Endogenous cleavage of annexin I generates a truncated protein with a reduced calcium requirement for binding to neutrophil secretory vesicles and plasma membrane

We have earlier shown that an N-terminal truncated annexin I molecule, annexin I(des1-8), is generated in human neutrophils through cleavage by a membrane localized metalloprotease. The truncated protein showed differences in membrane binding among the neutrophil granule populations as compared to full-length annexin I. In this study, we investigated the cleavage capabilities of isolated neutrophil secretory vesicles and plasma membrane, and the binding of full-length annexin I and annexin I(des1-8) to these membrane fractions. Translocations were performed in vitro to secretory vesicles and plasma membrane, respectively, at different Ca(2+) concentrations. We show that the annexin I-cleaving membrane localized metalloprotease is present both in the secretory vesicles and the plasma membrane. The N-terminal truncation of annexin I gives rise to a molecule with a decreased Ca(2+) requirement for binding, both to secretory vesicles and plasma membrane. There was, thus, no difference in binding of either full-length annexin I or annexin I(des1-8) to the secretory vesicles as compared to the plasma membrane.     

TatD is a cytoplasmic protein with DNase activity. No requirement for TatD family proteins in sec-independent protein export

The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.     

Isoprenoid requirement for intracellular transport and processing of murine leukemia virus envelope protein.

Lovastatin blocks the biosynthesis of the isoprenoid precursor, mevalonate. When Friend murine erythroleukemia (MEL) cells are cultured in medium containing lovastatin, the precursor of murine leukemia virus envelope glycoprotein (gPr90env) fails to undergo proteolytic processing, which normally occurs in the Golgi complex. Consequently, newly synthesized envelope proteins are not incorporated into viral particles that are shed into the culture medium. gPr90env appears to be localized in a pre-Golgi membrane compartment, based on its enrichment in subcellular fractions containing NADPH-cytochrome c reductase activity and the sensitivity of its carbohydrate chains to digestion with endoglycosidase H. Arrest of gPr90env processing occurs at concentrations of lovastatin that are not cytostatic, and the effect of the inhibitor is prevented by addition of mevalonate to the medium. The low molecular mass GTP-binding proteins, rab1p and rab6p, which are believed to function in early steps of the exocytic pathway, are normally modified posttranslationally by geranylgeranyl isoprenoids. However, in MEL cells treated with 1 microM lovastatin, nonisoprenylated forms of these proteins accumulate in the cytosol prior to arrest of gPr90env processing. These observations suggest that lovastatin may prevent viral envelope precursors from reaching the Golgi compartment by blocking the isoprenylation of rab proteins required for ER to Golgi transport.     

Differential requirement for Caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing

Activation of the cysteine protease Caspase-1 is a key event in the innate immune response to infections. Synthesized as a proprotein, Caspase-1 undergoes autoproteolysis within multiprotein complexes called inflammasomes. Activated Caspase-1 is required for proteolytic processing and for release of the cytokines interleukin-1β and interleukin-18, and it can also cause rapid macrophage cell death. We show that macrophage cell death and cytokine maturation in response to infection with diverse bacterial pathogens can be separated genetically and that two distinct inflammasome complexes mediate these events. Inflammasomes containing the signaling adaptor Asc form a single large "focus" in which Caspase-1 undergoes autoproteolysis and processes IL-1β/IL-18. In contrast, Asc-independent inflammasomes activate Caspase-1 without autoproteolysis and do not form any large structures in the cytosol. Caspase-1 mutants unable to undergo autoproteolysis promoted rapid cell death, but processed IL-1β/18 inefficiently. Our results suggest the formation of spatially and functionally distinct inflammasomes complexes in response to bacterial pathogens.     

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.     

Conformation and length of the signal sequence affect processing of secretory protein.

Processing of human lysozyme with artificially designed signal sequences was examined in an in vitro translation-translocation system and compared with their secretory capabilities in yeast. It has been shown that the conformation of the C-terminal region of the signal sequence and the length of the hydrophobic segment are important factors for efficient cleavage of the signal sequence.     

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. II. Identification of conserved sites for protein processing

The guinea pig seminal vesicle epithelium is an androgen-dependent tissue that synthesizes and secretes four major secretory proteins (SVP-1, SVP-2, SVP-3, and SVP-4). Sequencing of near full-length cDNA clones corresponding to the two most abundant mRNAs produced by the seminal vesicle reveals that all four secretory proteins are cleaved from two secretory protein precursors. Amino acid sequences from purified SVP-2 match the central region of the predicted amino acid sequences from the smaller cDNA clone, GP2 (581 nucleotides). Similar analysis demonstrates that the predicted amino acid sequence from the longer cDNA clone, GP1 (1368 nucleotides), codes for the related proteins SVP-3 and SVP-4 as well as SVP-1. The 43.2 kilodalton polyprotein precursor coded by GP1 contains two different sets of 24 amino acid tandemly repeated sequences. The two secretory protein precursors have extensive regions of peptide sequence homology, particularly in regions where protein processing must occur to produce the mature secretory proteins. Analysis of the predicted secondary structure of the two precursor polypeptides revealed a strong correlation between structural features and sites of protein processing.     

Regulated and constitutive secretion. Differential effects of protein synthesis arrest on transport of glycosaminoglycan chains to the two secretory pathways.

Many neural and endocrine cells possess two pathways of secretion: a regulated pathway and a constitutive pathway. Peptide hormones are stored in granules which undergo regulated release whereas other surface-bound proteins are externalized constitutively via a distinct set of vesicles. An important issue is whether proper function of these pathways requires continuous protein synthesis. Wieland et al. (Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) Cell 50, 289-300) have shown that a tripeptide containing the sequence Asn-Tyr-Thr can be glycosylated in intracellular compartments and secreted efficiently from Chinese hamster ovary and HepG2 cells, presumably via the constitutive secretory pathway. Secretion is not affected by cycloheximide, suggesting that operation of this pathway does not require components supplied by new protein synthesis. In this report we determined the effects of protein synthesis inhibitor on membrane traffic to the regulated secretory pathway in the mouse pituitary AtT-20 cells. We examined transport of glycosaminoglycan chains since previous studies have shown that these chains enter the regulated secretory pathways and are packaged along with the hormone adrenocorticotropin (ACTH). We found that cycloheximide treatment severely impairs the cell's ability to store and secrete glycosaminoglycan chains by the regulated secretory pathway. In marked contrast, constitutive secretion of glycosaminoglycan chains remains unhindered in the absence of protein synthesis. The differential requirements for protein synthesis indicate differences in the mechanisms for sorting and/or transport of molecules through the constitutive and the regulated secretory pathways. We discuss the possible mechanisms by which protein synthesis may influence trafficking of glycosaminoglycan chains to the regulated secretory pathway.     

A calcium requirement for movement of cultured cells.

When calcium is removed from culture medium, motility of cultured cells is decreased. The effect is rapid, reversible and pronounced. Decreased motility is observed with normal mouse Balb/c 3T3 cells, mouse L929 cells, rat kidney fibroblasts and chick embryo fibroblasts. The calcium dependence of movement can be observed both with individual cells and with the movement of the margin of a monolayer into a wound. Magnesium will not substitute for calcium to maintain motility. Strontium will substitute, but is not as effective as calcium for maintaining cell movement. Low concentrations of the divalent cation ionophore A23187 (0.5-1 micron) partially reverse the reduced migration observed at low calcium concentrations. These results are consistent with the hypothesis that movement of non-muscle cells occurs through mechanisms similar to those important in the contraction of muscle.     

Differential bile-pancreatic secretory effects of CCK-58 and CCK-8

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.     

Differential effects of extracellular calcium removal and nonspecific effects of Ca2+ antagonists on acid secretory activity in isolated gastric glands

The role of extracellular calcium in the action of the secretagogues, carbachol, histamine and forskolin, on parietal cell HCl secretion was investigated using glands isolated from rabbit gastric mucosa. Omission of calcium from the cellular incubation medium and chelation of a major portion of contaminating calcium with EGTA resulted in a disappearance of the initial transient response to carbachol (as measured by uptake of the weak base, amino[14C]pyrine), but the sustained response to carbachol persisted. Neither histamine nor forskolin-stimulated increase in amino[14C]pyrine uptake were affected by omission of extracellular calcium. Furthermore, the potentiating interactions between histamine and carbachol and between forskolin and carbachol appeared to occur independent of extracellular calcium. Attempts to assess the contribution of intracellular calcium to secretory activity using the Ca2+ antagonists, verapamil, nifedipine, nicardipine and lanthanum, and the putative intracellular Ca2+ antogonist, TMB-8 (3,4,5-trimethyloxybenzoic acid 8-(diethyl-amino)-octyl ester) were unsuccessful. Nifedipine had no effect on secretagogue stimulated amino[14C]pyrine accumulation even at concentration well above the pA2 reported for excitable tissues. Verapamil, nicardipine, lanthanum and TMB-8 all appeared to have nonspecific inhibitory effects on amino [14C]pyrine uptake. From these results we conclude that: (1) parietal cell HCl secretion can occur independent of extracellular Ca2+; (2) influx of extracellular Ca2+ enhances the response to carbachol but has little influence on the secretory response initiated by cAMP-dependent secretagogues; and (3) parietal cell Ca2+ channels have a different molecular configuration than Ca2+ channels in excitable cells.     

Structural requirement at the cleavage site for efficient processing of the lipoprotein secretory precursor of Escherichia coli

A phenotypically silent mutation in the signal peptide of the Escherichia coli outer membrane prolipoprotein was combined with other mutations in the mature lipoprotein structure. Under conditions where the individual mutations permit normal lipoprotein secretion, the prolipoprotein with both mutations was unable to be normally modified or processed. These results demonstrate that a given signal peptide is fully functional only if it is structurally compatible with the protein to be secreted. This structural compatibility between the signal peptide and the secretory protein is considered to be dependent on the secondary structure formed at or near the signal peptide cleavage site.     

Differential effects of monensin on the plant secretory pathway

The sodium ionophore monensin was used as an inhibitor of Golgifunction to study the secretory event in suspension-culturedsycamore cells. The morphological changes induced by monensinwere recorded. Concurrent studies of the disturbance causedby the drug were carried out by biochemical, cytochemical andimmunocytochemical techniques. Monensin induced accumulationof smooth and coated vesicles in the vicinity of the Golgi apparatus,swelling of Golgi cisternae and also provoked the formationof extracytoplasmic pockets between the plasma membrane andthe cell wall. Cytochemical tests for polysaccharides and immunolocalizationof secreted pectins, recognized by the monoclonal antibody JIM7, were performed, and the results indicated that the biosynthesisand/or processing machinery of polysaccharides was affectedby the drug. In contrast, immunolocalization of glycoproteins,recognized by the monoclonal antibodies JIM 84 and JIM 13 demonstratedthat the accumulation of vesicles in the cytoplasm were Golgiderived, and that the secretion of glycoproteins was not drasticallyaffected by monensin. Fluorography of radiolabelled proteinsdemonstrated that in this system the secretion of proteins wasnot qualitatively changed by monensin. The implications of theseresults are discussed in the light of the current hypotheseson the effects of monensin on the secretory pathway. Key words: Golgi apparatus, immunocytochemistry, monensin, secretion     

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. I. Evidence for androgen-regulated proteolytic processing

The guinea pig seminal vesicle epithelium synthesizes and secretes four major secretory proteins (SVP-1-4). Previous work has established that these four proteins are cleaved from two primary translation products in a complex series of protein processing reactions. The present studies suggest that these protein processing reactions are regulated by androgens. In vitro labeling of seminal vesicle proteins revealed significant differences in the patterns of secretory protein intermediates produced by tissue from intact and castrated animals. Seminal vesicle tissue explants from castrated animals secreted a subset of the processing intermediates secreted by tissue from intact animals. The changes in the patterns of secretory protein intermediates became more pronounced with increasing time after castration, and were fully reversible by treatment of castrated animals with testosterone, suggesting that androgens were affecting the processing or secretion of secretory protein precursors. Amino-terminal protein sequencing of secretory protein processing intermediates that accumulate in the seminal vesicle lumen after castration suggests that the guinea pig seminal vesicle contains an androgen-regulated proteolytic processing activity.     

Immediate entrance to the export pathway after synthesis as a requirement for export of the sak gene product in Escherichia coli.

Export through the cytoplasmic membrane and processing of the sak product in Escherichia coli cells were investigated with E. coli strains carrying pTS301, which produce large amounts of staphylokinase at 42 degrees C. High-level synthesis of the sak product caused transient accumulation not only of the staphylokinase precursor (pSAK) but also of the maltose-binding protein and outer membrane protein A precursors. Thus it was concluded that the sak product shares the export pathway with E. coli secreted proteins at least at a certain step. During high-level synthesis of the sak product, a significant amount of the newly synthesized pSAK remained unprocessed after a chase period, possibly causing the observed accumulation of pSAK. Accumulating pSAK did not mature for a long period, whereas the newly synthesized sak product was exclusively detected in the mature form. These results suggest that it is necessary for the sak product to enter the export pathway during or immediately after synthesis to be exported and processed normally.     

Loss of a calcium requirement for protein synthesis in pituitary cells following thermal or chemical stress

Ca2+ is required for the maintenance of high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). Following thermal stress at 46 degrees C or chemical stress from exposure to sodium arsenite or 8-hydroxyquinoline, rates of amino acid incorporation in Ca2+-restored GH3 cells were reduced acutely to those of unstressed, Ca2+-depleted control preparations. Sodium arsenite treatment resulted in loss of ability to accumulate polysomes in response to Ca2+. Stressed cells allowed to recover for 2-8 h either with or without Ca2+ in the medium exhibited comparable, increasing rates of amino acid incorporation and the induction of heat shock proteins (hsp). Abolition of the Ca2+-dependent component of translation was proportional to the intensity of the stress. Mild thermal stress (41 degrees C) resulted in the induction of hsp 68 and the retention of Ca2+-dependent protein synthesis; hsp 68 was synthesized in a Ca2+-dependent manner. After arsenite stress, restoration of the Ca2+ requirement for protein synthesis occurred by 24 h, and was preceded by a transitional period during which polysomes accumulated in response to Ca2+ without concomitant increased rates of incorporation. Responses to stress are proposed to include an acute inhibition of normal protein synthesis involving the destruction of Ca2+-stimulated initiation and a protracted period of recovery involving synthesis of the hsp accompanied by Ca2+-independent amino acid incorporation and slowed peptide chain elongation.     

Prostate secretory protein 94 inhibits sterol binding and export by the mammalian CAP protein CRISP2 in a calcium-sensitive manner

Members of the CAP protein superfamily are present in all kingdoms of life and have been implicated in many different processes, including pathogen defense, immune evasion, sperm maturation, and cancer progression. Most CAP proteins are secreted glycoproteins and share a unique conserved αβα sandwich fold. The precise mode of action of this class of proteins, however, has remained elusive. Saccharomyces cerevisiae has three CAP family members, termed pathogen related in yeast (Pry). We have previously shown that Pry1 and Pry2 export sterols in vivo and that they bind sterols in vitro. This sterol binding and export function of yeast Pry proteins is conserved in the mammalian CRISP proteins and other CAP superfamily members. CRISP3 is an abundant protein of the human seminal plasma and interacts with prostate secretory protein of 94 amino acids (PSP94), another major protein component in the seminal plasma. Here we examine whether the interaction between CRISP proteins and PSP94 affects the sterol binding function of CAP family members. We show that coexpression of PSP94 with CAP proteins in yeast abolished their sterol export function and the interaction between PSP94 and CAP proteins inhibits sterol binding in vitro. In addition, mutations that affect the formation of the PSP94–CRISP2 heteromeric complex restore sterol binding. Of interest, we found the interaction of PSP94 with CRISP2 is sensitive to high calcium concentrations. The observation that PSP94 modulates the sterol binding function of CRISP2 in a calcium-dependent manner has potential implications for the role of PSP94 and CRISP2 in prostate physiology and progression of prostate cancer.     

Insulin-like effects of dithiothreitol on isolated rat adipocytes.

The rate of 3H2O incorporation into lipid in vivo progressively decreased in liver but increased in parametrial adipose tissue during the last 3 days of gestation. These changes seem to be related to those occurring in plasma insulin and progesterone concentrations during the same period. Foetal liver showed a high rate of lipogenesis, which sharply decreased before parturition. foetal lung lipogenesis increased during days 20 and 21 of gestation.