A mathematical model for the dependence of keratin aggregate formation on the quantity of mutant keratin expressed in EGFP-K14 R125P keratinocytes

We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.

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Contribution of intermediate filaments to cell stiffness, stiffening, and growth

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.1.c188 ◽

2000 ◽

Vol 279 (1) ◽

pp. C188-C194 ◽

Cited By ~ 195

Author(s):

Ning Wang ◽

Dimitrije Stamenović

Keyword(s):

Mechanical Properties ◽

Applied Stress ◽

Extended Period ◽

Direct Consequence ◽

Cell Stiffness ◽

Cell Integrity ◽

Wild Type ◽

The Difference ◽

High Degree

It has been shown previously that intermediate filament (IF) gels in vitro exhibit stiffening at high-applied stress, and it was suggested that this stiffening property of IFs might be important for maintaining cell integrity at large deformations (Janmey PA, Evtenever V, Traub P, and Schliwa M, J Cell Biol 113: 155–160, 1991). In this study, the contribution of IFs to cell mechanical behavior was investigated by measuring cell stiffness in response to applied stress in adherent wild-type and vimentin-deficient fibroblasts using magnetic twisting cytometry. It was found that vimentin-deficient cells were less stiff and exhibited less stiffening than wild-type cells, except at the lowest applied stress (10 dyn/cm2) where the difference in the stiffness was not significant. Similar results were obtained from measurements on wild-type fibroblasts and endothelial cells after vimentin IFs were disrupted by acrylamide. If, however, cells were plated over an extended period of time (16 h), they exhibited a significantly greater stiffness before than after acrylamide, even at the lowest applied stress. A possible reason could be that the initially slack IFs became fully extended due to a high degree of cell spreading and thus contributed to the transmission of mechanical stress across the cell. Taken together, these findings were consistent with the notion that IFs play important roles in the mechanical properties of the cell during large deformation. The experimental data also showed that depleting or disrupting IFs reduced, but did not entirely abolish, cell stiffening. This residual stiffening might be attributed to the effect of geometrical realignment of cytoskeletal filaments in the direction of applied load. It was also found that vimentin-deficient cells exhibited a slower rate of proliferation and DNA synthesis than wild-type cells. This could be a direct consequence of the absence of the intracellular IFs that may be necessary for efficient mediation of mechanical signals within the cell. Taken together, results of this study suggest that IFs play important roles in the mechanical properties of cells and in cell growth.

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Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

Infection and Immunity ◽

10.1128/iai.02442-14 ◽

2014 ◽

Vol 83 (1) ◽

pp. 396-404 ◽

Cited By ~ 7

Author(s):

Jonathan F. Holt ◽

Megan R. Kiedrowski ◽

Kristi L. Frank ◽

Jing Du ◽

Changhui Guan ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Manduca Sexta ◽

Wild Type ◽

Genetic Determinants ◽

Content Type ◽

Polystyrene Plate ◽

Insect Gut ◽

Insight Into

Enterococcus faecalisis a commensal and pathogen of humans and insects. InManduca sexta,E. faecalisis an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigateE. faecalisfactors required for commensalism, we identifiedE. faecalisgenes that are upregulated in the gut ofM. sextausing recombinase-basedin vivoexpression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designatedpglA. ApglAdeletion mutant was impaired in both pathogenesis and gut persistence inM. sextaand produced enhanced biofilms compared with the wild type in anin vitropolystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants forE. faecaliscommensal and pathogenic interactions withM. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to preventE. faecalisinfections.

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Tyrosine 136 phosphorylation of α-synuclein aggregates in the Lewy body dementia brain: involvement of serine 129 phosphorylation by casein kinase 2

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01281-9 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Kazunori Sano ◽

Yasushi Iwasaki ◽

Yuta Yamashita ◽

Keiichi Irie ◽

Masato Hosokawa ◽

...

Keyword(s):

Lewy Body ◽

Cultured Cells ◽

Lewy Body Dementia ◽

Casein Kinase ◽

Casein Kinase 2 ◽

Central Feature ◽

Aggregate Formation ◽

Wild Type ◽

Brain Involvement

AbstractSerine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target.

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The soluble ectodomain of RetC634Y inhibits both the wild-type and the constitutively active Ret

Biochemical Journal ◽

10.1042/bj20021530 ◽

2003 ◽

Vol 372 (3) ◽

pp. 897-903 ◽

Cited By ~ 14

Author(s):

Laura CERCHIA ◽

Domenico LIBRI ◽

Maria Stella CARLOMAGNO ◽

Vittorio de FRANCISCIS

Keyword(s):

Molecular Basis ◽

Multiple Endocrine Neoplasia Type ◽

Cell System ◽

Biologically Active ◽

Tyrosine Kinase Receptor ◽

Wild Type ◽

Membrane Bound ◽

Ret Oncogene ◽

Insight Into

Substitution of Cys-634 in the extracellular domain of the Ret tyrosine kinase receptor causes its dimerization and activation of its transforming potential. To gain further insight into the molecular basis leading to Ret activation we purified a mutant protein consisting of the entire ectodomain of the Ret carrying a Cys-634→Tyr substitution (EC-RetC634Y). The protein is glycosylated, like the native one, and is biologically active. By using an in vitro cell system we show that EC-RetC634Y inhibits the membrane-bound receptor RetC634Y, interfering with its dimerization. Furthermore, we demonstrate that EC-RetC634Y competes with the wild-type Ret receptor for ligand binding. The results presented support the notion of the possible involvment of glial cell line-derived neurotrophic factor (GDNF) with multiple endocrine neoplasia type 2A (MEN2A) tumours, and describe a useful tool for generating molecular mimetics directed towards specific mutations of the ret oncogene.

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Insight into methanol synthesis from CO2 hydrogenation on Cu(111): Complex reaction network and the effects of H2O

Journal of Catalysis ◽

10.1016/j.jcat.2011.04.012 ◽

2011 ◽

Vol 281 (2) ◽

pp. 199-211 ◽

Cited By ~ 210

Author(s):

Ya-Fan Zhao ◽

Yong Yang ◽

Charles Mims ◽

Charles H.F. Peden ◽

Jun Li ◽

...

Keyword(s):

Methanol Synthesis ◽

Reaction Network ◽

Co2 Hydrogenation ◽

Complex Reaction ◽

Insight Into

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Abstract 226: Mechanism of LCAT Activation by Compound A

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.226 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Lita A Freeman ◽

Stephen J Demosky ◽

Monika Konaklieva ◽

Rostislav Kuskovsky ◽

Scott M Gordon ◽

...

Keyword(s):

Therapeutic Use ◽

Hek293 Cells ◽

Lcat Activity ◽

Wild Type ◽

Compound A ◽

Potential Binding ◽

Before And After ◽

Lcat Deficiency ◽

Insight Into

Background: Lecithin:cholesterol acyltransferase (LCAT) catalyzes cholesteryl ester (CE) production from free cholesterol (FC) and phosphatidylcholine (lecithin), promoting HDL formation. Objective: To investigate activation of LCAT by Compound A (Amgen), a previously described small-molecule activator of LCAT, with the ultimate goal of developing novel LCAT activators for therapeutic use. Methods: LCAT activity in plasma from Familial LCAT Deficiency (FLD) patients with different mutations was quantitated by TLC before and after addition of Compound A. HEK293 cells were transiently transfected with plasmids containing wild-type (WT) or mutant LCAT cDNA. Media was then incubated with either vehicle or Compound A and LCAT activity was quantitated using a novel plate assay utilizing Methylumbelliferyl Palmitate as a substrate. Results: Compound A increased LCAT activity for a subset of FLD mutations to a level above which renal disease may occur. Mutations of Cys31 in vitro strongly affected basal LCAT activity as well as activation by Compound A. Charged residues at position 31 profoundly decreased activity whereas bulky hydrophobic groups increased LCAT activity up to 3-fold (p < 0.005, all). Mass spectrometry of WT LCAT incubated with Compound A revealed a +103.017 m/z adduct to the tryptic peptide containing Cys31, indicative of a cyanopyrazine adduct to LCAT Cys31. Molecular modeling identified potential binding sites of Compound A to LCAT. Conclusions: Our findings yield important mechanistic insight into LCAT activation that can be used to design novel LCAT activators for therapeutic use.

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Tissue Transglutaminase Does Not Contribute to the Formation of Mutant Huntingtin Aggregates

The Journal of Cell Biology ◽

10.1083/jcb.153.1.25 ◽

2001 ◽

Vol 153 (1) ◽

pp. 25-34 ◽

Cited By ~ 42

Author(s):

Wanjoo Chun ◽

Mathieu Lesort ◽

Janusz Tucholski ◽

Christopher A. Ross ◽

Gail V.W. Johnson

Keyword(s):

Tissue Transglutaminase ◽

Intracellular Localization ◽

Mutant Huntingtin ◽

Polyglutamine Diseases ◽

Aggregate Formation ◽

Wild Type ◽

Human Neuroblastoma ◽

Neuronal Populations ◽

Antisense Construct

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the NH2-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated NH2-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates.

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A mechanistic insight into glucose conversion in subcritical water: Complex reaction network and the effects of acid-base catalysis

Fuel ◽

10.1016/j.fuel.2020.119969 ◽

2021 ◽

Vol 289 ◽

pp. 119969

Author(s):

Zhifeng Yan ◽

Jie Lian ◽

Yu Feng ◽

Miaoting Li ◽

Feng Long ◽

...

Keyword(s):

Subcritical Water ◽

Reaction Network ◽

Base Catalysis ◽

Complex Reaction ◽

Acid Base ◽

Water Complex ◽

Mechanistic Insight ◽

Insight Into

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A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

Hämostaseologie ◽

10.1055/s-0038-1660401 ◽

1999 ◽

Vol 19 (03) ◽

pp. 134-138

Author(s):

Gitta Kühnel ◽

A. C. Matzdorff

Keyword(s):

Flow Cytometry ◽

Platelet Count ◽

Blood Sample ◽

Platelet Activation ◽

Whole Blood ◽

Receptor Occupancy ◽

Aggregate Formation ◽

Inhibitor Activity ◽

Receptor Inhibitor

SummaryWe studied the effect of GPIIb/IIIa-inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GPIIb/IIIa-inhibitors (c7E3, DMP728, XJ757), then thrombin or ADP were added and after 1 min the sample was fixed. Samples without c7E3 but with 0.1 U/ml thrombin had a decrease in platelet count. Samples with increasing concentrations of c7E3 had a lesser or no decrease in platelet count. The two other inhibitors (DMP 725, XJ757) gave similar results. GPIIb/IIIa-inhibitors prevent aggregate formation and more single platelets remain in the blood sample. The agonist-induced decrease in platelet count correlates closely with the concentration of the GPIIb/IIIa inhibitor and receptor occupancy. This correlation may be used as a simple measure for inhibitor activity in whole blood.

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