfering with the HIF-1α signalling pathway, the amount of HO formation may be reduced. Echinomycin is a known inhibitor of HIF-1-alpha and was used in our study with the aim to prevent HO from forming. Methods We examined the effect of Echinomycin on HO formation in a murine model where an Achilles tenotomy was performed. This has previously been shown to reliably produce islets of heterotopic ossification within the soft tissue of mouse hind limbs at 10 weeks after surgery. The control group underwent Achilles tenotomy only, whereas the Echinomycin group additionally received Echinomycin subcutaneously. After trial completion, the limbs were harvested and Micro-CT was performed. Heterotopic bone volume was then identified in 3d images and quantified. Results We found a highly significant reduction in the bone volume following subcutaneous administration of Echinomycin compared to the control group. Conclusion Although a substantial reduction could be achieved, it was not possible to completely prevent heterotopic ossification from forming. Further studies have yet to be conducted to optimise the results by altering the dosage and duration of administration as well as investigate the mechanism by which Echinomycin led to the reduction of HO formation.
Members of the yeast p24 family, including Emp24p and Erv25p, form a heteromeric complex required for the efficient transport of selected proteins from the endoplasmic reticulum (ER) to the Golgi ...apparatus. The specific functions and sites of action of this complex are unknown. We show that Emp24p is directly required for efficient packaging of a lumenal cargo protein, Gas1p, into ER-derived vesicles. Emp24p and Erv25p can be directly cross-linked to Gas1p in ER-derived vesicles. Gap1p, which was not affected by emp24 mutation, was not cross-linked. These results suggest that the Emp24 complex acts as a cargo receptor in vesicle biogenesis from the ER.
The ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 is a mannose-specific membrane lectin operating as a cargo receptor for the transport of glycoproteins from the ER to the ERGIC. Lack of ...functional ERGIC-53 leads to a selective defect in secretion of glycoproteins in cultured cells and to hemophilia in humans. Beyond its interest as a transport receptor, ERGIC-53 is an attractive probe for studying numerous aspects of protein trafficking in the secretory pathway, including traffic routes, mechanisms of anterograde and retrograde traffic, retention of proteins in the ER, and the function of the ERGIC. Understanding these fundamental processes of cell biology will be crucial for the elucidation and treatment of many inherited and acquired diseases, such as cystic fibrosis, Alzheimer's disease and viral infections.
Soluble secretory proteins are transported from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) in vesicles coated with COP-II coat proteins. The sorting of secretory ...cargo into these vesicles is thought to involve transmembrane cargo-receptor proteins. Here we show that a cathepsin-Z-related glycoprotein binds to the recycling, mannose-specific membrane lectin ERGIC-53. Binding occurs in the ER, is carbohydrate- and calcium-ion-dependent and is affected by untrimmed glucose residues. Binding does not, however, require oligomerization of ERGIC-53, although oligomerization is required for exit of ERGIC-53 from the ER. Dissociation of ERGIC-53 occurs in the ERGIC and is delayed if ERGIC-53 is mislocalized to the ER. These results strongly indicate that ERGIC-53 may function as a receptor facilitating ER-to-ERGIC transport of soluble glycoprotein cargo.
Combined deficiency of factors V and VIII is an autosomal recessive bleeding disorder resulting from alterations in an unknown gene on chromosome 18q, distinct from the factor V and factor VIII ...genes. ERGIC-53, a component of the ER–Golgi intermediate compartment, was mapped to a YAC and BAC contig containing the critical region for the combined factors V and VIII deficiency gene. DNA sequence analysis identified two different mutations, accounting for all affected individuals in nine families studied. Immunofluorescence and Western analysis of immortalized lymphocytes from patients homozygous for either of the two mutations demonstrate complete lack of expression of the mutated gene in these cells. These findings suggest that ERGIC-53 may function as a molecular chaperone for the transport from ER to Golgi of a specific subset of secreted proteins, including coagulation factors V and VIII.
The positioning and dynamics of organelles in eukaryotic cells critically depend on membrane–cytoskeleton interactions. Motor proteins play an important role in the directed movement of organelle ...membranes along microtubules, but the basic mechanism by which membranes stably interact with the microtubule cytoskeleton is largely unknown. Here we report that p63, an integral membrane protein of the reticular subdomain of the rough endoplasmic reticulum (ER), binds microtubules in vivo and in vitro. Overexpression of p63 in cell culture led to a striking rearrangement of the ER and to concomitant bundling of microtubules along the altered ER. Mutational analysis of the cytoplasmic domain of p63 revealed two determinants responsible for these changes: an ER rearrangement determinant near the N‐terminus and a central microtubule‐binding region. The two determinants function independently of one another as indicated by deletion experiments. A peptide corresponding to the cytoplasmic tail of p63 promoted microtubule polymerization in vitro. p63 is the first identified integral membrane protein that can link a membrane organelle directly to microtubules. By doing so, it may contribute to the positioning of the ER along microtubules.
The endoplasmic reticulum (ER) of higher eukaryotic cells is a dynamic network of interconnected membrane tubules that pervades almost the entire cytoplasm. On the basis of the morphological changes ...induced by the disruption of the cytoskeleton or molecular motor proteins, the commonly accepted model has emerged that microtubules and conventional kinesin (kinesin‐1) are essential determinants in establishing and maintaining the structure of the ER by active membrane expansion. Surprisingly, very similar ER phenotypes have now been observed when the cytoskeleton‐linking ER membrane protein of 63 kDa (CLIMP‐63) is mutated, revealing stable attachment of ER membranes to the microtubular cytoskeleton as a novel requirement for ER maintenance. Additional recent findings suggest that ER maintenance also requires ongoing homotypic membrane fusion, possibly controlled by the p97/p47/VICP135 protein complex. Work on other proteins proposed to regulate ER structure, including huntingtin, the EF‐hand Ca2+‐binding protein p22, the vesicle‐associated membrane protein‐associated protein B and kinectin isoforms further contribute to the new emerging concept that ER shape is not only determined by motor driven processes but by a variety of different mechanisms.
ERGIC-53, a homo-oligomeric recycling protein associated with the ER-Golgi intermediate compartment (ERGIC), has properties of a mannose-selective lectin in vitro, suggesting that it may function as ...a transport receptor for glycoproteins in the early secretory pathway. To investigate if ERGIC-53 is involved in glycoprotein secretion, a mutant form of this protein was generated that is incapable of leaving the ER. If expressed in HeLa cells in a tetracycline-inducible manner, this mutant accumulated in the ER and retained the endogenous ERGIC-53 in this compartment, thus preventing its recycling. Mistargeting of ERGIC-53 to the ER did not alter the gross morphology of the early secretory pathway, including the distribution of β′-COP. However, it impaired the secretion of one major glycoprotein, identified as the precursor of the lysosomal enzyme cathepsin C, while overexpression of wild-type ERGIC-53 had no effect on glycoprotein secretion. Transport of two other lysosomal enzymes and three post-Golgi membrane glycoproteins was unaffected by inactivating the recycling of ERGIC-53. The results suggest that the recycling of ERGIC-53 is required for efficient intracellular transport of a small subset of glycoproteins, but it does not appear to be essential for the majority of glycoproteins.
The ER (endoplasmic reticulum) is a major protein folding and modification organelle. In its lumen, the ER processes a third of all newly synthesized proteins. To accomplish this task, numerous ...resident proteins capture the nascent and newly synthesized proteins. The underlying luminal protein-protein interactions, however, are inherently difficult to analyse, mainly due to their transient nature and the rather specialized environment of the ER. To overcome these limitations, we developed a PCA (protein fragment complementation assay) based on the citrine variant of YFP (yellow fluorescent protein). YFP PCA was successfully applied to visualize the protein interactions of the cargo transport receptor ERGIC-53 (endoplasmic reticulum-Golgi intermediate compartment protein of 53 kDa) with its luminal interaction partner MCFD2 (multiple coagulation factor deficiency protein 2) and its cargo proteins cathepsin Z and cathepsin C in a specific manner. With the prospect of screening cDNA libraries for novel protein-protein interactions, YFP PCA is a promising emerging technique for mapping protein interactions inside the secretory pathway in a genome-wide setting.
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