Biomedical subjects

D Shields

Publications and source records attributed to D Shields.

At least 37 records · Page 2Linked to original sources

PubMed1996

Formation of secretory vesicles in permeabilized cells: a salt extract from yeast membranes promotes budding of nascent secretory vesicles from the trans-Golgi network of endocrine cells.

The mechanism of secretory-vesicle formation from the trans-Golgi network (TGN) of endocrine cells is poorly understood. To identify cytosolic activities that facilitate the formation and fission of nascent secretory vesicles, we treated permeabilized pituitary GH3 cells with high salt to remove endogenous budding factors. Using this cell preparation, secretory-vesicle budding from the TGN required addition of exogenous cytosol and energy. Mammalian cytosols (GH3 cells and bovine brain) promoted post-TGN vesicle formation. Most significantly, a salt extract of membranes from the yeast Saccharomyces cerevisiae, a cell lacking a regulated secretory pathway, stimulated secretory vesicle budding in the absence of mammalian cytosolic factors. These results demonstrate that the factors which promote secretory-vesicle release from the TGN are conserved between yeast and mammalian cells.

Animals↗

PubMed1996

ADP-ribosylation factor-1 stimulates formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells.

ADP-ribosylation factor (ARF) is a small GTP-binding protein that has been implicated in intracellular vesicular transport. ARF regulates the budding of vesicles that mediate endoplasmic reticulum to Golgi and intra-Golgi transport. It also plays an important role in maintaining the function and morphology of the Golgi apparatus. Using a permeabilized cell system derived from GH3 cells, we provide evidence that ARF-1 regulates the formation of nascent secretory vesicles from the trans-Golgi network. Both myristoylated and non-myristoylated forms of recombinant human ARF-1 enhanced secretory vesicle budding about 2-fold. A mutant lacking the first 17 N-terminal residues, as well as one that preferentially binds GDP (T31N) did not stimulate vesicle formation. In contrast, a mutant defective in GTP hydrolysis (Q71L) promoted vesicle budding. Strikingly, a peptide corresponding to the N terminus of human ARF-1 (amino acids 2-17) also stimulated vesicle budding from the trans-Golgi network, in marked contrast to its inhibitory effect on vesicular transport from the endoplasmic reticulum to Golgi. These data demonstrate that in endocrine cells, ARF-1 and in particular its N terminus play an essential role in the formation of secretory vesicles.

ADP-Ribosylation Factors↗

PubMed1996

Prosomatostatin processing in permeabilized cells. Calcium is required for prohormone cleavage but not formation of nascent secretory vesicles.

Our laboratory has been using a permeabilized cell system derived from rat anterior pituitary GH3 cells expressing prosomatostatin (pro-SRIF) to study prohormone processing and nascent secretory vesicle formation in vitro. Because calcium is necessary for prohormone processing enzyme activity, secretory granule fusion with the plasma membrane, and possibly sorting to the regulated pathway, we treated permeabilized cells with the calcium ionophore A23187 to determine the role of calcium in pro-SRIF cleavage and nascent vesicle formation from the trans-Golgi network (TGN). Here we demonstrate that pro-SRIF cleavage was markedly inhibited when lumenal free calcium was chelated with EGTA in the presence of A23187. Surprisingly, submillimolar free calcium (approximately 15 microM) was sufficient to maintain prohormone cleavage efficiency, a value far lower than that estimated for total calcium levels in the TGN and secretory granules. Experiments using both A23187 and the protonophore CCCP revealed that free calcium is absolutely required for efficient pro-SRIF cleavage, even at the optimal pH of 6.1. Secretory vesicle formation by contrast was not inhibited by calcium chelation but rather by millimolar extralumenal free calcium. Together, these observations demonstrate that pro-SRIF processing and budding of nascent secretory vesicles from the TGN can be uncoupled and therefore have distinct biochemical requirements. Interestingly, our data using intact GH3 cells demonstrate that basal secretion of SRIF-related material is largely calcium-dependent and therefore cannot be equated with constitutive pathway secretion. These results underscore the importance of determining calcium requirements before assigning a secretion event to either the constitutive or regulated secretory pathway.

Amino Acid Sequence↗

PubMed1996

Fusion and confusion in the secretory pathway.

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Journal Article↗

PubMed1996

Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells.

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.

3T3 Cells↗

PubMed1996

Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.

ADP-Ribosylation Factor 1↗

PubMed1996

Topogenic effect of positively charged N-terminal amino acid in ER translocation of yeast alpha-factor precursor.

Expression of fusion proteins between prepro-alpha-factor and somatostatin (SRIF) in yeast, resulted in the correct processing and secretion of the heterologous 14-amino acid SRIF peptide (1). When the chimeric genes were placed under the control of yeast acid phosphatase (PHO5) promoter, significant amount of an unglycosylated form of the fusion precursor molecule accumulated intracellularly, suggesting disruption of an endoplasmic reticulum-mediated function. We report here that the appearance of the precursor is due to an alteration in the three amino terminal residues of the chimera, i.e., Met-Arg-Phe in native prepro-alpha-factor is changed to Met-Phe-Lys in the hybrids. The unglycosylated precursor represents a population of molecules that are disrupted at an early stage of targeting to or translocation across the endoplasmic reticulum membrane. Our data demonstrate that the N-terminus plays an important role in topogenesis. Furthermore, these results show that translocation and glycosylation can be uncoupled from protein synthesis in vivo, and therefore can be posttranslational events in yeast.

Amino Acids↗

PubMed1995

The propeptide of anglerfish preprosomatostatin-I rescues prosomatostatin-II from intracellular degradation.

Polypeptide hormones and neuropeptides are initially synthesized as precursors possessing one or several domains that constitute the propeptide. Previous work from our laboratory demonstrated that expression of anglerfish prosomatostatin-I (proSRIF-I) in rat anterior pituitary GH3 cells resulted in efficient and accurate cleavage of the prohormone to generate the mature 14-amino acid peptide, SRIF-I. We also implicated the propeptide in mediating intracellular sorting to the trans Golgi network where proteolytic processing is initiated. In contrast, expression of a second form of the precursor, proSRIF-II in GH3 cells resulted in its intracellular degradation in an acidic, post-trans Golgi network compartment, most probably lysosomes. To further investigate the positive sorting signal present in proSRIF-I, we constructed a chimera comprising the signal peptide and proregion of SRIF-I fused to proSRIF-II and expressed the cDNA in GH3 cells. Here we demonstrate that the propeptide of SRIF-I rescued proSRIF-II from intracellular degradation quantitatively and diverted it to secretory vesicles. Furthermore, the chimera was processed to SRIF-28, an amino-terminally extended form of the hormone that is the physiological cleavage product of proSRIF-II processing in vivo. Most significantly, the SRIF-I propeptide functioned only in cis as part of the fusion protein and not in trans when expressed as a separate polypeptide. These data suggest that the SRIF-I propeptide may possess a sorting signal for sequestration into the secretory pathway rather than functioning as an intramolecular chaperone to promote protein folding.

Amino Acid Sequence↗

PubMed1995

Inpatient and post-discharge wound infections in general surgery.

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Humans↗

PubMed1994

Prosomatostatin processing in pituitary GH3 cells. Identification and secretion of the intact propeptide.

Preprosomatostatin (preproSRIF) is a peptide hormone precursor that undergoes tissue-specific processing at either a single set of paired basic residues to yield SRIF-14 or, alternatively, at a monobasic site to produce SRIF-28, an NH2 terminally extended form of SRIF-14. Mammalian preproSRIFs are a family of precursors that are remarkably conserved from rat to humans. In five species, the signal peptide and propeptides are approximately 96% identical; this high degree of sequence identity may be indicative of functional conservation. Since the propeptide is approximately five times larger than SRIF-14, we hypothesized that it would be secreted as a separate polypeptide following proSRIF proteolytic processing. To test this idea, we raised polyclonal antibodies to the entire propeptide to follow its biosynthesis and secretion. Here we demonstrate that in transfected rat anterior pituitary GH3 cells both SRIF-14 and the intact 9.5-kDa propeptide were processed coordinately from proSRIF with identical kinetics. Treatment of the cells with chloroquine, a weak base which inhibits processing to mature SRIF-14, also inhibited the appearance of the 9.5-kDa propeptide. Approximately 40% of the propeptide was targeted to the regulated secretory pathway as determined by its quantitative secretion in response to secretagogues. We also examined the secretion of the SRIF propeptide independently of SRIF-14 by expressing a truncated "propeptide" in which SRIF-14 was deleted. Significantly, the SRIF propeptide was itself efficiently transported through the secretory pathway and secreted into the culture medium. This suggests that the propeptide possesses all the topogenic information necessary for intracellular transport. The coordinate secretion of the intact propeptide with mature SRIF-14 suggests that it might function as a novel bioactive peptide.

Amino Acid Sequence↗

PubMed1994

Role of gamma-glutamyltranspeptidase-mediated glutathione transport on the radiosensitivity of B16 melanoma variant cell lines.

PURPOSE: To elucidate the role of gamma-glutamyltranspeptidase-mediated glutathione transport on the radiosensitivity of B16 melanoma variant cell lines. METHODS AND MATERIALS: B16 melanoma variant cell lines were examined for their levels of gamma-glutamyltranspeptidase (GGTP; E.C. 2.3.2.2), a plasma membrane-associated ectoenzyme that is involved in the transport of extracellular glutathione, by flow cytometric and biochemical analysis. B16 cell lines were examined for rates of de novo glutathione synthesis from extracellular glutathione and for their sensitivity to gamma-irradiation and glutathione synthesis inhibition. The GGTP inhibitors were examined for their effect on the radiosensitivity of B16 melanoma cells. RESULTS: B16-F10-BL6 (BL6) melanoma cells were shown to express a 20-fold higher level of GGTP than the B16-F1 melanoma variant cells. Cultures of BL6 and B16-F1 cells depleted extracellular glutathione at rates of 2.4 and < 0.1 nmol glutathione/10(6) cells/h, respectively, and BL6 cells exported glutathione at a rate 7.2-fold higher than B16-F1 cells (710 and 98 pmol glutathione/10(6) cells/h, respectively). BL6 melanoma cells replenished exhausted intracellular glutathione levels from an extracellular glutathione source at a rate of 1.21 nmol glutathione/h (18% basal glutathione/h); however, B16-F1 cells lacked the capacity to replenish intracellular glutathione despite the presence of exogenous glutathione in the culture medium. BL6 melanoma cells were radioresistant compared to the B16-F1 cell line, exhibiting extrapolation numbers (ñ) of 14.9 and 1.0, respectively, and a lower surviving fraction to a wide range of radiation doses. The GGTP inhibitor combination of L-serine and sodium borate blocked the repletion of intracellular glutathione and in the presence or absence of buthionine sulfoximine-mediated depletion of glutathione reverses the radiation resistance in BL6 melanoma cells to near baseline levels observed with the B16-F1 parent clone. Serine-borate treatment of low-GGTP expressing B16-F1 cells had no effect on the ñ value or the surviving fraction of cells to a range of ionizing irradiation doses. CONCLUSIONS: These results suggest that GGTP plays an important role in the extracellular metabolism and transport of glutathione, which also provides radioresistance to BL6 melanoma cells in vitro.

Animals↗

PubMed1994

Prosomatostatin processing in permeabilized cells. Endoproteolytic cleavage is mediated by a vacuolar ATPase that generates an acidic pH in the trans-Golgi network.

To investigate the relationship between prohormone processing and sorting of mature polypeptides into nascent secretory vesicles, we recently developed a permeabilized cell system that supports both these reactions (Xu, H., and Shields, D. (1993) J. Cell Biol. 122, 1169-1184). Rat anterior pituitary GH3 cells expressing high levels of prosomatostatin (proSRIF) were incubated at 20 degrees C; this temperature prevented exit from the trans-Golgi network and inhibited proSRIF processing. Following the 20 degrees C block, the cells were mechanically permeabilized and incubated at 37 degrees C, and proSRIF processing was determined. Cleavage of proSRIF to the mature hormone required ATP hydrolysis and was inhibited by chloroquine, a weak base, or carbonyl cyanide m-chlorophenylhydrazone, a protonophore. This suggested that a proton gradient and/or an acidic pH facilitated by a vacuolar H(+)-ATPase was required for prohormone processing. We have now utilized the permeabilized cell system in conjunction with the antibiotic bafilomycin A1, a specific inhibitor of vacuolar H(+)-ATPases, to elucidate the role of acidic pH in prohormone processing. Here we report that (i) proSRIF processing was inhibited in vivo and in vitro by low concentrations of bafilomycin A1, confirming the involvement of a vacuolar type ATPase in prohormone processing; (ii) the ATP requirement for processing could be circumvented in vitro by incubating permeabilized cells at acidic pH in the presence of protonophores, indicating that an acidic pH rather than a H+ gradient is necessary for processing; and (iii) a pH of between 6 and 6.2 in the trans-Golgi network was optimal for proSRIF cleavage. We also demonstrate that prohormone convertase 2 exhibited temperature-dependent activity in which proSRIF processing was inhibited at 20 degrees C in vitro. This result explains our previous observation that prohormone processing is inhibited when intact cells are incubated at 20 degrees C.

Animals↗

PubMed1994

Prohormone processing in permeabilized cells: endoproteolytic cleavage of prosomatostatin in the trans-Golgi network.

Adenosine Triphosphate↗

PubMed1993

Intracellular degradation of prohormone-chloramphenicol-acetyl-transferase chimeras in a pre-lysosomal compartment.

Small peptide hormones (less than 50 amino acids) are synthesized as larger inactive precursors. Work from several laboratories, including our own, has implicated the propeptide of various precursors in mediating intracellular transport and targeting to secretory granules. We previously demonstrated that the proregion of prosomatostatin, one of the simplest peptide hormone precursors, when fused to alpha-globin, enabled the globin polypeptide to be transported to the regulated secretory pathway. To identify sorting motifs in this propeptide, we have now constructed a chimera comprising the somatostatin signal peptide and proregion fused to chloramphenicol acetyl transferase (CAT) and a control protein consisting of the signal peptide fused to CAT, both of which were expressed in rat anterior-pituitary GH3 cells. Both molecules were translocated into the endoplasmic reticulum (ER) efficiently and core-glycosylated on the single cryptic N-linked glycosylation site present in CAT. Surprisingly, the glycosylated propeptide-CAT and signal without CAT were degraded intracellularly with half-lives of 30 min and 90 min, respectively. Based on the kinetics of degradation, temperature sensitivity, and resistance to lysosomotrophic agents, we suggest that degradation occurred in the ER. Our data imply that the pro-region is not an a priori universal sorter, but only directs heterologous peptides to the secretory pathway when the passenger peptide assumes a secretion-competent conformation.

Animals↗

PubMed1993

Choice of agent for peripheral thrombolysis.

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Humans↗

PubMed1993

Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells.

Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the TGN or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin (proSRIF) (Stoller, T. J., and D. Shields. J. Cell Biol. 1988. 107:2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by preincubating the permeabilized cells with low concentrations of N-ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP-binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.

Adenosine Triphosphate↗

PubMed1993

2-Alkylsulfonyloxy-3-hydroxy-1,4-naphthoquinones: a novel class of radio- and chemosensitizers of V79 cells.

Four 2-alkysulfonyloxy-3-hydroxy-1,4-naphthoquinone derivatives, VH(1-4), have been synthesized as a novel class of bioreductive alkylating agents and tested as hypoxic cytotoxins, radiosensitizers, and chemosensitizers of V79 cells. In this series, the naphthoquinone is the bioreductive moiety while the sulfonyloxy group, as a good leaving group, could lead to alkylations of nucleophilic substrates in the cells. All VH-compounds are moderately selective cytotoxins of V79 cells with a differential aerobic-hypoxic toxicity of approximately 2, i.e. ratio of drug concentrations to give 50% killing for aerobic versus hypoxic cells. All derivatives are radiosensitizers of hypoxic V79 cells but only VH-3 can give a sensitization enhancement ratio of 2.05 at a nontoxic concentration. Finally, all VH-derivatives are good chemosensitizers of V79 cells to the aerobic toxicity of melphalan, either under hypoxic preincubation without melphalan or under aerobic coincubation with melphalan. The dose modification factors were approximately 2, at low, non-toxic concentrations.

Absorption↗

PubMed1992

Translocation of preproinsulin across the endoplasmic reticulum membrane. The relationship between nascent polypeptide size and extent of signal recognition particle-mediated inhibition of protein synthesis.

Signal recognition particle (SRP) induces elongation arrest of nascent presecretory proteins as the signal peptide protrudes from the large ribosomal subunit. To examine the relationship between the size of the precursor and extent of SRP mediated inhibition of polypeptide chain elongation, we performed in vitro translation experiments in the presence of SRP using a series of truncated preproinsulin mRNA molecules. These precursors possessed the same NH2 terminus as native preproinsulin followed by progressively shorter COOH termini. SRP inhibited translation of precursors as short as 64 amino acids in length, however, the extent of inhibition diminished for shorter precursors. This correlated with a reduction in the time required for ribosomes to transit through the mRNA encoding the shortened precursors. By exploiting a chimeric protein comprising the first 71 residues of preproinsulin fused to the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase, we demonstrate that the largest size a nascent chain can reach and still be susceptible to SRP-mediated elongation arrest is approximately 17 kDa. Our data support the model that SRP binding to the signal peptide is a reversible process even in the absence of microsomal membranes, and that SRP can arrest polypeptide chain elongation at multiple stages during translation.

Animals↗