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.

A neuroprotective role for Ca 2+ -binding proteins in neurodegenerative conditions ranging from ischemia to Alzheimer's disease has been suggested in several studies. A key phenomenon in neurodegeneration is the Ca 2+ -mediated excitotoxicity brought about by the neurotransmitter glutamate. To evaluate the relative ability to resist excitotoxicity of neurons containing the slow-onset Ca 2+ -binding protein parvalbumin (PV), we injected the glutamate agonist ibotenic acid (IBO) into the striatum of adult mice ectopically expressing PV in neurons. Striatal ibotenic acid injection results in local nerve cell loss and reactive astrogliosis. Light microscopic evaluation, carried out after a delay of 2 and 4 weeks, reveals an enlarged and accelerated neurodegenerative process in mice ectopically expressing neuronal PV. Thus, PV is not neuroprotective, it rather enhances nerve cell death. This result implicates that the increase in cytosolic Ca 2+ -buffering capacity in the transgenic mice impairs other systems involved in Ca 2+ sequestration. In addition, ultrastructural morphometric analysis shows that in neurons the mitochondrial volume is reduced in mice ectopically expressing neuronal PV. This is paralleled by a reduction in the amount of the mitochondrial marker enzyme cytochrome c oxidase subunit I (COXI). We conclude that alterations in the Ca 2+ homeostasis present in mice ectopically expressing neuronal PV are more deleterious under excitotoxic stress and largely outweigh the potential benefits of an increased Ca 2+ -buffering capacity resulting from PV.

shuttle towards intracellular Ca 2+ sinks, like mitochondria and the endoplasmic reticulum. Constitutively, it is present in a subset of inhibitory neurones. In transgenic mice expressing pan-neuronal PV, the mitochondrial volume is reduced. We tested whether elevated levels of intraneuronal [Ca 2+ ] and reduced mitochondrial volume in the neurone interfere with the generation of parenchymal microcalcification. Methods: The striatum of wild type and transgenic mice was injected with the glutamate receptor agonist ibotenic acid (IBO), which is known to induce not only excitotoxic neurodegeneration, but also parenchymal calcification. Sections were studied by light and electron microscopy at various time points after IBO application. Results: Morphometric analysis 2, 4 and 20 weeks after IBO application revealed microcalcification in transgenic and wild type mice; the calcification process, however, was enhanced and accelerated in the transgenic animals. Ultrastructural analyses suggest neuronal mitochondria as the nucleators of the deposits which consist of hydroxyapatite. The time-dependent changes in size and surface structure of the deposits indicate the presence of biological mechanisms in the brain promoting regression of bioapatites. Conclusions: The overload of intraneuronal [Ca 2+ ] in combination with impaired mitochondrial function activates neuronal microcalcification. It is hypothesized that this process is an alternative/adaptive mechanism of the neurone to reduce further brain damage.

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Proinsulin is usually targetted to the regulated secretory pathway of beta cells, and converted to insulin in beta granules. Under certain pathological situations, a significant amount of proinsulin becomes diverted to the constitutive pathway. To study the kinetics of proinsulin conversion in the constitutive pathway, FAO (hepatoma) cells, which secrete proteins uniquely via this pathway and not the regulated pathway, were stably transfected with cDNA encoding human, rat I or rat II proinsulin. Products released to the medium of transfected cells were analysed by reversed phase HPLC and radioimmunoassay. For human proinsulin, des 31,32 split proinsulin (the conversion intermediate resulting from cleavage only at the B-chain/C-peptide junction followed by trimming of C-terminal basic residues by carboxypeptidase) was the only detectable conversion intermediate; for rat proinsulin II it was des 64,65 split proinsulin (cleaved and trimmed only at the C-peptide/A-chain junction); for rat proinsulin I, both intermediates were seen. Complete processing to insulin occurred for all three, but was most extensive for rat proinsulin I. When considered with the corresponding proinsulin sequences, these data show that a -4 basic residue (i.e. 4 residues N-terminal to the site of cleavage) facilitates proinsulin conversion in the constitutive pathway, and that arginine is preferred over lysine.

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the parts of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions. Transfected cells were incubated for four successive 2-h periods. The media were pooled, and pro-insulin, conversion intermediates, and insulin were separated by reverse-phase high-performance liquid chromatography to monitor conversion activity. There was little conversion of any proinsulin in COS cells without cotransfection of an exogenous endoprotease. When furin or PC3 was cotransfected with any of the three proinsulins, there was extensive processing, with insulin as the major conversion product. PC2, by contrast, failed to cleave human proinsulin but was able to cleave both human proinsulin Arg62 and rat proinsulin II. Cleavage by PC2 of these proinsulins was predominantly at the C-peptide-A-chain junction, generating the conversion intermediate des-64,65-split proinsulin as the major product and only very small amounts of insulin itself.

No abstract

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.