Lysosomotropic agents ammonium chloride and chloroquine inhibit both the synthesis and secretion of procollagen by freshly isolated embryonic chick tendon cells

Neblock, Donald S.; Berg, Richard A.

doi:10.1016/0006-291x(82)91055-5

Cited by 28 publications

(8 citation statements)

References 12 publications

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“…B ). Note that chloroquine also caused additional procollagen accumulation in WT cells, likely by inhibiting secretion . The effect of bafilomycin A1 on secretion was much smaller.…”

Section: Resultsmentioning

confidence: 95%

“…Note that chloroquine also caused additional procollagen accumulation in WT cells, likely by inhibiting secretion. (24) The effect of bafilomycin A1 on secretion was much smaller. Other commonly used autophagy (3-methyladenine, aka 3MA) and lysosome (NH 4 Cl/leupeptin) inhibitors did not work in our pOB cultures, as they did not result in the accumulation of an autophagy cargo protein, p62 (Supplemental Fig.…”

Section: Intracellular Accumulation and Degradation Of Misfolded Procmentioning

confidence: 97%

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Osteoblast Malfunction Caused by Cell Stress Response to Procollagen Misfolding in α2(I)-G610C Mouse Model of Osteogenesis Imperfecta

Mirigian

Makareeva

Mertz

et al. 2016

Journal of Bone and Mineral Research

125

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Glycine (Gly) substitutions in collagen Gly-X-Y repeats disrupt folding of type I procollagen triple helix and cause severe bone fragility and malformations (osteogenesis imperfecta, aka OI). However, these mutations do not elicit the expected Endoplasmic Reticulum (ER) stress response, in contrast to other protein folding diseases. Thus, it has remained unclear whether cell stress and osteoblast malfunction contribute to the bone pathology caused by Gly substitutions. Here we used a mouse with a Gly610 to cysteine (Cys) substitution in the procollagen α2(I) chain to show that misfolded procollagen accumulation in the ER leads to an unusual form of cell stress, which is neither a conventional unfolded protein response stress nor ER overload. Despite pronounced ER dilation, there is no upregulation of BIP expected in the former and no activation of NFκB signaling expected in the latter. Altered expression of ER chaperones αB crystalline and HSP47, phosphorylation of EIF2α, activation of autophagy, upregulation of general stress response protein CHOP, and osteoblast malfunction reveal some other adaptive response to the ER disruption. We show how this response alters differentiation and function of osteoblasts in culture and in vivo. We demonstrate that bone matrix deposition by cultured osteoblasts is rescued by activation of misfolded procollagen autophagy, suggesting a new therapeutic strategy for OI.

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“…B ). Note that chloroquine also caused additional procollagen accumulation in WT cells, likely by inhibiting secretion . The effect of bafilomycin A1 on secretion was much smaller.…”

Section: Resultsmentioning

confidence: 95%

Section: Intracellular Accumulation and Degradation Of Misfolded Procmentioning

confidence: 97%

Osteoblast Malfunction Caused by Cell Stress Response to Procollagen Misfolding in α2(I)-G610C Mouse Model of Osteogenesis Imperfecta

Mirigian

Makareeva

Mertz

et al. 2016

Journal of Bone and Mineral Research

125

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“…6B). Neutralization of the secretory pathway significantly decreases protein secretion (39,40), and, indeed, viral production from WT-infected cells was decreased ϳ3 logs by bafilomycin treatment. While no mutant virus was produced from untreated cultures, virus production was rescued in the presence of bafilomycin, with mutant-and WT-infected cells producing similar titers under these conditions.…”

Section: Analysis Of Interactions At the Chikv E3-e2 Interfacementioning

confidence: 95%

The Role of E3 in pH Protection during Alphavirus Assembly and Exit

Uchime

Fields

Kielian

2013

J Virol

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Alphaviruses are small enveloped viruses whose surface is covered by spikes composed of trimers of E2/E1 glycoprotein heterodimers. During virus entry, the E2/E1 dimer dissociates within the acidic endosomal environment, freeing the E1 protein to mediate fusion of the viral and endosome membranes. E2 is synthesized as a precursor, p62, which is cleaved by furin in the late secretory pathway to produce mature E2 and a small peripheral glycoprotein, E3. The immature p62/E1 dimer is acid resistant, but since p62 is cleaved before exit from the acidic secretory pathway, low pH-dependent binding of E3 to the spike complex is believed to prevent premature fusion. Based on analysis of the structure of the Chikungunya virus E3/E2/E1 complex, we hypothesized that interactions of E3 residues Y47 and Y48 with E2 are important in this binding. We then directly tested the in vivo role of E3 in pH protection by alanine substitutions of E3 Y47 and Y48 (Y47/48A) in Semliki Forest virus. The mutant was nonviable and was blocked in E1 transport to the plasma membrane and virus production. Although the Y47/48A mutant initially formed the p62/E1 heterodimer, the dimer dissociated during transport through the secretory pathway. Neutralization of the pH in the secretory pathway successfully rescued dimer association, E1 transport, and infectious particle production. Further mutagenesis identified the critical contact as the cation-interaction of E3 Y47 with E2. Thus, E3 mediates pH protection of E1 during virus biogenesis via interactions strongly dependent on Y47 at the E3-E2 interface. E nveloped viruses use membrane fusion to deliver their genomes into host cells (reviewed in references 1, 2, and 3). These viral fusion reactions can be triggered by various combinations of receptor/coreceptor binding and/or by the low-pH environment of the endocytic pathway. Fusion is mediated by specialized viral fusion proteins, which are highly regulated for deployment at the correct time and place during entry. Suppression of the fusion reaction during virus biogenesis is as crucial as its correct triggering during virus entry. Viruses with low-pH-triggered fusion reactions have evolved several mechanisms to protect their membrane fusion proteins from the low pH of the exocytic pathway. Influenza virus (4) and hepatitis C virus (5) express small membrane proteins, termed viroporins, that act as ion channels and can neutralize the exocytic pathway as a protection mechanism. The alphavirus and flavivirus fusion proteins are synthesized with "companion" proteins whose dimeric interactions protect the fusion protein from low pH (reviewed in reference 6). Maturation of the companion protein by furin cleavage primes the virus to respond to low pH during entry (7,8).Alphaviruses such as Semliki Forest virus (SFV) are plusstrand RNA viruses enveloped in a lipid bilayer containing a lattice of trimeric spikes of E1 and E2 glycoprotein heterodimers (9). The companion protein E2 binds receptors on the cell surface, leading to clathrin-mediated endocytosis...

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“…3b, using (4). Procollagen synthesis is repressed by inhibition of secretion (19) or by a peptide derived from the amino terminus of collagen (8). In Escherichia coli, mutations in secA or secC genes, encoding components of the secretory machinery, specifically repress the synthesis of secretory proteins, and the repression can be alleviated by signal sequence mutations (11).…”

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confidence: 99%

Blockage of tropoelastin secretion by monensin represses tropoelastin synthesis at a pretranslational level in rat smooth muscle cells.

Frisch¹,

Davidson²,

Werb³

1985

Mol. Cell. Biol.

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The blockage of protein secretion in the R22 cultured rat aortic smooth muscle cell strain with monensin repressed tropoelastin gene expression at the mRNA level by ca. 50-fold as measured by biosynthetic pulse-labeling, in vitro translation, and hybridization with a tropoelastin genomic DNA probe. These results suggest that tropoelastin gene expression is autoregulated, and they represent the first reported effect of monensin on gene expression.Elastin, a major protein of the extracellular matrix of elastic tissues such as aorta and lung, is assembled by the covalent cross-linking of tropoelastin (TE), its 72-kilodalton (kDa) soluble precursor. Cells committed to elastogenesis devote as much as 25 to 40% of their total protein synthesis to TE in the later part of fetal life and in the newborn animal, yet they synthesize little or no detectable TE in adult connective tissues. These changes are reflected in altered levels of TE mRNA (1,5,24), although the nature of the regulation is unknown.There is evidence that the synthesis of another secreted connective tissue macromolecule, procollagen, is subject to feedback repression by fragments of the protein itself (8). A similar general mechanism may be involved in the negative autoregulation of synthesis of tubulin (2) and simian virus 40 T antigen (22). We have used vascular smooth muscle cells as a model system in which to investigate the regulation of TE synthesis. To block protein secretion, we used monensin, a polycyclic ionophore that disrupts monovalent cation gradients across biological membranes and arrests the transit of secretory proteins from the early (proximal) to late (distal) Golgi subcompartments, causing an accumulation of secretory proteins within the cell (25). In this report, we demonstrate that monensin repressed TE synthesis at the mRNA level, suggesting that TE gene expression may be autoregulated.The aortic smooth muscle cell strain, R22, which was isolated by Jones et al. (9) from embryonic rat heart, synthesizes and secretes several extracellular matrix components in large amounts (6,29,30), devoting 10 to 30% of its total protein synthesis to TE. We identified the TE gene product by its preferential labeling with [3H]valine, complete resistance to CNBr cleavage owing to its lack of methionine (23) (Fig. la), comigration in two-dimensional gel electrophoresis with purified chick TE (Fig. lb), and partial chymotryptic peptide mapping in comparison with chick TE (data not shown).First, we showed that TE is accumulated within cells treated with monensin. R22 cells were incubated with 20 ,uCi * Corresponding author. of [3H]valine and 50 p,g of 3-aminopropionitrile per ml in minimal Eagle medium without nonradioactive valine in the presence of 0 to 20 ,uM monensin (Sigma Chemical Co.) for 4 or 10 h, and intracellular proteins were analyzed on sodium dodecyl sulfate (SDS)-polyacrylamide gels (Fig. 2a). Because a significant amount of secreted TE spontaneously coacervated into an insoluble form in aqueous buffer despite the presence of P-aminoprop...

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Lysosomotropic agents ammonium chloride and chloroquine inhibit both the synthesis and secretion of procollagen by freshly isolated embryonic chick tendon cells

Cited by 28 publications

References 12 publications

Osteoblast Malfunction Caused by Cell Stress Response to Procollagen Misfolding in α2(I)-G610C Mouse Model of Osteogenesis Imperfecta

Osteoblast Malfunction Caused by Cell Stress Response to Procollagen Misfolding in α2(I)-G610C Mouse Model of Osteogenesis Imperfecta

The Role of E3 in pH Protection during Alphavirus Assembly and Exit

Blockage of tropoelastin secretion by monensin represses tropoelastin synthesis at a pretranslational level in rat smooth muscle cells.

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