scholarly journals Optineurin mediates delivery of membrane from transferrin receptor-positive endosomes to autophagosomes

2022 ◽  
Author(s):  
Megha Bansal ◽  
Kapil Sirohi ◽  
Shivranjani C Moharir ◽  
Ghanshyam Swarup
Keyword(s):  
Transferrin Receptor ◽  
Control Mechanism ◽  
Adaptor Protein ◽  
Wild Type ◽  
Autophagosome Formation ◽  
Embryonic Fibroblasts ◽  
Over Expression ◽  
Ubiquitin Binding ◽  
Quality Control Mechanism

Autophagy is a conserved quality control mechanism that removes damaged proteins, organelles and invading bacteria through lysosome-mediated degradation. During autophagy several organelles including endoplasmic reticulum, mitochondria, plasma membrane and endosomes contribute membrane for autophagosome formation. However, the mechanisms and proteins involved in membrane delivery to autophagosomes are not clear. Optineurin (OPTN), a cytoplasmic adaptor protein, is involved in promoting maturation of phagophores into autophagosomes; it is also involved in regulating endocytic trafficking and recycling of transferrin receptor (TFRC). Here, we have examined the role of optineurin in the delivery of membrane from TFRC-positive endosomes to autophagosomes. Only a small fraction of autophagosomes was positive for TFRC, indicating that TFRC-positive endosomes could contribute membrane to a subset of autophagosomes. The percentage of TFRC-positive autophagosomes was reduced in Optineurin knockout mouse embryonic fibroblasts (Optn-/-MEFs) in comparison with normal MEFs. Upon over-expression of optineurin, the percentage of TFRC-positive autophagosomes was increased in Optn-/- MEFs. Unlike wild-type optineurin, a disease-associated mutant, E478G, defective in ubiquitin binding, was not able to enhance formation of TFRC-positive autophagosomes in Optn-/- MEFs. TFRC degradation mediated by autophagy was decreased in optineurin deficient cells. Our results suggest that optineurin mediates delivery of TFRC and perhaps associated membrane from TFRC-positive endosomes to autophagosomes, and this may contribute to autophagosome formation.

scholarly journals Novel Insights into the Role of UBE3A in Regulating Apoptosis and Proliferation

2020 ◽  
Vol 9 (5) ◽  
pp. 1573 ◽  
Author(s):  
Lilach Simchi ◽  
Julia Panov ◽  
Olla Morsy ◽  
Yonatan Feuermann ◽  
Hanoch Kaphzan
Keyword(s):  
Angelman Syndrome ◽  
Critical Role ◽  
Embryonic Fibroblasts ◽  
Ubiquitin E3 Ligase ◽  
Proliferation And Apoptosis ◽  
Ube3a Gene ◽  
Ubiquitin Binding ◽  
Molecular Effects ◽  
Substrate Proteins

The UBE3A gene codes for a protein with two known functions, a ubiquitin E3-ligase which catalyzes ubiquitin binding to substrate proteins and a steroid hormone receptor coactivator. UBE3A is most famous for its critical role in neuronal functioning. Lack of UBE3A protein expression leads to Angelman syndrome (AS), while its overexpression is associated with autism. In spite of extensive research, our understanding of UBE3A roles is still limited. We investigated the cellular and molecular effects of Ube3a deletion in mouse embryonic fibroblasts (MEFs) and Angelman syndrome (AS) mouse model hippocampi. Cell cultures of MEFs exhibited enhanced proliferation together with reduced apoptosis when Ube3a was deleted. These findings were supported by transcriptome and proteome analyses. Furthermore, transcriptome analyses revealed alterations in mitochondria-related genes. Moreover, an analysis of adult AS model mice hippocampi also found alterations in the expression of apoptosis- and proliferation-associated genes. Our findings emphasize the role UBE3A plays in regulating proliferation and apoptosis and sheds light into the possible effects UBE3A has on mitochondrial involvement in governing this balance.

scholarly journals The J-related Segment of Tim44 Is Essential for Cell Viability: A Mutant Tim44 Remains in the Mitochondrial Import Site, but Inefficiently Recruits mtHsp70 and Impairs Protein Translocation

1999 ◽  
Vol 145 (5) ◽  
pp. 961-972 ◽  
Author(s):  
Alessio Merlin ◽  
Wolfgang Voos ◽  
Ammy C. Maarse ◽  
Michiel Meijer ◽  
Nikolaus Pfanner ◽  
...  
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
J Proteins

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Δ18 in addition to the endogenous wild-type Tim44. Tim44Δ18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Δ18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Δ18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

Role of HFE in Hepatic Iron Overload: Novel Findings Demonstrate Direct Effects of Mutant C282Y on Hepcidin and Calreticulin mRNA Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1549-1549
Author(s):  
Jorge P. Pinto ◽  
Pedro Ramos ◽  
Sergio de Almeida ◽  
Susana Oliveira ◽  
Laura Breda ◽  
...  
Keyword(s):  
Protective Effect ◽  
Cell Line ◽  
Mononuclear Cells ◽  
Hepatic Cell ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Mutant Proteins ◽  
Over Expression ◽  
Blood Mononuclear Cells

Abstract Studies done in non-hepatic cell lines, focusing on the interaction between HFE with TFR1 and β-2M proved insufficient to explain the discrepancies found in the clinical penetrance of hemochromatosis in subjects carrying the C282Y mutation. Our first goal was to investigate the role of HFE wild type (wt) and mutant proteins (C282Y and H63D) in a human hepatic cell line, focusing on the cellular localization and interaction of HFE with the expression of other iron related proteins. HFE mutant C282Y was found to be retained in the endoplasmic reticulum (ER). Thus, in addition, we investigated the effect of HFE wt and mutant proteins on Calreticulin, which is a chaperon protein that responds to ER stress and has a protective effect on oxidative damage in some cell lines. Here we report setting up a stable transfection of wt- and mutant-HFE in a hepatic cell line (HepG2) and examine the intracellular distribution of wt- and HFE mutants, their effect on iron intake independently of TFR1 and on the expression of other iron and ER stress response genes, namely Hepcidin and Calreticulin. In addition, we validated some of the novel effects of HFE on Calreticulin using peripheral blood mononuclear cells from HFE patients. The localization of the HFE variants was analyzed using KDEL and Golgin-97 as ER and the Golgi complex markers, respectively. HFE C282Y shows a high degree of overlap with the ER markers, confirming a retention of this variant in this organelle. Over-expression of the HFE wt impaired the intake of 55Fe relatively to transfected control cells (P<0.008) independently of TFR1, as demonstrated by RNAi silencing. Hamp RNA expression was decreased in cells over expressing C282Y in comparison to HFE wt cells (P<0.011). Finally over-expression of HFE wt decreases Calreticulin mRNA, whereas the C282Y had an opposite effect, compared to the control cell line. A similar result was observed in peripheral blood mononuclear cells (PMBC) of C282Y homozygous HFE patients, compared to wild type blood donors (P<0.006). Interestingly, this data suggest that synthesis of the HFE mutant C282Y triggers a protective effect on oxidative damage mediated by Calreticulin. In fact, HepG2 cells over-expressing C282Y showed lower levels of ROS than HFE wt (P<0.004). This observation might contribute to explain some of the discrepancies seen in the clinical penetrance of the disease in C282Y carrying subjects. The direct effect of the mutant HFE C282Y on mRNA expression of hepcidin also demonstrated here for the first time corroborates and provides a molecular basis for earlier reports of low hepcidin levels in HH patients and in Hfe-KO mice.

scholarly journals The mammalian ULK1 complex and autophagy initiation

2017 ◽  
Vol 61 (6) ◽  
pp. 585-596 ◽  
Author(s):  
Maria Zachari ◽  
Ian G. Ganley
Keyword(s):  
Control Mechanism ◽  
Degradation Pathway ◽  
Autophagosome Formation ◽  
Interacting Protein ◽  
Initial Event ◽  
Protein Partners ◽  
Recycling Pathway ◽  
Quality Control Mechanism ◽  
Cellular Components ◽  
Membrane Organelle

Autophagy is a vital lysosomal degradation pathway that serves as a quality control mechanism. It rids the cell of damaged, toxic or excess cellular components, which if left to persist could be detrimental to the cell. It also serves as a recycling pathway to maintain protein synthesis under starvation conditions. A key initial event in autophagy is formation of the autophagosome, a unique double-membrane organelle that engulfs the cytosolic cargo destined for degradation. This step is mediated by the serine/threonine protein kinase ULK1 (unc-51-like kinase 1), which functions in a complex with at least three protein partners: FIP200 (focal adhesion kinase family interacting protein of 200 kDa), ATG (autophagy-related protein) 13 (ATG13), and ATG101. In this artcile, we focus on the regulation of the ULK1 complex during autophagy initiation. The complex pattern of upstream pathways that converge on ULK1 suggests that this complex acts as a node, converting multiple signals into autophagosome formation. Here, we review our current understanding of this regulation and in turn discuss what happens downstream, once the ULK1 complex becomes activated.

Tumor Suppressor Microrna-29a/b and Microrna-34a Mediate Small Molecule PRIMA-1Met-Induced Aoptosis In Multiple Myeloma Cells By Targeting c-Myc

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1919-1919
Author(s):  
Manujendra N. Saha ◽  
Yijun Yang ◽  
Hong Chang
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Mutant P53 ◽  
Pcr Array ◽  
Wild Type ◽  
Down Regulation ◽  
Over Expression ◽  
P53 Status ◽  
Low Expression

Abstract PRIMA-1Met/APR246 (p53 reactivation and induction of massive apoptosis), is a small molecule with remarkable anti-tumor activities in various human tumor cells, and is currently under phase I/II clinical trial. We have previously demonstrated anti-tumor activity of PRIMA-1Met in multiple myeloma (MM) cells irrespective of p53 status. In addition, we have shown that PRIMA-1Met alone or in combination with dexamethasone triggers significant tumor growth inhibition in vivo in a murine xenograft model of human MM. However, the molecular mechanism underlying anti-myeloma activity of PRIMA-1Met has not been fully elucidated. MicroRNAs (miRNAs) are non-coding small RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Since the role of miRNAs and their regulation in response to PRIMA-1Met in MM is not known, here we investigated the relationship between PRIMA-1Met-induced apoptosis and miRNA expression in MM cells. Using a miRNA PCR array platform (Human Cancer Pathway Finder miScript miRNA PCR array, MIHS-102Z, Qiagen Inc), we analyzed the miRNA profiles in two MM cell lines of different p53 status (MM.1S with wild type p53 and 8226 with mutant p53) treated with either PRIMA-1Met or DMSO control. After normalization to a set of housekeeping genes, differential expressions of the miRNAs were analysed. miRNA-29a, miRNA-29b, and miRNA-34a were found significantly up-regulated (more than 2 fold, p<0.05) in cells treated with PRIMA-1Met compared to DMSO-treated cells. To evaluate the effect of over-expression of these miRNAs, we transfected two MM cell lines (MM.1S and 8226) with either miR-29a/b or miR-34a. Cells transfected with scramble miRNA were used as control. Over-expression of the miRNAs resulted in a dose-dependent inhibition of viability and increase in apoptosis of MM.1S or 8226 cells. Next, we examined the endogenous expression of these miRNAs in 5 primary MM samples by qPCR. Results showed a significant low expression of miR-29a/b and miR-34a in 3 of the 5 samples. Treatment of the two primary MM samples with low expression for miR-29a/b and miR-34a with PRIMA-1Met resulted in up-regulation of these miRNAs leading to inhibition of the viability and induction of apoptosis. To identify the possible targets of these miRNAs, we performed bioinformatics analysis. Results obtained from different searches by miRanda and TargetScan algorithm predicted c-Myc as a potential target for miRNA-29a/b and miRNA-34a. c-Myc is an oncogene whose over-expression has been associated with resistance to current chemotherapy in MM. Global gene expression profiling by microarray showed significant down-regulation of c-Myc in two MM cell lines with either wild type or mutant p53 treated with PRIMA-1Met compare to cells treated with DMSO. Importantly, down-regulation of c-Myc (∼2.6-fold) by PRIMA-1Met was also observed in a MM cell line (8226R5) lacking p53 expression suggesting an important role of c-Myc in p53-independent apoptosis of MM cells induced by PRIMA-1Met. By qPCR and Western blot analysis, we confirmed significant down-regulation of c-Myc in PRIMA-1Met-treated MM cells. These data provided the evidence for an inverse correlation between the expression of these miRNAs and c-Myc indicating that apoptosis of MM cells induced by PRIMA-1Met is regulated by miRNAs29a/b or miRNA34a targeting c-Myc. Our results suggest a novel mechanism for PRIMA-1Met-induced apoptotic signaling in MM cells mediated by up-regulation of miR-29a/b and miR-34a targeting c-Myc. Our findings also provide a preclinical framework for development of therapeutic strategies in combination of PRIMA-1Met and miRNA (miR-29a/b or miR-34a) mimics for the treatment of MM patients, especially for those with high c-Myc expressions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Essential Role of Nuclear Factor (NF)-κB–Inducing Kinase and Inhibitor of κb (Iκb) Kinase α in Nf-κb Activation through Lymphotoxin β Receptor, but Not through Tumor Necrosis Factor Receptor I

2001 ◽  
Vol 193 (5) ◽  
pp. 631-636 ◽  
Author(s):  
Akemi Matsushima ◽  
Tsuneyasu Kaisho ◽  
Paul D. Rennert ◽  
Hiroyasu Nakano ◽  
Kyoko Kurosawa ◽  
...  
Keyword(s):  
Tumor Necrosis ◽  
Essential Role ◽  
Nuclear Factor ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Iκb Kinase ◽  
Β Receptor ◽  
Deficient Mice ◽  
Necrosis Factor

Both nuclear factor (NF)-κB–inducing kinase (NIK) and inhibitor of κB (IκB) kinase (IKK) have been implicated as essential components for NF-κB activation in response to many external stimuli. However, the exact roles of NIK and IKKα in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKα in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin β receptor (LTβR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IκBα in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTβR stimulation induced NF-κB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKα-deficient mice. Consistent with the essential role of IKKα in LTβR signaling, we found that development of Peyer's patches was defective in IKKα-deficient mice. These results demonstrate that both NIK and IKKα are essential for the induction of NF-κB through LTβR, whereas the NIK–IKKα pathway is dispensable in TNFR-I signaling.

scholarly journals Essential Roles of Receptor-Interacting Protein and TRAF2 in Oxidative Stress-Induced Cell Death

2004 ◽  
Vol 24 (13) ◽  
pp. 5914-5922 ◽  
Author(s):  
Han-Ming Shen ◽  
Yong Lin ◽  
Swati Choksi ◽  
Jamie Tran ◽  
Tian Jin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Tumor Necrosis Factor Receptor ◽  
Membrane Lipid ◽  
Critical Event ◽  
Wild Type ◽  
Embryonic Fibroblasts ◽  
Interacting Protein ◽  
Necrosis Factor

ABSTRACT Oxidative stress and reactive oxygen species (ROS) can elicit and modulate various physiological and pathological processes, including cell death. However, the mechanisms controlling ROS-induced cell death are largely unknown. Data from this study suggest that receptor-interacting protein (RIP) and tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), two key effector molecules of TNF signaling, are essential for ROS-induced cell death. We found that RIP−/− or TRAF2−/− mouse embryonic fibroblasts (MEF) are resistant to ROS-induced cell death when compared to wild-type cells, and reconstitution of RIP and TRAF2 gene expression in their respective deficient MEF cells restored their sensitivity to H2O2-induced cell death. We also found that RIP and TRAF2 form a complex upon H2O2 exposure, but without the participation of TNFR1. The colocalization of RIP with a membrane lipid raft marker revealed a possible role of lipid rafts in the transduction of cell death signal initiated by H2O2. Finally, our results demonstrate that activation of c-Jun NH2-terminal kinase 1 is a critical event downstream of RIP and TRAF2 in mediating ROS-induced cell death. Therefore, our study uncovers a novel signaling pathway regulating oxidative stress-induced cell death.

scholarly journals Cardiac-Specific PID1 Overexpression Enhances Pressure Overload-Induced Cardiac Hypertrophy in Mice

2015 ◽  
Vol 35 (5) ◽  
pp. 1975-1985 ◽  
Author(s):  
Yaoqiu Liu ◽  
Yahui Shen ◽  
Jingai Zhu ◽  
Ming Liu ◽  
Xing Li ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Mapk Pathway ◽  
Pressure Overload ◽  
Wild Type Mouse ◽  
Heart Tissue ◽  
Wild Type ◽  
Heart Transplants ◽  
Over Expression

Background/Aims: PID1 was originally described as an insulin sensitivity relevance protein, which is also highly expressed in heart tissue. However, its function in the heart is still to be elucidated. Thus this study aimed to investigate the role of PID1 in the heart in response to hypertrophic stimuli. Methods: Samples of human failing hearts from the left ventricles of dilated cardiomyopathy (DCM) patients undergoing heart transplants were collected. Transgenic mice with cardiomyocyte-specific overexpression of PID1 were generated, and cardiac hypertrophy was induced by transverse aortic constriction (TAC). The extent of cardiac hypertrophy was evaluated by echocardiography as well as pathological and molecular analyses of heart samples. Results: A significant increase in PID1 expression was observed in failing human hearts and TAC-treated wild-type mouse hearts. When compared with TAC-treated wild-type mouse hearts, PID1-TG mouse showed a significant exacerbation of cardiac hypertrophy, fibrosis, and dysfunction. Further analysis of the signaling pathway in vivo suggested that these adverse effects of PID1 were associated with the inhibition of AKT, and activation of MAPK pathway. Conclusion: Under pathological conditions, over-expression of PID1 promotes cardiac hypertrophy by regulating the Akt and MAPK pathway.

scholarly journals Phosphorylation of Tyrosine Residues 31 and 118 on Paxillin Regulates Cell Migration through an Association with Crk in Nbt-II Cells

2000 ◽  
Vol 148 (5) ◽  
pp. 957-970 ◽  
Author(s):  
Valérie Petit ◽  
Brigitte Boyer ◽  
Delphine Lentz ◽  
Christopher E. Turner ◽  
Jean Paul Thiery ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Critical Role ◽  
Adaptor Protein ◽  
Signaling Molecules ◽  
Sh2 Domain ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Pathological Conditions

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin–Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin–Crk complex in the collagen-induced cell motility.

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