scholarly journals Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes.

1996 ◽  
Vol 134 (3) ◽  
pp. 615-623 ◽  
Author(s):  
D Kasper ◽  
F Dittmer ◽  
K von Figura ◽  
R Pohlmann
Keyword(s):  
Lysosomal Enzymes ◽  
Single Type ◽  
Minor Importance ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Mannose 6 Phosphate ◽  
Partial Correction ◽  
Mpr 46 ◽  
Lysosomal Proteins

Mouse embryonic fibroblasts that are deficient in the two mannose 6-phosphate receptors (MPRs) MPR 46 and MPR 300 missort the majority (> or = 85%) of soluble lysosomal proteins into the medium. Human MPR 46 and MPR 300 were expressed in these cells to test whether overexpression of a single type of MPR can restore transport of lysosomal proteins to lysosomes. Only a partial correction of the missorting was observed after overexpression of MPR 46. Even at MPR 46 levels that are five times higher than the wild-type level, more than one third of the newly synthesized lysosomal proteins accumulates in the secretions. Two-fold overexpression of MPR 300 completely corrects the missorting of lysosomal enzymes. However, at least one fourth of the lysosomal enzymes are transported along a secretion-recapture pathway that is sensitive to mannose 6-phosphate in medium. In control fibroblasts that express both types of MPR, the secretion-recapture pathway is of minor importance. These results imply that neither overexpression of MPR 46 nor MPR 300 is sufficient for targeting of lysosomal proteins along intracellular routes.

scholarly journals Specific alterations in levels of mannose 6-phosphorylated glycoproteins in different neuronal ceroid lipofuscinoses

1998 ◽  
Vol 334 (3) ◽  
pp. 547-551 ◽  
Author(s):  
David E. SLEAT ◽  
Istvan SOHAR ◽  
Premila S. PULLARKAT ◽  
Peter LOBEL ◽  
Raju K. PULLARKAT
Keyword(s):  
Lysosomal Enzyme ◽  
Neurodegenerative Disorders ◽  
Enzyme Activities ◽  
Lysosomal Enzymes ◽  
Vesicular Transport ◽  
Cell Types ◽  
Neuronal Ceroid Lipofuscinoses ◽  
Mannose 6 Phosphate ◽  
Normal Controls ◽  
Lysosomal Proteins

Mannose 6-phosphate (Man-6-P) is a carbohydrate modification that is generated on newly synthesized lysosomal proteins. This modification is specifically recognized by two Man-6-P receptors that direct the vesicular transport of the lysosomal enzymes from the Golgi to a prelysosomal compartment. The Man-6-P is rapidly removed in the lysosome of most cell types; however, in neurons the Man-6-P modification persists. In this study we have examined the spectrum of Man-6-P-containing glycoproteins in brain specimens from patients with different neuronal ceroid lipofuscinoses (NCLs), which are progressive neurodegenerative disorders with established links to defects in lysosomal catabolism. We find characteristic alterations in the Man-6-P glycoproteins in specimens from late-infantile (LINCL), juvenile (JNCL) and adult (ANCL) patients. Man-6-P glycoproteins in LINCL patients were similar to controls, with the exception that the band corresponding to CLN2, a recently identified lysosomal enzyme whose deficiency results in this disease, was absent. In an ANCL patient, two Man-6-P glycoproteins were elevated in comparison with normal controls, suggesting that this disease also results from a perturbation in lysosomal hydrolysis. In JNCL, total levels of Man-6-P glycoproteins were 7-fold those of controls. In general this was reflected by increased lysosomal enzyme activities in JNCL but three Man-6-P glycoproteins were elevated to an even greater degree. These are CLN2 and the unidentified proteins that are also highly elevated in the ANCL.

Differential expression of prohibitin and regulation of apoptosis in wild-type and COX-2 null mouse embryonic fibroblasts

2009 ◽  
Vol 53 (2) ◽  
pp. 157-159 ◽  
Author(s):  
Young-Hoon Kim ◽  
Hyangkyu Lee ◽  
Tae-Yoon Kim ◽  
Hyang-Ran Hwang ◽  
Sang Chul Lee
Keyword(s):  
Differential Expression ◽  
Null Mouse ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Cox 2

scholarly journals Caspase3-deficient cells require fibronectin for protection against autophagy-dependent death

2021 ◽  
Author(s):  
David B. Weir ◽  
Lawrence H. Boise
Keyword(s):  
Cell Death ◽  
Survival Advantage ◽  
Deficient Mouse ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Serum Withdrawal ◽  
Presence And Absence ◽  
Induced Apoptosis

ABSTRACTCaspases are required for execution of apoptosis. However, in their absence, signals that typically induce apoptosis can still result in cell death. Our laboratory previously demonstrated that Casp3-deficient mouse embryonic fibroblasts (MEFs) have increased fibronectin (FN) secretion, and an adhesion-dependent survival advantage compared to wild type (WT) MEFs. Here, we show that FN is required for survival of Casp3-deficient MEFs following serum withdrawal. Furthermore, when FN is silenced, serum withdrawal-induced death is caspase-independent. However, procaspase-7 is cleaved, suggesting that MOMP is taking place. Indeed, in the absence of FN, cytochrome c release is increased following serum withdrawal in Casp3-deficient MEFs. Yet death does not correspond to cytochrome c release in Casp3-deficient MEFs. This is true both in the presence and absence of FN. Additionally, caspase-independent death is inhibited by Bcl-XL overexpression. These findings suggest that Bcl-XL is not inhibiting death through regulation of Bax/Bak insertion into the mitochondria, but through a different mechanism. One such possibility is autophagy and induction of autophagy is associated with caspase-independent death in Casp3-deficient cells. Importantly, when ATG5 is ablated in Casp3-deficient cells, autophagy is blocked and death is largely inhibited. Taken together, our data indicate that Casp3-deficient cells incapable of undergoing canonical serum withdrawal-induced apoptosis, are protected from autophagy-dependent death by FN-mediated adhesion.

scholarly journals Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts

2020 ◽  
Vol 21 (8) ◽  
pp. 2772
Author(s):  
Qiang Zhao ◽  
Kathleen A Coughlan ◽  
Ming-Hui Zou ◽  
Ping Song
Keyword(s):  
Molecular Mechanisms ◽  
Adenosine Monophosphate ◽  
Centrosome Amplification ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Cellular Energy ◽  
Chromosome Missegregation ◽  
Cell Mitosis ◽  
Metaphase Chromosome Spread

Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1−/−) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34–66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.

scholarly journals The Absence of Ser389 Phosphorylation in p53 Affects the Basal Gene Expression Level of Many p53-Dependent Genes and Alters the Biphasic Response to UV Exposure in Mouse Embryonic Fibroblasts

2008 ◽  
Vol 28 (6) ◽  
pp. 1974-1987 ◽  
Author(s):  
Wendy Bruins ◽  
Oskar Bruning ◽  
Martijs J. Jonker ◽  
Edwin Zwart ◽  
Tessa V. van der Hoeven ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Uv Irradiation ◽  
Target Genes ◽  
Late Response ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Endogenous Gene ◽  
Cellular Processes

ABSTRACT Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53−/− mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53−/−, possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53−/− but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.

NF-κB Protection against Apoptosis Induced by HEMA

2004 ◽  
Vol 83 (11) ◽  
pp. 837-842 ◽  
Author(s):  
G. Spagnuolo ◽  
C. Mauro ◽  
A. Leonardi ◽  
M. Santillo ◽  
R. Paternò ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Protective Role ◽  
Oxygen Species ◽  
Wild Type ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Primary Fibroblasts ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Dose Dependent

The cytotoxicity of dental monomers has been widely investigated, but the underlying mechanisms have not been elucidated. We studied the molecular mechanisms involved in cell death induced by HEMA. In human primary fibroblasts, HEMA induced a dose-dependent apoptosis that was confirmed by the activation of caspases-8, -9, and -3. We found an increase of reactive oxygen species (ROS) and NF-κB activation after HEMA exposure. Blocking of ROS production by anti-oxidants had no direct influence on apoptosis caused by HEMA, but inhibition of NF-κB increased the fraction of apoptotic cells. Accordingly, mouse embryonic fibroblasts (MEF) from p65−/− mice were more susceptible to HEMA-induced apoptosis than were wild-type controls. Our results indicate that exposure to HEMA triggers apoptosis and that this mechanism is not directly dependent upon redox signaling. Nevertheless, ROS induction by HEMA activates NF-κB, which exerts a protective role in counteracting apoptosis.

scholarly journals Mitochondrial LonP1 protease is implicated in the degradation of unstable Parkinson's disease-associated DJ-1/PARK 7 missense mutants

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raúl Sánchez-Lanzas ◽  
José G. Castaño
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Mitochondrial Pathway ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Strong Reduction ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Biochemical Fractionation ◽  
Early Onset Parkinson’S Disease

AbstractDJ-1/PARK7 mutations are linked with familial forms of early-onset Parkinson's disease (PD). We have studied the degradation of untagged DJ-1 wild type (WT) and missense mutants in mouse embryonic fibroblasts obtained from DJ-1-null mice, an approach closer to the situation in patients carrying homozygous mutations. The results showed that the mutants L10P, M26I, A107P, P158Δ, L166P, E163K, and L172Q are unstable proteins, while A39S, E64D, R98Q, A104T, D149A, A171S, K175E, and A179T are as stable as DJ-1 WT. Inhibition of proteasomal and autophagic-lysosomal pathways had little effect on their degradation. Immunofluorescence and biochemical fractionation studies indicated that M26I, A107P, P158Δ, L166P, E163K, and L172Q mutants associate with mitochondria. Silencing of mitochondrial matrix protease LonP1 produced a strong reduction of the degradation of the mitochondrial-associated DJ-1 mutants A107P, P158Δ, L166P, E163K, and L172Q but not of mutant L10P. These results demonstrated a mitochondrial pathway of degradation of those DJ-1 missense mutants implicated in PD pathogenesis.

Dictyostelium lysosomal proteins with different sugar modifications sort to functionally distinct compartments

1997 ◽  
Vol 110 (18) ◽  
pp. 2239-2248 ◽  
Author(s):  
G.M. Souza ◽  
D.P. Mehta ◽  
M. Lammertz ◽  
J. Rodriguez-Paris ◽  
R. Wu ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Lysosomal Enzymes ◽  
Cysteine Proteinases ◽  
Retrieval Mechanism ◽  
Mannose 6 Phosphate ◽  
Lysosomal Proteins

Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P- and Man-6-P-bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation.

Sign in / Sign up

Export Citation Format

Share Document