Post-translational processing of bovine chondromodulin-I

Chondromodulin-I (ChM-I) is a small glycoprotein that is abundant in fetal cartilage. Mature chondromodulin-I is processed from a larger precursor form, presumably at a proteolytic site RERR-ELVR. The precursor, mature chondromodulin-I and two processed products, the remnant left after removal of mature chondromodulin-I and a smaller, unglycosylated form, were identified using antipeptide antisera. The products of chondromodulin-I precursor processing were seen in cultured chondrocytes, a stable long-term culture chondrosarcoma cell line, as well as Chinese hamster ovary (CHO) cells transfected with an expression plasmid that contained cDNA coding for the chondromodulin-I precursor. Pulse-chase analysis allowed a processing pathway to be analyzed for chondromodulin-I. To further dissect the processing events, three constructs that express recombinant wild-type or mutant chondromodulin-I were transfected into CHO cells. We showed that chondromodulin-I is cleaved intracellularly at the predicted cleavage site, and that the mature glycopeptide is rapidly secreted immediately after processing. The chondromodulin-1 precursor has a short half-life and is not readily apparent in tissue samples, suggesting that chondromodulin is not a member of the juxtacrine family of growth factors, despite some similarities. The smaller unglycosylated form of chondromodulin-I was only observed in cartilage and not in short-term cultures or transfected cells, suggesting an extracellular processing event. No processing occurred when the precursor cleavage site was mutated to RERQ-SLVR or when precursor chondromodulin-I was expressed in the furin-deficient CHO cell line, suggesting the involvement of furin in processing.     

神经肽前体的翻译后加工

神经肽前体的翻译后加工是神经肽生物合成中的重要环节,它具有顺序专一性、组织特异性以及生理调控性。     

Post-translational processing and secretory pathway of human atriopeptin in rat pheochromocytoma cells.

Atriopeptin (AP) is expressed in several tissues with each tissue capable of specific differences in processing of the prohormone (pro-AP) to mature low molecular forms of the peptide. Since pro-AP has low biological activity, processing into mature AP is a critical activation event. This observation prompted us to study whether granule storage or regulated secretion of AP is essential for cleavage of mature peptide. We examined the processing of AP in adrenal medulla derived cells, using the rat pheochromocytoma cell line (PC12 cell) stably transfected with a genomic human AP DNA in the presence and absence of nerve growth factor (NGF), and also examined the mechanism of AP secretion and compared the results with those obtained using transfected chinese hamster ovary cells (CHO cells). The amount of prohormone was 5-10 fold higher than that of low molecular form of AP in the transfected PC12 cells. This ratio was essentially unchanged in differentiated PC12 cells after NGF treatment of the cells. Potassium depolarization of the transfected PC12 cells caused a 5-fold increase in AP release into the medium primarily as the intact prohormone. On the other hand, transfected CHO cells only exhibited constitutive AP release which is non-response to depolarization. These results suggest that the AP prohormone is sorted into secretory granules as the prohormone in PC12 cells and undergoes regulated release in response to depolarization indicating granule storage or release is not the critical determinant of AP prohormone cleavage.     

Post-translational processing of beta-secretase in Alzheimer's disease

Beta-amyloid is released into the brains of Alzheimer's patients, where it aggregates and causes damage to neurons. It is cleaved proteolytically from a large transmembrane glycoprotein amyloid precursor protein by a membrane-bound protease, known as beta-secretase identified previously as the acid protease, Asp-2. We have shown previously that beta-secretase is up-regulated by increased intracellular cholesterol, and down-regulated by cholesterol biosynthesis inhibition. Here we show using mass spectrometry that discrete changes in the glycosylation and palmitoylation of beta-secretase occur when cells expressing it are treated with statins.     

GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation

In rat frontal cortex, extracellular levels of glutamate are raised by the anti-psychotic drug clozapine. We have recently shown that a significant reduction in the levels of the glutamate transporter GLT-1 may be one of the mechanisms responsible for this elevation. Here we studied whether GLT-1 down-regulation induced by chronic clozapine treatment is associated with changes in the expression of synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicular glutamate transporter 1 (VGLUT1), three major presynaptic proteins involved in neurotransmitter release. Quantitative high-resolution confocal microscopy studies in vivo showed that GLT-1 down-regulation is closely associated with a significant increase in synaptophysin, but not SNAP-25 and VGLUT1, expression. This was confirmed in vitro studies, and in western blotting studies of synaptophysin, SNAP-25 and VGLUT1. In addition, our results show that, following clozapine treatment, synaptophysin expression increases in the very cortical regions in which GLT-1 expression is down-regulated. These findings suggest that part of the effects of clozapine may be exerted via an action on the presynaptic machinery involved in neurotransmitter release.     

Post-translational processing of the porcine gastrin precursor by phosphorylation of the COOH-terminal fragment

The gene sequence encoding porcine preprogastrin is known; in order to clarify pathways of post-translational processing of the predicted precursor peptide we have characterized material reacting with antibodies to a synthetic peptide corresponding to the expected extreme COOH-terminal portion of the precursor. Radioimmunoassay was used to identify and monitor the purification of peptides in porcine antral mucosa. Two peptides (I and II) were isolated to homogeneity by steps involving gel filtration, ion exchange, and reversed-phase high performance liquid chromatography. The two co-eluted on gel filtration but were separated on anion-exchange chromatography. The more acidic peptide (II) was less hydrophobic on high performance liquid chromatography. Automated gas-phase microsequencing revealed the less acidic peptide (I) to have the sequence of porcine preprogastrin 96-104 (SAEEGDQRP); it would be produced by tryptic-like cleavage of Arg95-Ser96. The second peptide did not yield a phenylthiohydantoin-derivative on the first cycle but thereafter it sequenced as the first peptide (i.e. -AEEGDQRP). Incubation in alkali liberated almost equimolar amounts of phosphate from peptide II but not from I. In addition, alkaline phosphatase liberated phosphate and converted the acidic peptide to the less acidic one. The results suggest that serine in the first position is phosphorylated in peptide II but not I. The tripeptide -Ser(P)-Ala-Glu- also occurs in adrenocorticotropic hormone; this tripeptide is a substrate for physiological casein kinase. Potential phosphorylation sites occur at comparable positions in the precursors of a number of regulatory peptides.     

Post-translational processing of Drosophila nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDPK) was purified from Drosophila melanogaster by a combination of anion-exchange, hydroxyapatite, and reversed-phase chromatography. The identity of the purified enzyme was confirmed by sequencing internal peptides (the N-terminus appeared to be blocked). Post-translational modifications were investigated by using protein chemical and mass spectrometric methods. Analysis by nanoelectrospray ionization-mass spectrometry revealed that the mass of the enzyme was considerably smaller than that predicted from its amino acid sequence. Although its open-reading frame predicts a 153-residue polypeptide, the mature enzyme was found to comprise 152 amino acids, being modified by proteolytic removal of the initiator Met and N-acetylation of Ala2. This explains why the observed pI of the Drosophila enzyme is more acidic than that predicted from its amino acid sequence. No additional post-translational modifications such as glycosylation or O-phosphorylation, which have been identified on homologous NDPKs from other organisms, were detected on the Drosophila enzyme.     

Peptide correction of memory disrupted by food deprivation in the rat

Food deprivation worsens elaboration of conditioned active avoidance in white rats. Arginil-vasopressin positively influences their conditioned memory disturbed by food deprivation. Changes of specific content of hybrid-bound and general RNA occur in the neocortex and cerebellum under influences different by their functional specificity, such as elaboration of active avoidance, food deprivation, administration of vasopressin and their combinations.     

Post-translational processing in Xenopus oocytes includes carboxyl-terminal amidation

Xenopus oocytes are versatile cells capable of carrying out many post-translational processes. Although previously reported not to be capable of C-terminal amidation, this report demonstrates that Xenopus oocytes do indeed have an amidating enzyme. The amidating activity from Xenopus ovaries is compared to the known amidating activity found in porcine pituitaries. The demonstration of C-terminal amidation by Xenopus oocytes extends their usefulness in studying post-translational events.     

Post-translational processing of urea amidolyase in Saccharomyces cerevisiae.

Urea amidolyase catalyzes the two reactions (urea carboxylase and a allophanate hydrolase) associated with urea degradation in Saccharomyces cerevisiae. Past work has shown that both reactions are catalyzed by a 204-kilodalton, multifunctional protein. In view of these observations, it was surprising to find that on induction at 22 degrees C, approximately 2 to 6 min elapsed between the appearance of allophanate hydrolase and urea carboxylase activities. In search of an explanation for this apparent paradox, we determined whether or not a detectable period of time elapsed between the appearance of allophanate hydrolase activity and activation of the urea carboxylase domain by the addition of biotin. We found that a significant portion of the protein produced immediately after the onset of induction lacked the prosthetic group. A steady-state level of biotin-free enzyme was reached 16 min after induction and persisted indefinitely thereafter. These data are consistent with the suggestion that sequential induction of allophanate hydrolase and urea carboxylase activities results from the time required to covalently bind biotin to the latter domain of the protein.     

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.     

Parallel processing in the mammalian retina

Our eyes send different 'images' of the outside world to the brain - an image of contours (line drawing), a colour image (watercolour painting) or an image of moving objects (movie). This is commonly referred to as parallel processing, and starts as early as the first synapse of the retina, the cone pedicle. Here, the molecular composition of the transmitter receptors of the postsynaptic neurons defines which images are transferred to the inner retina. Within the second synaptic layer - the inner plexiform layer - circuits that involve complex inhibitory and excitatory interactions represent filters that select 'what the eye tells the brain'.     

Filipin-sterol complexes in disrupted myelin in the rat

Using filipin as a cytochemical probe for cholesterol we have compared the distribution of filipin labelling in mildly disrupted myelin and normal myelin. The myelin lamellae in rat sciatic nerve were separated either by hypotonic saline (0.035-0.07 M) or nerve section (24-32 h) before aldehyde fixation and filipin treatment. Myelin separation was assessed in ultrathin sections and filipin distribution in freeze-fracture replicas. In separated myelin lamellae filipin labelling was similar throughout the myelin sheath while in normal control myelin filipin occurred most in the outer (abaxonal), least in the inner (adaxonal) and intermediate in the middle lamellae. It is concluded that this heterogeneous filipin labelling in normal myelin is a result of diffusion gradients to filipin within the myelin sheath and that in vivo cholesterol is uniformly distributed throughout all the lamellae of the myelin sheath. The site of the diffusion barrier to filipin within normal myelin is considered.     

Post-translational processing of Schizosaccharomyces pombe YPT proteins.

ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation. These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization. The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic. These proteins end in XCC or CXC motifs. We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe. We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from [3H]mevalonic acid when expressed in transfected COS cells in vivo. Furthermore, prenylation was necessary for membrane binding in vivo. The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S. pombe and in COS cells in vivo. However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein. YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras.     

Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis.

We have studied the post-translational processing of p21ras proteins. The primary translation product pro-p21 is cytosolic and is rapidly converted to a cytosolic form (c-p21) of higher mobility on SDS-PAGE. c-p21 is converted in turn to the membrane-bound mature palmitoylated form (m-p21) of slightly higher mobility. These processing steps are accompanied by increases in isoelectric point and in hydrophobicity as judged by Triton X-114 partitioning. Although the increases in electrophoretic mobility and hydrophobicity precede acylation we show that mutation of Cys186, which has been shown to block acylation, also abolishes the pro-p21 to c-p21 conversion. Thus the Cys186 residue is involved in the processing steps prior to acylation. We have identified two processing events which contribute to the pro-p21 conversion. Site-directed mutagenesis to insert tryptophan, which is not present in the wild type, followed by metabolic labelling with [3H]tryptophan has allowed us to map a proteolytic processing event which removes the three C-terminal residues. In addition, both the c-p21 and m-p21 forms are carboxyl-methylated. Approximately one methyl group is incorporated per molecule of p21 at steady state, which can partially account for the increase in isoelectric point. Unlike palmitate, methyl group turnover is not observed.     

Post-translational modification and proteolytic processing of urinary osteopontin

H(2)O(2) is a relatively long-lived reactive oxygen species that signals between cells and organisms. H(2)O(2) signalling in plants is essential for response to stress, defence against pathogens and the regulation of programmed cell death. Although H(2)O(2) diffusion across membranes is often considered as a passive property of lipid bilayers, native membranes represent significant barriers for H(2)O(2). In the present study we addressed the question of whether channels might facilitate H(2)O(2) conduction across plasma membranes. The expression of several plant plasma membrane aquaporins in yeast, including PIP2;1 from Arabidopsis (where PIP is plasma membrane intrinsic protein), enhanced the toxicity of H(2)O(2) and increased the fluorescence of dye-loaded yeast when exposed to H(2)O(2). The sensitivity of aquaporin-expressing yeast to H(2)O(2) was altered by mutations that alter gating and the selectivity of the aquaporins. The conduction of water, H(2)O(2) and urea was compared, using molecular dynamics simulations based on the crystal structure of SoPIP2;1 from spinach. The calculations identify differences in the conduction between the substrates and reveal channel residues critically involved in H(2)O(2) conduction. The results of the calculations on tetramers and monomers are in agreement with the biochemical data. Taken together, the results strongly suggest that plasma membrane aquaporin pores determine the efficiency of H(2)O(2) signalling between cells. Aquaporins are present in most species and their capacity to facilitate the diffusion of H(2)O(2) may be of physiological significance in many organisms and particularly in communication between different species.