Regulation of Vg1 biogenesis during mesendoderm induction

AbstractThe TGF-beta signals Vg1 and Nodal form heterodimers to induce the vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal. Here we investigate the mechanisms underlying Vg1 retention, processing, secretion and signaling in zebrafish. First, using a newly devised Synthetic Processing (SynPro) system, we find that Vg1 can be processed by intra- or extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. Our results establish SynPro as a new in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active Vg1-Nodal heterodimers. These observations suggest two strategies for rapid mesendoderm induction: chaperone-binding motifs help store Vg1 as an inactive but ready-to-heterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intra- and extracellularly.

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The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes

Biochemical Journal ◽

10.1042/bj20060990 ◽

2007 ◽

Vol 401 (3) ◽

pp. 701-709 ◽

Cited By ~ 13

Author(s):

Matthew P. A. Henderson ◽

Yeen Ting Hwang ◽

John M. Dyer ◽

Robert T. Mullen ◽

David W. Andrews

Keyword(s):

Endoplasmic Reticulum ◽

Subcellular Localization ◽

Lipid Bilayers ◽

Fusion Proteins ◽

Molecular Mechanisms ◽

Cytochrome B5 ◽

Terminal Sequence ◽

C Terminus

The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.

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The pattern of Nodal morphogen signaling is shaped by co-receptor expression

10.1101/2019.12.30.891101 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nathan D. Lord ◽

Adam N. Carte ◽

Philip B. Abitua ◽

Alexander F. Schier

Keyword(s):

Travelling Wave ◽

Receptor Expression ◽

Target Cells ◽

Nodal Signaling ◽

Normal Range ◽

Localized Source ◽

Gradient Formation ◽

Zebrafish Embryogenesis ◽

Signaling Activity

AbstractEmbryos must communicate instructions to their constituent cells over long distances. These instructions are often encoded in the concentration of signals called morphogens. In the textbook view, morphogen molecules diffuse from a localized source to form a concentration gradient, and target cells adopt fates by measuring the local morphogen concentration. However, natural patterning systems often incorporate numerous co-factors and extensive signaling feedback, suggesting that embryos require additional mechanisms to generate signaling patterns. Here, we examine the mechanisms of signaling pattern formation for the mesendoderm inducer Nodal during zebrafish embryogenesis. We find that Nodal signaling activity spans a normal range in the absence of signaling feedback, suggesting that diffusion is sufficient for Nodal gradient formation. We further show that the range of endogenous Nodal ligands is set by the EGF-CFC co-receptor Oep: in the absence of Oep, Nodal ligands spread to form a nearly uniform distribution throughout the embryo. In turn, increasing Oep levels sensitizes cells to Nodal ligands. We recapitulate these experimental results with a computational model in which Oep regulates the diffusive spread of Nodal ligands by setting the rate of capture by target cells. This model predicts, and we confirm in vivo, the surprising observation that a failure to replenish Oep during patterning transforms the Nodal signaling gradient into a travelling wave. These results reveal that patterns of Nodal morphogen signaling are shaped by co-receptor-mediated restriction of ligand spread and cell sensitization.

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Bring it back, bring it back, don't take it away from me – the sorting receptor RER1

Journal of Cell Science ◽

10.1242/jcs.231423 ◽

2020 ◽

Vol 133 (17) ◽

pp. jcs231423

Author(s):

Wim Annaert ◽

Christoph Kaether

Keyword(s):

Endoplasmic Reticulum ◽

Cell Biology ◽

Mammalian Cells ◽

Future Directions ◽

Binding Motifs ◽

Sorting Receptor ◽

Intermediate Compartment ◽

The Brain

ABSTRACTThe quote “bring it back, bring it back, don't take it away from me” from Queen's Love of my life describes the function of the sorting receptor RER1, a 23 kDa protein with four transmembrane domains (TMDs) that localizes to the intermediate compartment and the cis-Golgi. From there it returns escaped proteins that are not supposed to leave the endoplasmic reticulum (ER) back to it. Unique about RER1 is its ability to recognize its ligands through binding motifs in TMDs. Among its substrates are ER-resident proteins, as well as unassembled subunits of multimeric complexes that are retrieved back into the ER, this way guarding the full assembly of their respective complexes. The basic mechanisms for RER1-dependent retrieval have been already elucidated some years ago in yeast. More recently, several important cargoes of RER1 have been described in mammalian cells, and the in vivo role of RER1 is being unveiled by using mouse models. In this Review, we give an overview of the cell biology of RER1 in different models, discuss its controversial role in the brain and provide an outlook on future directions for RER1 research.

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CaMKII as a pathological mediator of ER stress, oxidative stress, and mitochondrial dysfunction in a murine model of nephronophthisis

AJP Renal Physiology ◽

10.1152/ajprenal.00426.2015 ◽

2016 ◽

Vol 310 (11) ◽

pp. F1414-F1422 ◽

Cited By ~ 17

Author(s):

Christina Bracken ◽

Philippe Beauverger ◽

Olivier Duclos ◽

Ryan J. Russo ◽

Kelly A. Rogers ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Oxidative Damage ◽

Molecular Mechanisms ◽

Kidney Diseases ◽

Cystic Kidney ◽

Effective Inhibition ◽

Stress Oxidative

Polycystic kidney diseases (PKDs) are genetic diseases characterized by renal cyst formation with increased cell proliferation, apoptosis, and transition to a secretory phenotype at the expense of terminal differentiation. Despite recent progress in understanding PKD pathogenesis and the emergence of potential therapies, the key molecular mechanisms promoting cystogenesis are not well understood. Here, we demonstrate that mechanisms including endoplasmic reticulum stress, oxidative damage, and compromised mitochondrial function all contribute to nephronophthisis-associated PKD. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is emerging as a critical mediator of these cellular processes. Therefore, we reasoned that pharmacological targeting of CaMKII may translate into effective inhibition of PKD in jck mice. Our data demonstrate that CaMKII is activated within cystic kidney epithelia in jck mice. Blockade of CaMKII with a selective inhibitor results in effective inhibition of PKD in jck mice. Mechanistic experiments in vitro and in vivo demonstrated that CaMKII inhibition relieves endoplasmic reticulum stress and oxidative damage and improves mitochondrial integrity and membrane potential. Taken together, our data support CaMKII inhibition as a new and effective therapeutic avenue for the treatment of cystic diseases.

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Disruption of transforming growth factor beta signaling by a mutation that prevents transphosphorylation within the receptor complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1573 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1573-1581 ◽

Cited By ~ 160

Author(s):

J Cárcamo ◽

A Zentella ◽

J Massagué

Keyword(s):

Growth Factor ◽

Ligand Binding ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Signal Propagation ◽

Tgf Beta ◽

Wild Type ◽

Growth Factor Beta ◽

Signaling Activity

T beta R-II (transforming growth factor beta [TGF-beta] type II receptor) is a transmembrane serine/threonine kinase that acts as the primary TGF-beta receptor. Ligand binding to T beta R-II leads to the recruitment and phosphorylation of T beta R-I, a distantly related transmembrane kinase that acts as a downstream signaling component. T beta R-I phosphorylation by T beta R-II is shown here to be essential for signaling. A mutant T beta R-II that binds ligand but lacks signaling activity was identified. This mutant was identified by screening with a TGF-beta-inducible vector a series of mink lung epithelial cell clones that have normal TGF-beta binding activity but have lost antiproliferative and transcriptional responses to TGF-beta. When transiently cotransfected with T beta R-II, one of these cell lines, S-21, recovered TGF-beta responsiveness. cDNA cloning and sequencing of T beta R-II from S-21 cells revealed a point mutation that changes proline 525 to leucine in kinase subdomain XI. A recombinant receptor containing this mutation, T beta R-II(P525L), is similar to wild-type T beta R-II in its abilities to bind ligand, support ligand binding to T beta R-I, and form a complex with T beta R-I in vivo. T beta R-II(P525L) has autophosphorylating activity in vitro and in vivo; however, unlike the wild-type receptor, it fails to phosphorylate an associated T beta R-I. These results suggest that T beta R-II(P525L) is a catalytically active receptor that cannot recognize T beta R-I as a substrate. The close link between T beta R-I transphosphorylation and signaling activity argues that transphosphorylation is essential for signal propagation via T beta R-I.

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Transforming growth factor-beta dynamically regulates vascular smooth muscle differentiation in vivo

Journal of Cell Science ◽

10.1242/jcs.111.19.2977 ◽

1998 ◽

Vol 111 (19) ◽

pp. 2977-2988 ◽

Cited By ~ 3

Author(s):

D.J. Grainger ◽

J.C. Metcalfe ◽

A.A. Grace ◽

D.E. Mosedale

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Muscle Differentiation ◽

Tgf Beta ◽

Differentiation Markers ◽

Smooth Muscle Tissue ◽

Smooth Muscle Differentiation

Variations in the levels of smooth muscle-specific isoforms of contractile proteins have been reported to occur in many different vascular diseases. However, although much work has been done in vitro to investigate the regulation of smooth muscle cell differentiation, the molecular mechanisms which regulate the differentiation of vascular smooth muscle tissue in vivo are unknown. Using quantitative immunofluorescence, we show that in rat arteries levels of smooth muscle differentiation markers correlate with the levels of the cytokine TGF-beta. In young mice with one allele of the TGF-beta1 gene deleted, the levels of both TGF-beta1 and smooth muscle differentiation markers are reduced compared to wild-type controls. This regulation of smooth muscle differentiation by TGF-beta during post-natal development also occurs dynamically in the adult animal. Following various pharmacological or surgical interventions, including treatment of mice with tamoxifen and balloon injury of rat carotid arteries, there is a strong correlation between the changes in the levels of TGF-beta and changes in the levels of smooth muscle differentiation markers (r=0. 9, P<0.0001 for n=26 experiments). We conclude that TGF-beta dynamically regulates smooth muscle differentiation in rodent arteries in vivo.

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P5438Cardiac abnormalities after induction of endoplasmic reticulum stress are associated with mitochondrial dysfunction and connexin43 expression

European Heart Journal ◽

10.1093/eurheartj/ehz746.0394 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

T Liu ◽

J He ◽

M Gong ◽

C Luo ◽

Y Zhao ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Connexin 43 ◽

Protein Unfolding ◽

Molecular Mechanisms ◽

Diastolic Pressure ◽

Systolic Pressure ◽

Calcium Handling ◽

Stress Induction

Abstract Background The endoplasmic reticulum (ER) is responsible for protein synthesis and calcium storage. ER stress, reflected by protein unfolding and calcium handling abnormalities, has been studied as a pathogenetic factor in cardiovascular diseases. Purpose The aim of this study is to examine the effects of ER stress on mechanical and electrophysiological functions in the heart and explore the underlying molecular mechanisms. Methods A total of 30 rats were randomly divided into control, ER stress induction (tunicamycin) and ER stress inhibition (4-phenylbutyric acid, 4-PBA) groups. Results ER stress induction led to significantly systolic and diastolic dysfunction as reflected by maximal increasing/decreasing rate of left intraventricular pressure (±dp/dt), LV peak systolic pressure, LV development pressure and LV end-diastolic pressure. Epicardial electrical mapping performed in vivo revealed reduced conduction velocity, increased conduction heterogeneity and spontaneous ventricular tachycardia. Masson's trichrome staining revealed marked fibrosis in the myocardial interstitium and sub-pericardium and thickened epicardium. Western blot analysis revealed increased pro-fibrotic factor TGF-β1, decreased mitochondrial biogenesis protein PGC-1a, decreased mitochondrial fusion protein MFN2. These changes were associated with decreased mitochondrial membrane potential (MMP) and connexin 43 translocation to mitochondria. These abnormalities can be partially prevented by the ER stress inhibitor 4-PBA. Conclusions Our study shows that ER stress induction can produce cardiac electrical and mechanism dysfunction as well as structural remodeling. Mitochondrial function alterations are contributed by CX43 transposition to mitochondria. These abnormalities can be partially prevented by the ER stress inhibitor 4-PBA. Acknowledgement/Funding National Natural Science Foundation of China (No. 81570298 to T.L.)

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Lithium Chloride Induces Apoptosis in Human Choroidal Melanoma Cells via the NOXA/Mcl-1 Axis and Induction of ER Stress

10.21203/rs.3.rs-64030/v1 ◽

2020 ◽

Author(s):

Qiuqiu Zhang ◽

Qianwei Zhang ◽

Huiyuan Li ◽

Xiaofei Zhao ◽

Han Zhang

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Cell Survival ◽

Lithium Chloride ◽

Molecular Mechanisms ◽

Choroidal Melanoma ◽

Xenograft Model ◽

Melanoma Cells ◽

Induced Apoptosis

Abstract Background: Choroidal melanoma is the most common primary intraocular malignancy that occurs in adults. Lithium chloride (LiCl) has been safely used in the clinic to treat psychiatric disorders for several decades. In this study, we aimed to understand whether LiCl exerts anticancer effects on choroidal melanoma cells and elucidate the underlying molecular mechanisms. Methods: Human choroidal melanoma cells were treated with LiCl, and cell survival was assessed with MTT assays. Cell proliferation was measured by plate colony formation assays. Cell apoptosis was evaluated using flow cytometry, and proteins were detected using western blotting. A human choroidal melanoma xenograft model was established to demonstrate the effect of LiCl on human choroidal melanoma in vivo. An unpaired t-test was used to compare differences between two groups, and one-way ANOVA was used to compare differences among more than two groups.Results: We found that LiCl inhibited cell survival and clonogenic potential and induced apoptosis in human choroidal melanoma cells. LiCl also reduced the proliferation of choroidal melanoma cells in vivo. Moreover, the upregulation of NOXA and downregulation of Mcl-1 were responsible for LiCl-induced apoptosis. Mcl-1 overexpression obviously impaired LiCl-induced apoptosis and cleavage of casp8, casp9, casp3 and PARP. Moreover, the protein expression of endoplasmic reticulum stress markers, including IRE1α, Bip, p-eIF2α, ATF4 and CHOP, was upregulated following treatment with LiCl. Conclusions: In summary, LiCl induced an endoplasmic reticulum stress response while activating intrinsic apoptosis. Furthermore, the NOXA/Mcl-1 axis contributed to LiCl-induced apoptosis both in vitro and in vivo. The present study provides important mechanistic insight into potential cancer treatments involving LiCl and enhances the understanding of human choroidal melanoma.

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GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

npj Breast Cancer ◽

10.1038/s41523-021-00340-7 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Xiaolin Li ◽

Sixuan Liu ◽

Xuan Chen ◽

Run Huang ◽

Lisi Ma ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Er Stress ◽

Molecular Mechanisms ◽

Ovarian Function ◽

Mtor Signaling ◽

Unfolded Protein ◽

Hormone Analogs

AbstractChemotherapy-induced ovarian dysfunction is a serious adverse effect in premenopausal patients with cancer. Gonadotrophin-releasing hormone analogs (GnRHa) protect ovarian function, but its molecular mechanisms have not yet been determined. In this study, we attempted to determine the previously unknown molecular mechanism by which such protection occurs. Serum anti-Müllerian hormone (AMH) levels were tested in tumor-bearing nude mice, a series of exploratory experiments were conducted. We discovered that GnRHa protects granulosa cells from chemotherapeutic toxicity in vivo and in vitro. We also showed that CTX-induced endoplasmic reticulum stress inhibits the secretion of AMH, and treatment with GnRHa relieves ER stress and the subsequent unfolded-protein response by modulating mTOR signaling to induce autophagy. The results of mechanistic studies indicated that GnRHa-modulated mTOR signaling to induce autophagy, which alleviated CTX-induced ER stress and promoted the secretion of AMH.

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LiCl induces apoptosis via CHOP/NOXA/Mcl-1 axis in human choroidal melanoma cells

10.21203/rs.3.rs-64030/v2 ◽

2021 ◽

Author(s):

Qiuqiu Zhang ◽

Qianwei Zhang ◽

Huiyuan Li ◽

Xiaofei Zhao ◽

Han Zhang

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Cell Survival ◽

Molecular Mechanisms ◽

Glycogen Synthase ◽

Choroidal Melanoma ◽

Melanoma Cells ◽

Human Leukemia ◽

Induced Apoptosis

Abstract Background: Choroidal melanoma is the most common primary intraocular malignancy that occurs in adults. Lithium Chloride Promotes Apoptosis in Human Leukemia NB4 Cells by Inhibiting Glycogen Synthase Kinase-3 Beta. In this study, we aimed to understand whether LiCl exerts anticancer effects on choroidal melanoma cells and elucidate the underlying molecular mechanisms. Methods: Human choroidal melanoma cells were treated with LiCl, and cell survival was assessed with MTT assays. Cell reproductive viability was measured by plate colony formation assays. Cell apoptosis was evaluated using flow cytometry, and proteins were detected using western blotting. A human choroidal melanoma xenograft model was established to demonstrate the effect of LiCl on human choroidal melanoma in vivo. Results: We found that LiCl inhibited cell survival and clonogenic potential and induced apoptosis in human choroidal melanoma cells. LiCl also reduced the proliferation of choroidal melanoma cells in vivo. Moreover, the upregulation of NOXA and downregulation of Mcl-1 were responsible for LiCl-induced apoptosis. Mcl-1 overexpression obviously impaired LiCl-induced apoptosis and cleavage of caspase8, caspase9, caspase3 and PARP. Moreover, the protein expression of endoplasmic reticulum stress markers, including IRE1α, Bip, p-eIF2α, ATF4 and CHOP, was upregulated following treatment with LiCl. When CHOP expression was knocked down and cells were treated with LiCl, the protein level of NOXA was partially increased, and Mcl-1 expression was increased, while the cleavage of caspase8, caspase9, caspase3 and PARP that was induced by the LiCl was reduced compared with the vehicle treated group. Prolonged ER stress results in the activation of the apoptotic pathway.Conclusions: In summary, LiCl induced an endoplasmic reticulum stress response while activating intrinsic apoptosis. Furthermore, the CHOP/NOXA/Mcl-1 axis contributed to LiCl-induced apoptosis both in vitro and in vivo. The present study provides important mechanistic insight into potential cancer treatments involving LiCl and enhances the understanding of human choroidal melanoma.

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