The Mammalian Cytosolic Thioredoxin Reductase Pathway Acts via a Membrane Protein to Reduce ER-localised Proteins

SummaryFolding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfide bonds. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is critical for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER lumen. These results provide compelling evidence for the critical role of the cytosol in regulating ER redox homeostasis to ensure correct protein folding and to facilitate the degradation of misfolded ER proteins.

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Passive Sorting in Maturing Granules of AtT-20 Cells: The Entry and Exit of Salivary Amylase and Proline-rich Protein

The Journal of Cell Biology ◽

10.1083/jcb.138.1.45 ◽

1997 ◽

Vol 138 (1) ◽

pp. 45-54 ◽

Cited By ~ 35

Author(s):

Anna M. Castle ◽

Amy Y. Huang ◽

J. David Castle

Keyword(s):

Secretory Pathway ◽

Critical Role ◽

Endogenous Hormones ◽

Salivary Amylase ◽

Regulated Secretion ◽

J 13 ◽

Regulated Secretory Pathway ◽

Granule Maturation ◽

Proline Rich Protein

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093– 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711– 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

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Oxidative Stress and Thrombosis during Aging: The Roles of Oxidative Stress in RBCs in Venous Thrombosis

International Journal of Molecular Sciences ◽

10.3390/ijms21124259 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4259

Author(s):

Qinhong Wang ◽

Rahima Zennadi

Keyword(s):

Oxidative Stress ◽

Venous Thrombosis ◽

Life Stage ◽

Redox Homeostasis ◽

Critical Role ◽

Membrane Integrity ◽

Accelerated Aging ◽

Coagulation Factors ◽

Membrane Structure And Function

Mid-life stage adults are at higher risk of developing venous thrombosis (VT)/thromboembolism (VT/E). Aging is characterized by an overproduction of reactive oxygen species (ROS), which could evoke a series of physiological changes involved in thrombosis. Here, we focus on the critical role of ROS within the red blood cell (RBC) in initiating venous thrombosis during aging. Growing evidence has shifted our interest in the role of unjustifiably unvalued RBCs in blood coagulation. RBCs can be a major source of oxidative stress during aging, since RBC redox homeostasis is generally compromised due to the discrepancy between prooxidants and antioxidants. As a result, ROS accumulate within the RBC due to the constant endogenous hemoglobin (Hb) autoxidation and NADPH oxidase activation, and the uptake of extracellular ROS released by other cells in the circulation. The elevated RBC ROS level affects the RBC membrane structure and function, causing loss of membrane integrity, and decreased deformability. These changes impair RBC function in hemostasis and thrombosis, favoring a hypercoagulable state through enhanced RBC aggregation, RBC binding to endothelial cells affecting nitric oxide availability, RBC-induced platelet activation consequently modulating their activity, RBC interaction with and activation of coagulation factors, increased RBC phosphatidylserine exposure and release of microvesicles, accelerated aging and hemolysis. Thus, RBC oxidative stress during aging typifies an ultimate mechanism in system failure, which can affect major processes involved in the development of venous thrombosis in a variety of ways. The reevaluated concept of the critical role of RBC ROS in the activation of thrombotic events during aging will help identify potential targets for novel strategies to prevent/reduce the risk for VT/E or VT/E recurrences in mid-life stage adults.

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The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction

Journal of Virology ◽

10.1128/jvi.01604-20 ◽

2020 ◽

Vol 94 (21) ◽

Author(s):

Victoria J. Madigan ◽

Garrett E. Berry ◽

Tyne O. Tyson ◽

Dasean Nardone-White ◽

Jonathan Ark ◽

...

Keyword(s):

Gene Transfer ◽

Conformational Changes ◽

Secretory Pathway ◽

Critical Role ◽

Calcium Ionophore ◽

Calcium Atpase ◽

Cellular Calcium ◽

Golgi Compartment ◽

Genome Transcription

ABSTRACT Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigated the role of cellular calcium in the AAV infectious pathway. First, we demonstrated a critical role for the host Golgi compartment-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1 [SPCA1]) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake, or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate dispersed and punctate trafficking distinct from the perinuclear, trans-Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro. Conversely, the calcium ionophore ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrated that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo. Taking these data together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription. IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigated the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency.

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Role of Phytochemicals in Perturbation of Redox Homeostasis in Cancer

Antioxidants ◽

10.3390/antiox10010083 ◽

2021 ◽

Vol 10 (1) ◽

pp. 83

Author(s):

Shreyas Gaikwad ◽

Sanjay K. Srivastava

Keyword(s):

Treatment Options ◽

Redox Homeostasis ◽

Critical Role ◽

Threshold Values ◽

Cellular Death ◽

External Stimulation ◽

The Past ◽

Oncogenic Pathways ◽

Ros Homeostasis

Over the past few decades, research on reactive oxygen species (ROS) has revealed their critical role in the initiation and progression of cancer by virtue of various transcription factors. At certain threshold values, ROS act as signaling molecules leading to activation of oncogenic pathways. However, if perturbated beyond the threshold values, ROS act in an anti-tumor manner leading to cellular death. ROS mediate cellular death through various programmed cell death (PCD) approaches such as apoptosis, autophagy, ferroptosis, etc. Thus, external stimulation of ROS beyond a threshold is considered a promising therapeutic strategy. Phytochemicals have been widely regarded as favorable therapeutic options in many diseased conditions. Over the past few decades, mechanistic studies on phytochemicals have revealed their effect on ROS homeostasis in cancer. Considering their favorable side effect profile, phytochemicals remain attractive treatment options in cancer. Herein, we review some of the most recent studies performed using phytochemicals and, we further delve into the mechanism of action enacted by individual phytochemicals for PCD in cancer.

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Critical Role of Caveolin-1 Loss/Dysfunction in Pulmonary Hypertension

Medical Sciences ◽

10.3390/medsci9040058 ◽

2021 ◽

Vol 9 (4) ◽

pp. 58

Author(s):

Rajamma Mathew

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Pulmonary Hypertension ◽

Membrane Protein ◽

Critical Role ◽

High Morbidity ◽

Caveolin 1 ◽

Enhanced Expression ◽

Medial Hypertrophy

Pulmonary hypertension (PH) is a rare disease with a high morbidity and mortality rate. A number of systemic diseases and genetic mutations are known to lead to PH. The main features of PH are altered vascular relaxation responses and the activation of proliferative and anti-apoptotic pathways, resulting in pulmonary vascular remodeling, elevated pulmonary artery pressure, and right ventricular hypertrophy, ultimately leading to right heart failure and premature death. Important advances have been made in the field of pulmonary pathobiology, and several deregulated signaling pathways have been shown to be associated with PH. Clinical and experimental studies suggest that, irrespective of the underlying disease, endothelial cell disruption and/or dysfunction play a key role in the pathogenesis of PH. Endothelial caveolin-1, a cell membrane protein, interacts with and regulates several transcription factors and maintains homeostasis. Disruption of endothelial cells leads to the loss or dysfunction of endothelial caveolin-1, resulting in reciprocal activation of proliferative and inflammatory pathways, leading to cell proliferation, medial hypertrophy, and PH, which initiates PH and facilitates its progression. The disruption of endothelial cells, accompanied by the loss of endothelial caveolin-1, is accompanied by enhanced expression of caveolin-1 in smooth muscle cells (SMCs) that leads to pro-proliferative and pro-migratory responses, subsequently leading to neointima formation. The neointimal cells have low caveolin-1 and normal eNOS expression that may be responsible for promoting nitrosative and oxidative stress, furthering cell proliferation and metabolic alterations. These changes have been observed in human PH lungs and in experimental models of PH. In hypoxia-induced PH, there is no endothelial disruption, loss of endothelial caveolin-1, or enhanced expression of caveolin-1 in SMCs. Hypoxia induces alterations in membrane composition without caveolin-1 or any other membrane protein loss. However, caveolin-1 is dysfunctional, resulting in cell proliferation, medial hypertrophy, and PH. These alterations are reversible upon removal of hypoxia, provided there is no associated EC disruption. This review examined the role of caveolin-1 disruption and dysfunction in PH.

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The membrane protein Raw regulates dendrite pruning via the secretory pathway

Development ◽

10.1242/dev.191155 ◽

2020 ◽

Vol 147 (19) ◽

pp. dev191155

Author(s):

Menglong Rui ◽

Shufeng Bu ◽

Liang Yuh Chew ◽

Qiwei Wang ◽

Fengwei Yu

Keyword(s):

Nervous System ◽

Cell Adhesion ◽

Membrane Protein ◽

Sensory Neurons ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Jnk Signaling

ABSTRACTNeuronal pruning is essential for proper wiring of the nervous systems in invertebrates and vertebrates. Drosophila ddaC sensory neurons selectively prune their larval dendrites to sculpt the nervous system during early metamorphosis. However, the molecular mechanisms underlying ddaC dendrite pruning remain elusive. Here, we identify an important and cell-autonomous role of the membrane protein Raw in dendrite pruning of ddaC neurons. Raw appears to regulate dendrite pruning via a novel mechanism, which is independent of JNK signaling. Importantly, we show that Raw promotes endocytosis and downregulation of the conserved L1-type cell-adhesion molecule Neuroglian (Nrg) prior to dendrite pruning. Moreover, Raw is required to modulate the secretory pathway by regulating the integrity of secretory organelles and efficient protein secretion. Mechanistically, Raw facilitates Nrg downregulation and dendrite pruning in part through regulation of the secretory pathway. Thus, this study reveals a JNK-independent role of Raw in regulating the secretory pathway and thereby promoting dendrite pruning.

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The Critical Role of Redox Homeostasis in Shikonin-Induced HL-60 Cell Differentiation via Unique Modulation of the Nrf2/ARE Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2012/781516 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 10

Author(s):

Bo Zhang ◽

Na Chen ◽

Hongmei Chen ◽

Zhenhua Wang ◽

Qiusheng Zheng

Keyword(s):

Drug Exposure ◽

Target Genes ◽

Redox Homeostasis ◽

Critical Role ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Downstream Target ◽

Dose Dependent Increase

Among various cancer cell lines, the leukemia cell line HL-60 was most sensitive to Shikonin, with evidence showing both the prooxidative activities and proapoptotic effects of micromolar concentrations of Shikonin. However, the mechanism involved in the cytotoxicity of Shikonin in the submicromolar range has not been fully characterized. Using biochemical and free radical biological experimentsin vitro, we identified the prodifferentiated profiles of Shikonin and evaluated the redox homeostasis during HL-60 differentiation. The data showed a strong dose-response relationship between Shikonin exposure and the characteristics of HL-60 differentiation in terms of morphology changes, nitroblue tetrazolium (NBT) reductive activity, and the expression level of surface antigens CD11b/CD14. During drug exposure, intercellular redox homeostasis changes towards oxidation are necessary to support Shikonin-induced differentiation, which was proven by additional enzymatic and non-enzymatic redox modulators. A statistically significant and dose-dependent increase (P<0.05) was recorded with regard to the unique expression levels of the Nrf2/ARE downstream target genes in HL-60 cells undergoing late differentiation, which were restored with further antioxidants employed with the Shikonin treatment. Our research demonstrated that Shikonin is a differentiation-inducing agent, and its mechanisms involve the Nrf2/ARE pathway to modulate the intercellular redox homeostasis, thus facilitating differentiation.

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Intracellular Redox-Modulated Pathways as Targets for Effective Approaches in the Treatment of Viral Infection

International Journal of Molecular Sciences ◽

10.3390/ijms22073603 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3603

Author(s):

Alessandra Fraternale ◽

Carolina Zara ◽

Marta De Angelis ◽

Lucia Nencioni ◽

Anna Teresa Palamara ◽

...

Keyword(s):

Viral Infection ◽

Disulfide Bonds ◽

Inflammatory Responses ◽

Redox Homeostasis ◽

Effective Immune Response ◽

Cellular Pathways ◽

Key Events ◽

Protein Disulfide ◽

Host Genes

Host-directed therapy using drugs that target cellular pathways required for virus lifecycle or its clearance might represent an effective approach for treating infectious diseases. Changes in redox homeostasis, including intracellular glutathione (GSH) depletion, are one of the key events that favor virus replication and contribute to the pathogenesis of virus-induced disease. Redox homeostasis has an important role in maintaining an appropriate Th1/Th2 balance, which is necessary to mount an effective immune response against viral infection and to avoid excessive inflammatory responses. It is known that excessive production of reactive oxygen species (ROS) induced by viral infection activates nuclear factor (NF)-kB, which orchestrates the expression of viral and host genes involved in the viral replication and inflammatory response. Moreover, redox-regulated protein disulfide isomerase (PDI) chaperones have an essential role in catalyzing formation of disulfide bonds in viral proteins. This review aims at describing the role of GSH in modulating redox sensitive pathways, in particular that mediated by NF-kB, and PDI activity. The second part of the review discusses the effectiveness of GSH-boosting molecules as broad-spectrum antivirals acting in a multifaceted way that includes the modulation of immune and inflammatory responses.

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