scholarly journals Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3118
Author(s):  
Supansa Pantoom ◽  
Adam Pomorski ◽  
Katharina Huth ◽  
Christina Hund ◽  
Janine Petters ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Copper Metabolism ◽  
Central Protein ◽  
Potential Binding ◽  
Normal Human ◽  
Autophagy Pathway ◽  
Interaction Regions ◽  
Cellular Copper

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B−/−) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

scholarly journals Direct Interaction of ATP7B and LC3B Proteins Suggests a Cooperative Role of Copper Transportation and Autophagy

2021 ◽  
Author(s):  
Supansa Pantoom ◽  
Adam Pomorski ◽  
Katharina Huth ◽  
Christina Hund ◽  
Janine Petters ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Copper Metabolism ◽  
Central Protein ◽  
Potential Binding ◽  
Normal Human ◽  
Autophagy Pathway ◽  
Interaction Regions ◽  
Cellular Copper

Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in autophagy pathway, so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B-/-) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.

Neuronal plasticity in chronic pancreatitis is mediated via the neurturin/GFRα2 axis

2012 ◽  
Vol 303 (9) ◽  
pp. G1017-G1028 ◽  
Author(s):  
Ihsan Ekin Demir ◽  
Kun Wang ◽  
Elke Tieftrunk ◽  
Nathalia A. Giese ◽  
Baocai Xing ◽  
...  
Keyword(s):  
Chronic Pancreatitis ◽  
Glial Cell ◽  
Neurotrophic Factor ◽  
Human Pancreas ◽  
Parasympathetic Innervation ◽  
Tissue Extracts ◽  
Normal Human ◽  
The Impact

The glial cell line-derived neurotrophic factor (GDNF) family member neurturin (NRTN) and its receptor GFRα2 play a deciding role in the normal development of pancreatic parasympathetic innervation. In this study, we aimed at investigating the role of NRTN/GFRα2 axis in pancreatic neuropathy in human chronic pancreatitis (CP). Expression of NRTN/GFRα2 was compared between normal human pancreas (NP) and CP tissues via immunohistochemistry, immunoblotting, and quantitative RT-PCR and correlated to abdominal pain sensation. To elucidate the impact of NRTN in pancreatic neuroplasticity, neuronal phenotype and glial density were quantified via an in vitro neuroplasticity assay in dissociated newborn rat dorsal root ganglia (DRG) cultured 1) in CP tissue extracts depleted from NRTN, 2) in NP, 3) in untreated CP tissue extracts, and 4) CP extracts in which nerve growth factor, glial cell derived-neurotrophic factor, or TGF-β1was depleted. NRTN and GFRα2 were highly upregulated in CP, especially in intrapancreatic nerves and the extracellular matrix. CP tissue demonstrated increased amounts of mature multimeric NRTN and elevated levels of GFRα2. The noticeable neurotrophic effect of CP tissue extracts on DRG neurons was diminished upon blockade of NRTN from these extracts. However, blockade of NRTN from CP extracts did not influence the density of DRG glia cells. In conclusion, the NRTN/GFRα2 axis is activated during the course of CP and represents a major key player in the reactive neural alterations in CP. This is the first study to provide functional evidence for the contribution of neurotrophic factors to neuroplasticity in CP.

scholarly journals Glioma malignancy is linked to interdependent and inverse AMOG and L1 adhesion molecule expression

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Qiong Jiang ◽  
Qing Xie ◽  
Chengliang Hu ◽  
Zhai Yang ◽  
Peizhi Huang ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Glioma Cells ◽  
Tissue Microarrays ◽  
Human Gliomas ◽  
Glial Tumors ◽  
Therapeutic Choice ◽  
Degree Of Malignancy ◽  
Normal Human

Abstract Background Gliomas account for the majority of primary human brain tumors and remain a challenging neoplasm for cure due to limited therapeutic options. Cell adhesion molecules play pivotal roles in the growth and progression of glial tumors. Roles of the adhesion molecules on glia (AMOG) and L1CAM (L1) in glioma cells have been shown to correlate with tumorigenesis: Increased expression of L1 and decreased expression of AMOG correlate with degree of malignancy. Methods We evaluated the interdependence in expression of these molecules by investigating the role of AMOG in vitro via modulation of L1 expression and analyzing apoptosis and cell senescence of glioma cells. Results Immunohistochemical staining of normal human cortical and glioma tissue microarrays demonstrated that AMOG expression was lower in human gliomas compared to normal tissue and is inversely correlated with the degree of malignancy. Moreover, reduction of AMOG expression in human glioblastoma cells elevated L1 expression, which is accompanied by decreased cell apoptosis as well as senescence. Conclusion AMOG and L1 interdependently regulate their expression levels not only in U-87 MG cells but also in U251 and SHG44 human glioma cell lines. The capacity of AMOG to reduce L1 expression suggests that methods for increasing AMOG expression may provide a therapeutic choice for the management of glial tumors with high expression of L1.

scholarly journals Structural basis of the interplay between α-synuclein and Tau in regulating pathological amyloid aggregation

2020 ◽  
Vol 295 (21) ◽  
pp. 7470-7480 ◽  
Author(s):  
Jinxia Lu ◽  
Shengnan Zhang ◽  
Xiaojuan Ma ◽  
Chunyu Jia ◽  
Zhenying Liu ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Neurodegenerative Diseases ◽  
Direct Interaction ◽  
Structural Basis ◽  
Amyloid Aggregation ◽  
C Terminus ◽  
Amyloid Fibrillation

Amyloid aggregation of pathological proteins is closely associated with a variety of neurodegenerative diseases, and α-synuclein (α-syn) deposition and Tau tangles are considered hallmarks of Parkinson's disease and Alzheimer's disease, respectively. Intriguingly, α-syn and Tau have been found to co-deposit in the brains of individuals with dementia and parkinsonism, suggesting a potential role of cross-talk between these two proteins in neurodegenerative pathologies. Here we show that monomeric α-syn and the two variants of Tau, Tau23 and K19, synergistically promote amyloid fibrillation, leading to their co-aggregation in vitro. NMR spectroscopy experiments revealed that α-syn uses its highly negatively charged C terminus to directly interact with Tau23 and K19. Deletion of the C terminus effectively abolished its binding to Tau23 and K19 as well as its synergistic effect on promoting their fibrillation. Moreover, an S129D substitution of α-syn, mimicking C-terminal phosphorylation of Ser129 in α-syn, which is commonly observed in the brains of Parkinson's disease patients with elevated α-syn phosphorylation levels, significantly enhanced the activity of α-syn in facilitating Tau23 and K19 aggregation. These results reveal the molecular basis underlying the direct interaction between α-syn and Tau. We proposed that this interplay might contribute to pathological aggregation of α-syn and Tau in neurodegenerative diseases.

CIAPIN1 is a potential target for apoptosis of multiple myeloma

2019 ◽  
Vol 9 (9) ◽  
pp. 1106-1111
Author(s):  
Xiao-Bo Wang ◽  
Le-Ping Yan ◽  
Li-Hua Yuan ◽  
Bo Lu ◽  
Dong-Jun Lin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Xenograft Model ◽  
Soft Agar ◽  
Normal Human ◽  
Related Proteins

This study firstly aimed to reveal the gene expression differences of CIAPIN1 between myelomas cells from bone marrow cells of multiple myeloma patients and normal human, and subsequently investigate the regulation role of this gene on tumorigenicity ability of multiple myeloma (MM) cell line U266 via in vitro colony formation and in vivo xenograft studies. RT-PCR results obtained from 18 MM patients and 10 health people showed that the expression of CIAPIN1 gene was 4 times higher in normal human compared to MM patients. Besides, CIAPIN1 siRNA (si-CIAPIN1) transfected U266 cells presented higher proliferation ratio and superior colony forming ability than U266 cells and U266 cells transfected with non-coding siRNA (controls) evaluated by CCK8 test and soft agar colony formation assay, respectively. In a mice MM xenograft model, the si-CIAPIN1 transfected U266 cells induced the biggest tumor compared to the controls. Furthermore, CIAPIN1 overexpressed U266 cells were developed and compared with the si-CIAPIN1 transfected U266 cells to study the role of CIAPIN1 in the production of apoptosis related proteins in U266 cells. Results indicated that CIAPIN1 facilitated apoptosis promoting proteins expression in U266 cells, such as upregulation of BAX, BAK, Bcl-xs and BIM, and downregulation of p38, PKC, Bcl-2 and Bcl-xl proteins. Therefore, CIAPIN1 can be a potential suppression target gene in multiple myeloma.

scholarly journals Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake

2007 ◽  
Vol 407 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Peter V. E. van den Berghe ◽  
Dineke E. Folmer ◽  
Helga E. M. Malingré ◽  
Ellen van Beurden ◽  
Adriana E. M. Klomp ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Fluorescent Protein ◽  
Copper Metabolism ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Copper Uptake ◽  
Copper Transporter ◽  
Late Endosomes ◽  
Cellular Copper

High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2–vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2–vsvG. Furthermore, hCTR2–vsvG was co-immunoprecipitated with hCTR2–FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2–eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)–luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE–luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 μM CuCl2, consistent with the Km of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE–luciferase reporter activation in a copper-dependent manner at 40 and 100 μM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.

scholarly journals Cells with Fc gamma receptors from normal donors suppress granulocytic macrophage colony formation

Blood ◽  
1982 ◽  
Vol 60 (3) ◽  
pp. 758-766 ◽  
Author(s):  
G Spitzer ◽  
DS Verma
Keyword(s):  
Blood Cells ◽  
Colony Formation ◽  
Regulatory Mechanism ◽  
Peripheral Blood Cells ◽  
Normal Human ◽  
Macrophage Colony ◽  
Mitogen Activation ◽  
Marrow Cells

Abstract We investigated the role of normal human marrow cells with Fc receptors for IgG (Fc gamma+) on autologous granulocyte-macrophage colony (GM- CFC) formation. It was found that Fc gamma+ normal human marrow cells, both with (E+) or without receptors for sheep erythrocytes suppressed GM-CFC at as low a concentration as 0.25 X 10(5) cells/ml of culture. A similar effect was observed with E- Fc gamma+ but not E+ Fc gamma+ peripheral blood cells. Suppression by Fc gamma+ cells did not require mitogen activation and was not inactivated by irradiation (2000 R). This report presents a new in vitro regulatory mechanism for GM-CFC growth in normal donors.

Effect of Benoxaprofen on Release of Slow-Reacting Substances from Human Lung Tissue In Vitro

1982 ◽  
Vol 63 (2) ◽  
pp. 219-221 ◽  
Author(s):  
V. Y. Lee ◽  
J. Margaret Hughes ◽  
J. P. Seale ◽  
Diana M. Temple
Keyword(s):  
Lung Cancer ◽  
Lung Tissue ◽  
Human Lung ◽  
Calcium Ionophore ◽  
Inhibitory Effects ◽  
Human Lung Tissue ◽  
Normal Human ◽  
Clinical Asthma

1. Macroscopically normal human lung tissue was obtained from operative specimens removed for lung cancer and challenged with antigen or calcium ionophore. The release of histamine and slow-reacting substances was measured by fluorimetric and bioassay techniques respectively. 2. Benoxaprofen, a drug with inhibitory effects on the lipoxygenase and cyclo-oxygenase pathways, caused a dose-related reduction of release of slow-reacting substances without affecting histamine release. 3. These results with human lung tissue in vitro suggest that benoxaprofen may be used to investigate the role of slow-reacting substances in experimental and clinical asthma.

scholarly journals Role of a Cdc42p Effector Pathway in Recruitment of the Yeast Septins to the Presumptive Bud Site

2006 ◽  
Vol 17 (3) ◽  
pp. 1110-1125 ◽  
Author(s):  
Masayuki Iwase ◽  
Jianying Luo ◽  
Satish Nagaraj ◽  
Mark Longtine ◽  
Hyong Bai Kim ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Ring Formation ◽  
Cell Cortex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Distinct Structure ◽  
Effector Pathway ◽  
Mutant Phenotypes ◽  
Bud Site

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p.

scholarly journals In vivo reconstitution of autophagy in Saccharomyces cerevisiae

2008 ◽  
Vol 182 (4) ◽  
pp. 703-713 ◽  
Author(s):  
Yang Cao ◽  
Heesun Cheong ◽  
Hui Song ◽  
Daniel J. Klionsky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Networks ◽  
In Vitro System ◽  
Atg Genes ◽  
Minimum Requirements ◽  
Autophagy Pathway ◽  
Cellular Dysfunction

Autophagy is a major intracellular degradative pathway that is involved in various human diseases. The role of autophagy, however, is complex; although the process is generally considered to be cytoprotective, it can also contribute to cellular dysfunction and disease progression. Much progress has been made in our understanding of autophagy, aided in large part by the identification of the autophagy-related (ATG) genes. Nonetheless, our understanding of the molecular mechanism remains limited. In this study, we generated a Saccharomyces cerevisiae multiple-knockout strain with 24 ATG genes deleted, and we used it to carry out an in vivo reconstitution of the autophagy pathway. We determined minimum requirements for different aspects of autophagy and studied the initial protein assembly steps at the phagophore assembly site. In vivo reconstitution enables the study of autophagy within the context of the complex regulatory networks that control this process, an analysis that is not possible with an in vitro system.

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