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Seminars in Cell & Developmental Biology Nov 2020In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and... (Review)
Review
In eukaryotic cells, protein sorting is a highly regulated mechanism important for many physiological events. After synthesis in the endoplasmic reticulum and trafficking to the Golgi apparatus, proteins sort to many different cellular destinations including the endolysosomal system and the extracellular space. Secreted proteins need to be delivered directly to the cell surface. Sorting of secreted proteins from the Golgi apparatus has been a topic of interest for over thirty years, yet there is still no clear understanding of the machinery that forms the post-Golgi carriers. Most evidence points to these post-Golgi carriers being tubular pleomorphic structures that bud from the -face of the Golgi. In this review, we present the background studies and highlight the key components of this pathway, we then discuss the machinery implicated in the formation of these carriers, their translocation across the cytosol, and their fusion at the plasma membrane.
Topics: Animals; Cell Membrane; Golgi Apparatus; Humans; Lipid Metabolism; Membrane Fusion; Protein Transport; Secretory Pathway
PubMed: 32317144
DOI: 10.1016/j.semcdb.2020.04.001 -
Autophagy Dec 2021We propose that beyond its role in WNT secretion, WLS/GPR177 (wntless, WNT ligand secretion mediator) acts as an essential regulator controlling protein glycosylation,...
We propose that beyond its role in WNT secretion, WLS/GPR177 (wntless, WNT ligand secretion mediator) acts as an essential regulator controlling protein glycosylation, endoplasmic reticulum (ER) homeostasis, and dendritic cell (DC)-mediated immunity. WLS deficiency in bone marrow-derived DCs (BMDCs) resulted in poor growth and an inability to mount cytokine and T-cell responses , phenotypes that were irreversible by the addition of exogenous WNTs. In fact, WLS was discovered to integrate a protein complex in N-glycan-dependent and WLS domain-selective manners, comprising ER stress sensors and lectin chaperones. WLS deficiency in BMDCs led to increased ER stress response and macroautophagy/autophagy, decreased calcium efflux from the ER, and the loss of CALR (calreticulin)-CANX (calnexin) cycle, and hence protein hypo-glycosylation. Consequently, DC-specific -null mice were unable to develop both Th1-, Th2- and Th17-associated responses in the respective autoimmune and allergic disease models. These results suggest that WLS is a critical chaperone in maintaining ER homeostasis, glycoprotein quality control and calcium dynamics in DCs.: ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; ATP2A1/SERCA1: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1; BALF: bronchoalveolar lavage fluid; BFA: brefeldin A; BMDC: bone marrow-derived dendritic cell; CALR: calreticulin; CANX: calnexin; CCL2/MCP-1: C-C motif chemokine ligand 2; CNS: central nervous system; CT: C-terminal domain; DTT: dithiothreitol; DNAJB9/ERDJ4: DnaJ heat shock protein family (Hsp40) member B9; EAE: experimental autoimmune encephalomyelitis; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GFP: green fluorescent protein; HSPA5/GRP78/BiP: heat shock protein A5; IFNA: interferon alpha; IFNAR1: interferon alpha and beta receptor subunit 1; IFNB: interferon beta; IFNG/INFγ: interferon gamma; IFNGR2: interferon gamma receptor 2; IL6: interleukin 6; IL10: interleukin 10; IL12A: interleukin 12A; IL23A: interleukin 23 subunit alpha; ITGAX/CD11c: integrin subunit alpha X; ITPR1/InsP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; OVA: ovalbumin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLF: predicted lipocalin fold; PPP1R15A/GADD34: protein phosphatase 1 regulatory subunit 15A; RYR1/RyanR1: ryanodine receptor 1, skeletal muscle; SD: signal domain; TGFB/TGF-β: transforming growth factor beta family; Th1: T helper cell type 1; Th17: T helper cell type 17; TM: tunicamycin; TNF/TNF-α: tumor necrosis factor; UPR: unfolded protein response; WLS/wntless: WNT ligand secretion mediator.
Topics: Animals; Autophagy; Dendritic Cells; Endoplasmic Reticulum Stress; Homeostasis; Mice; Wnt Signaling Pathway
PubMed: 33853474
DOI: 10.1080/15548627.2021.1907516 -
International Journal of Molecular... Aug 2023Melanoma-associated antigen D2 (MAGED2) plays an essential role in activating the cAMP/PKA pathway under hypoxic conditions, which is crucial for stimulating renal salt...
Melanoma-associated antigen D2 (MAGED2) plays an essential role in activating the cAMP/PKA pathway under hypoxic conditions, which is crucial for stimulating renal salt reabsorption and thus explaining the transient variant of Bartter's syndrome. The cAMP/PKA pathway is also known to regulate autophagy, a lysosomal degradation process induced by cellular stress. Previous studies showed that two members of the melanoma-associated antigens MAGE-family inhibit autophagy. To explore the potential role of MAGED2 in stress-induced autophagy, specific MAGED2-siRNA were used in HEK293 cells under physical hypoxia and oxidative stress (cobalt chloride, hypoxia mimetic). Depletion of MAGED2 resulted in reduced p62 levels and upregulation of both the autophagy-related genes (ATG5 and ATG12) as well as the autophagosome marker LC3II compared to control siRNA. The increase in the autophagy markers in MAGED2-depleted cells was further confirmed by leupeptin-based assay which concurred with the highest LC3II accumulation. Likewise, under hypoxia, immunofluorescence in HEK293, HeLa and U2OS cell lines demonstrated a pronounced accumulation of LC3B puncta upon MAGED2 depletion. Moreover, LC3B puncta were absent in human fetal control kidneys but markedly expressed in a fetal kidney from a MAGED2-deficient subject. Induction of autophagy with both physical hypoxia and oxidative stress suggests a potentially general role of MAGED2 under stress conditions. Various other cellular stressors (brefeldin A, tunicamycin, 2-deoxy-D-glucose, and camptothecin) were analyzed, which all induced autophagy in the absence of MAGED2. Forskolin (FSK) inhibited, whereas GNAS Knockdown induced autophagy under hypoxia. In contrast to other MAGE proteins, MAGED2 has an inhibitory role on autophagy only under stress conditions. Hence, a prominent role of MAGED2 in the regulation of autophagy under stress conditions is evident, which may also contribute to impaired fetal renal salt reabsorption by promoting autophagy of salt-transporters in patients with MAGED2 mutation.
Topics: Humans; HEK293 Cells; Autophagy; Oxidative Stress; Autophagosomes; Sodium Chloride; Sodium Chloride, Dietary; Melanoma; Antigens, Neoplasm; Adaptor Proteins, Signal Transducing
PubMed: 37686237
DOI: 10.3390/ijms241713433 -
American Journal of Physiology. Cell... Aug 2021Endothelial hyperpermeability is the hallmark of acute respiratory distress syndrome (ARDS). Laborious efforts in the investigation of the molecular pathways involved in...
Endothelial hyperpermeability is the hallmark of acute respiratory distress syndrome (ARDS). Laborious efforts in the investigation of the molecular pathways involved in the regulation of the vascular barrier shall reveal novel therapeutic targets toward that respiratory disorder. Herein, we investigate in vitro the effects of the α-1,2-mannosidase 1 inhibitor kifunensine (KIF) and brefeldin A (BFA) in the lipopolysaccharides (LPS)-induced endothelial breakdown. Our results suggest that BFA opposes the deteriorating effects of KIF [unfolded protein response (UPR) suppressor] toward the lung microvasculature. Since KIF is a UPR suppressor, and brefeldin A is a UPR inducer, we suggest that a carefully devised UPR manipulation may deliver novel therapeutic avenues in diseases related to endothelial barrier dysfunction (e.g., ARDS and sepsis).
Topics: Alkaloids; Animals; Brefeldin A; Cattle; Endothelial Cells; Endothelium, Vascular; Humans; Lipopolysaccharides; Lung; Microvessels; Permeability; Respiratory Distress Syndrome
PubMed: 34161151
DOI: 10.1152/ajpcell.00142.2021 -
Cell Death & Disease Jul 2021All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress...
All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress survival mechanism is the stringent response triggered by the accumulation "alarmone" (p)ppGpp, whose level is regulated by RelA and SpoT. While metazoan genomes encode MESH1 (Metazoan SpoT Homolog 1) with ppGpp hydrolase activity, neither ppGpp nor the stringent response is found in metazoa. The deletion of Mesh1 in Drosophila triggers a transcriptional response reminiscent of the bacterial stringent response. However, the function of MESH1 remains unknown until our recent discovery of MESH1 as the first cytosolic NADPH phosphatase that regulates ferroptosis. To further understand whether MESH1 knockdown triggers a similar transcriptional response in mammalian cells, here, we employed RNA-Seq to analyze the transcriptome response to MESH1 knockdown in human cancer cells. We find that MESH1 knockdown induced different genes involving endoplasmic reticulum (ER) stress, especially ATF3, one of the ATF4-regulated genes in the integrative stress responses (ISR). Furthermore, MESH1 knockdown increased ATF4 protein, eIF2a phosphorylation, and induction of ATF3, XBPs, and CHOP mRNA. ATF4 induction contributes to ~30% of the transcriptome induced by MESH1 knockdown. Concurrent ATF4 knockdown re-sensitizes MESH1-depleted RCC4 cells to ferroptosis, suggesting its role in the ferroptosis protection mediated by MESH1 knockdown. ATF3 induction is abolished by the concurrent knockdown of NADK, implicating a role of NADPH accumulation in the integrative stress response. Collectively, these results suggest that MESH1 depletion triggers ER stress and ISR as a part of its overall transcriptome changes to enable stress survival of cancer cells. Therefore, the phenotypic similarity of stress tolerance caused by MESH1 removal and NADPH accumulation is in part achieved by ISR to regulate ferroptosis.
Topics: Activating Transcription Factor 4; Brefeldin A; Cell Cycle; Cell Line; Endoplasmic Reticulum Stress; Ferroptosis; Gene Knockdown Techniques; Humans; Neoplasm Proteins; Phosphorylation; Pyrophosphatases; Stress, Physiological; Transcription, Genetic; Tunicamycin
PubMed: 34294679
DOI: 10.1038/s41419-021-04018-7 -
Journal of Human Genetics Sep 2021Breast cancer is a heterogeneous disease that develops through a multistep process via the accumulation of genetic/epigenetic alterations in various cancer-related... (Review)
Review
Breast cancer is a heterogeneous disease that develops through a multistep process via the accumulation of genetic/epigenetic alterations in various cancer-related genes. Current treatment options for breast cancer patients include surgery, radiotherapy, and chemotherapy including conventional cytotoxic and molecular-targeted anticancer drugs for each intrinsic subtype, such as endocrine therapy and antihuman epidermal growth factor receptor 2 (HER2) therapy. However, these therapies often fail to prevent recurrence and metastasis due to resistance. Overall, understanding the molecular mechanisms of breast carcinogenesis and progression will help to establish therapeutic modalities to improve treatment. The recent development of comprehensive omics technologies has led to the discovery of driver genes, including oncogenes and tumor-suppressor genes, contributing to the development of molecular-targeted anticancer drugs. Here, we review the development of anticancer drugs targeting cancer-specific functional therapeutic targets, namely, MELK (maternal embryonic leucine zipper kinase), TOPK (T-lymphokine-activated killer cell-originated protein kinase), and BIG3 (brefeldin A-inhibited guanine nucleotide-exchange protein 3), as identified through comprehensive breast cancer transcriptomics.
Topics: Antineoplastic Agents; Breast Neoplasms; Drug Discovery; Female; Genomics; Humans
PubMed: 34285339
DOI: 10.1038/s10038-021-00962-6 -
Nature Communications Jan 2021Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of...
Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.
Topics: Animals; Brefeldin A; COP-Coated Vesicles; Cell Membrane; Endoplasmic Reticulum; Fibroblasts; Golgi Apparatus; HEK293 Cells; Homeostasis; Humans; Lipoylation; Luciferases; Membrane Proteins; Mice; Nucleotidyltransferases; Protein Binding; Protein Transport
PubMed: 33397928
DOI: 10.1038/s41467-020-20234-9 -
Marine Drugs Dec 2021Brefeldin A (), a potent cytotoxic natural macrolactone, was produced by the marine fungus sp. (HS-N-29) from the medicinal mangrove . Series of its ester derivatives -...
Brefeldin A (), a potent cytotoxic natural macrolactone, was produced by the marine fungus sp. (HS-N-29) from the medicinal mangrove . Series of its ester derivatives - were designed and semi-synthesized, and their structures were characterized by spectroscopic methods. Their cytotoxic activities were evaluated against human chronic myelogenous leukemia K562 cell line in vitro, and the preliminary structure-activity relationships revealed that the hydroxy group played an important role. Moreover, the monoester derivatives exhibited stronger cytotoxic activity than the diester derivatives. Among them, brefeldin A 7--2-chloro-4,5-difluorobenzoate () exhibited the strongest inhibitory effect on the proliferation of K562 cells with an IC value of 0.84 µM. Further evaluations indicated that induced cell cycle arrest, stimulated cell apoptosis, inhibited phosphorylation of BCR-ABL, and thereby inactivated its downstream AKT signaling pathway. The expression of downstream signaling molecules in the AKT pathway, including mTOR and p70S6K, was also attenuated after -treatment in a dose-dependent manner. Furthermore, molecular modeling of docked into binding site of an ARF1-GDP-GEF complex represented well-tolerance. Taken together, had the potential to be served as an effective antileukemia agent or lead compound for further exploration.
Topics: Animals; Antineoplastic Agents; Aquatic Organisms; Brefeldin A; Cell Proliferation; Humans; K562 Cells; Penicillium; Structure-Activity Relationship; Wetlands
PubMed: 35049881
DOI: 10.3390/md20010026 -
Molecules (Basel, Switzerland) May 2023Brefeldin A has a wide range of anticancer activity against a variety of tumor cells. Its poor pharmacokinetic properties and significant toxicity seriously hinder its...
Brefeldin A has a wide range of anticancer activity against a variety of tumor cells. Its poor pharmacokinetic properties and significant toxicity seriously hinder its further development. In this manuscript, 25 brefeldin A-isothiocyanate derivatives were designed and synthesized. Most derivatives showed good selectivity between HeLa cells and L-02 cells. In particular, exhibited potent antiproliferative activity against HeLa cells (IC = 1.84 μM) with no obvious cytotoxic activity to L-02 (IC > 80 μM). Further cellular mechanism tests indicated that induced HeLa cell cycle arrest at G1 phase. Cell nucleus fragmentation and decreased mitochondrial membrane potential suggested could induce apoptosis in HeLa cells through the mitochondrial-dependent pathway.
Topics: Female; Humans; HeLa Cells; Uterine Cervical Neoplasms; Brefeldin A; Cell Proliferation; Antineoplastic Agents; Apoptosis; Isothiocyanates; Drug Screening Assays, Antitumor; Cell Line, Tumor; Structure-Activity Relationship
PubMed: 37298761
DOI: 10.3390/molecules28114284 -
Redox Biology Nov 2023We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the...
We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 μM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 μU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control μU/mg). Interestingly, free iron (Fe and Fe) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 μM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.
Topics: Animals; Mice; Xanthine Dehydrogenase; Hemin; Iron; NF-kappa B; Heme; Hepatocytes
PubMed: 37703667
DOI: 10.1016/j.redox.2023.102866