scholarly journals Lysosomal degradation ensures accurate chromosomal segregation to prevent genomic instability

2019 ◽  
Author(s):  
Eugènia Almacellas ◽  
Charles Day ◽  
Santiago Ambrosio ◽  
Albert Tauler ◽  
Caroline Mauvezin
Keyword(s):  
Genomic Instability ◽  
Chromosomal Instability ◽  
Regulatory Protein ◽  
Genetic Material ◽  
Mitotic Division ◽  
Mitotic Progression ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Chromosomal Segregation ◽  
Interphase Cells

ABSTRACTLysosomes, as primary degradative organelles, are the end-point of different converging pathways including macroautophagy. To date, lysosome function has mainly focused on interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition does not only rely on proteasome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel p62-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent genomic instability.

scholarly journals Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability

Autophagy ◽  
2020 ◽  
pp. 1-18
Author(s):  
Eugènia Almacellas ◽  
Joffrey Pelletier ◽  
Charles Day ◽  
Santiago Ambrosio ◽  
Albert Tauler ◽  
...  
Keyword(s):  
Chromosomal Instability ◽  
Lysosomal Degradation ◽  
Chromosomal Segregation

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R

2007 ◽  
Vol 292 (5) ◽  
pp. F1303-F1313 ◽  
Author(s):  
Xianhua Yi ◽  
Richard Bouley ◽  
Herbert Y. Lin ◽  
Shaliha Bechoua ◽  
Tian-xiao Sun ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Human Kidney ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Parathyroid Hormone Receptor ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
G Protein Coupled ◽  
Regulatory Event

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK1 epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

scholarly journals SKIP Downregulation Increases TGF-β1-Induced Matrix Metalloproteinase-9 Production in Transformed Keratinocytes

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Jelena Kocić ◽  
Victor Villar ◽  
Aleksandra Krstić ◽  
Juan F. Santibanez
Keyword(s):  
Matrix Metalloproteinase ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Regulatory Protein ◽  
Mek Inhibitor ◽  
Matrix Metalloproteinase 9 ◽  
Transformed Cells ◽  
Interacting Protein ◽  
Metalloproteinase 9

Transforming growth factor-beta (TGF-β1) is a potent inductor of matrix metalloproteinase-9 (MMP-9) in transformed cells. Recently, Ski-interacting protein (SKIP) has been described as a regulator of TGF-β1 signal transduction, but its role in the induction of cell malignance by TGF-β1 has not been fully elucidated so far. In the present study, we analyzed the role of SKIP on TGF-β1-induced MMP-9 production. Mouse transformed keratinocytes (PDV) were stably transfected with SKIP antisense construct. We observed that SKIP depletion provoked an enhancement in the expression of MMP-9 in response to TGF-β1 treatment. The downregulation of SKIP produced an enhancement in TGF-β1-activated ERK1,2 MAP kinase as well as increased transactivation of downstream Elk1 transcription factor. The increased MMP-9 production in response to TGF-β1 was dependent of MAPK activation as PD98059, an MEK inhibitor, reduced MMP-9 expression in SKIP antisense transfected cells. Thus, we propose SKIP as a regulatory protein in TGF-β1-induced MMP-9 expression acting by controlling ERK1,2 signaling in transformed cells.

scholarly journals RICK Promotes Inflammation and Lethality after Gram-Negative Bacterial Infection in Mice Stimulated with Lipopolysaccharide

2009 ◽  
Vol 77 (4) ◽  
pp. 1569-1578 ◽  
Author(s):  
Jong-Hwan Park ◽  
Yun-Gi Kim ◽  
Gabriel Núñez
Keyword(s):  
Protein Kinase ◽  
Regulatory Protein ◽  
Mitogen Activated Protein Kinase ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Proinflammatory Response ◽  
Interacting Protein ◽  
Gram Negative ◽  
Mitogen Activated Protein

ABSTRACT RICK (receptor-interacting protein-like interacting caspase-like apoptosis regulatory protein kinase), a serine-threonine kinase, functions downstream of the pattern recognition receptors Nod1 and Nod2 to mediate NF-κB and mitogen-activated protein kinase (MAPK) activation in response to specific microbial stimuli. However, the function of RICK in the recognition and host defense of gram-negative bacteria remains poorly understood. We report here that infection of wild-type and RICK-deficient macrophages with Pseudomonas aeruginosa and Escherichia coli elicited comparable activation of NF-κB and MAPKs as well as secretion of proinflammatory cytokines. However, production of interleukin 6 (IL-6) and IL-1β induced by these gram-negative bacteria was impaired in RICK-deficient macrophages when the cells had previously been stimulated with lipopolysaccharide (LPS) or E. coli. The diminished proinflammatory response of RICK-deficient macrophages to bacteria was associated with reduced activation of NF-κB and MAPKs. Importantly, mutant mice deficient in RICK were less susceptible than wild-type mice to P. aeruginosa infection when the animals had previously been stimulated with LPS. The reduced lethality of RICK-deficient mice infected with P. aeruginosa was independent of pathogen clearance but was associated with diminished production of proinflammatory molecules in vivo. These results demonstrate that RICK contributes to the induction of proinflammatory responses and susceptibility to gram-negative bacteria after exposure to LPS, a condition that is associated with reduced Toll-like receptor signaling.

Abstract 1462: The oncogene MYC induces chromosomal instability through dysregulating mitotic progression evoking a dependency on TPX2

2018 ◽  
Author(s):  
Julia Rohrberg ◽  
Alexandra Corella ◽  
Moufida Taileb ◽  
Marie-Lena Jokisch ◽  
Roman Camarda ◽  
...  
Keyword(s):  
Chromosomal Instability ◽  
Mitotic Progression

scholarly journals Evidence for Regulation of Mitotic Progression through Temporal Phosphorylation and Dephosphorylation of CK2α

2009 ◽  
Vol 29 (8) ◽  
pp. 2068-2081 ◽  
Author(s):  
Nicole A. St-Denis ◽  
D. Richard Derksen ◽  
David W. Litchfield
Keyword(s):  
Protein Kinase Ck2 ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Catastrophe ◽  
Mitotic Progression ◽  
Proliferating Cells ◽  
Cultured Fibroblasts ◽  
Catalytic Subunits ◽  
Chromosomal Segregation ◽  
Mitotic Phosphorylation ◽  
Kinase Ck2

ABSTRACT Proper mitotic progression is crucial for maintenance of genomic integrity in proliferating cells and is regulated through an intricate series of events, including protein phosphorylation governed by a complex network of protein kinases. One kinase family implicated in the regulation of mitotic progression is protein kinase CK2, a small family of enzymes that is overexpressed in cancer and induces transformation in mice and cultured fibroblasts. CK2α, one isoform of the catalytic subunits of CK2, is maximally phosphorylated at four sites in nocodazole-treated cells. To investigate the effects of CK2α phosphorylation on mitotic progression, we generated phosphospecific antibodies against its mitotic phosphorylation sites. In U2OS cells released from S-phase arrest, these antibodies reveal that CK2α is most highly phosphorylated in prophase and metaphase. Phosphorylation gradually decreases during anaphase and becomes undetectable during telophase and cytokinesis. Stable expression of phosphomimetic CK2α (CK2α-4D, CK2α-4E) results in aberrant centrosome amplification and chromosomal segregation defects and loss of mitotic cells through mitotic catastrophe. Conversely, cells expressing nonphosphorylatable CK2α (CK2α-4A) show a decreased ability to arrest in mitosis following nocodazole treatment, suggesting involvement in the spindle assembly checkpoint. Collectively, these studies indicate that reversible phosphorylation of CK2α requires precise regulation to allow proper mitotic progression.

scholarly journals Mechanisms and Clinical Applications of Genome Instability in Multiple Myeloma

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Antonia Cagnetta ◽  
Davide Lovera ◽  
Raffaella Grasso ◽  
Nicoletta Colombo ◽  
Letizia Canepa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Genomic Instability ◽  
Molecular Basis ◽  
Genome Instability ◽  
Dna Recombination ◽  
Clinical Applications ◽  
Response To Treatment ◽  
Chromosomal Segregation ◽  
Telomere Function ◽  
Mitotic Regulation

Ongoing genomic instability represents a hallmark of multiple myeloma (MM) cells, which manifests largely as whole chromosome- or translocation-based aneuploidy. Importantly, although it supports tumorigenesis, progression and, response to treatment in MM patients, it remains one of the least understood components of malignant transformation in terms of molecular basis. Therefore these aspects make the comprehension of genomic instability a pioneering strategy for novel therapeutic and clinical speculations to use in the management of MM patients. Here we will review mechanisms mediating genomic instability in MM cells with an emphasis placed on pathogenic mutations affecting DNA recombination, replication and repair, telomere function and mitotic regulation of spindle attachment, centrosome function, and chromosomal segregation. We will discuss the mechanisms by which genetic aberrations give rise to multiple pathogenic events required for myelomagenesis and conclude with a discussion of the clinical applications of these findings in MM patients.

scholarly journals An essential role for the Glut1 PDZ-binding motif in growth factor regulation of Glut1 degradation and trafficking

2009 ◽  
Vol 418 (2) ◽  
pp. 345-367 ◽  
Author(s):  
Heather L. Wieman ◽  
Sarah R. Horn ◽  
Sarah R. Jacobs ◽  
Brian J. Altman ◽  
Sally Kornbluth ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Glucose Uptake ◽  
Pdz Domain ◽  
Binding Motif ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Surface Localization ◽  
Cell Surface Localization ◽  
Growth Factor Regulation

Cell surface localization of the Glut (glucose transporter), Glut1, is a cytokine-controlled process essential to support the metabolism and survival of haemopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. In the present study, we show that a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence through deletion or point mutation sharply decreased surface Glut1 levels and led to rapid targeting of internalized Glut1 to lysosomes for proteolysis, particularly in growth factor-deprived cells. The PDZ-domain protein, GIPC (Gα-interacting protein-interacting protein, C-terminus), bound to Glut1 in part via the Glut1 C-terminal PDZ-binding motif, and we found that GIPC deficiency decreased Glut1 surface levels and glucose uptake. Unlike the Glut1 degradation observed on mutation of the Glut1 PDZ-binding domain, however, GIPC deficiency resulted in accumulation of intracellular Glut1 in a pool distinct from the recycling pathway of the TfR (transferrin receptor). Blockade of Glut1 lysosomal targeting after growth factor withdrawal also led to intracellular accumulation of Glut1, a portion of which could be rapidly restored to the cell surface after growth factor stimulation. These results indicate that the C-terminal PDZ-binding motif of Glut1 plays a key role in growth factor regulation of glucose uptake by both allowing GIPC to promote Glut1 trafficking to the cell surface and protecting intracellular Glut1 from lysosomal degradation after growth factor withdrawal, thus allowing the potential for a rapid return of intracellular Glut1 to the cell surface on restimulation.

scholarly journals Actin-binding Protein-1 Interacts with WASp-interacting Protein to Regulate Growth Factor-induced Dorsal Ruffle Formation

2010 ◽  
Vol 21 (1) ◽  
pp. 186-197 ◽  
Author(s):  
Christa L. Cortesio ◽  
Benjamin J. Perrin ◽  
David A. Bennin ◽  
Anna Huttenlocher
Keyword(s):  
Growth Factor ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Structural Similarity ◽  
Actin Binding ◽  
Interacting Protein ◽  
Actin Binding Protein ◽  
Mediated Effects ◽  
Actin Binding Domain ◽  
Calpain 2

Growth factor stimulation induces the formation of dynamic actin structures known as dorsal ruffles. Mammalian actin-binding protein-1 (mAbp1) is an actin-binding protein that has been implicated in regulating clathrin-mediated endocytosis; however, a role for mAbp1 in regulating the dynamics of growth factor–induced actin-based structures has not been defined. Here we show that mAbp1 localizes to dorsal ruffles and is necessary for platelet-derived growth factor (PDGF)-mediated dorsal ruffle formation. Despite their structural similarity, we find that mAbp1 and cortactin have nonredundant functions in the regulation of dorsal ruffle formation. mAbp1, like cortactin, is a calpain 2 substrate and the preferred cleavage site occurs between the actin-binding domain and the proline-rich region, generating a C-terminal mAbp1 fragment that inhibits dorsal ruffle formation. Furthermore, mAbp1 directly interacts with the actin regulatory protein WASp-interacting protein (WIP) through its SH3 domain. Finally, we demonstrate that the interaction between mAbp1 and WIP is important in regulating dorsal ruffle formation and that WIP-mediated effects on dorsal ruffle formation require mAbp1. Taken together, these findings identify a novel role for mAbp1 in growth factor–induced dorsal ruffle formation through its interaction with WIP.

scholarly journals LUBAC Formation Is Impaired in the Livers of Mice with MCD-Dependent Nonalcoholic Steatohepatitis

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Yasuka Matsunaga ◽  
Yusuke Nakatsu ◽  
Toshiaki Fukushima ◽  
Hirofumi Okubo ◽  
Misaki Iwashita ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Nonalcoholic Steatohepatitis ◽  
Molecular Mechanisms ◽  
Regulatory Protein ◽  
Gel Filtration ◽  
Native Page ◽  
Ubiquitin Chain ◽  
Interacting Protein ◽  
Hepatic Lipid Accumulation ◽  
Pathway Activation

Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic lipid accumulation followed by the inflammation-induced death of hepatocytes and fibrosis. In this process, oxidative stress contributes to the induction of several inflammatory cytokines including TNF-αand　IL-1βin macrophages, while, in hepatocytes, NF-κB reportedly induces the expressions of cell survival genes for protection from apoptosis. Recently, it was reported that the new ubiquitin ligase complex termed linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on NF-κB essential modulator (NEMO) and thereby induces NF-κB pathway activation. In this study, we demonstrated the formation of LUBAC to be impaired in the livers of NASH rodent models produced by methionine and choline deficient (MCD) diet feeding, first by either gel filtration or Blue Native-PAGE, with subsequent confirmation by western blotting. The reduction of LUBAC is likely to be attributable to markedly reduced expression of SHARPIN, one of its components. Thus, impaired LUBAC formation, which would result in insufficient NF-κB activation, may be one of the molecular mechanisms underlying the enhanced apoptotic response of hepatocytes in MCD diet-induced NASH livers.

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