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

2001 ◽  
Vol 153 (1) ◽  
pp. 25-34 ◽  
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.

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

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

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.

scholarly journals Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Hui Qin Wang ◽  
Ensar Halilovic ◽  
Xiaoyan Li ◽  
Jinsheng Liang ◽  
Yichen Cao ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Human Neuroblastoma Cell ◽  
Wild Type ◽  
Combination Strategy ◽  
Human Neuroblastoma ◽  
Xenograft Models ◽  
Neuroblastoma Cell Lines

The efficacy of ALK inhibitors in patients with ALK-mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of TP53 wild-type neuroblastoma cells harboring ALK amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for TP53 wild-type neuroblastoma with ALK aberrations.

Tissue transglutaminase is not involved in the aggregate formation of stably expressed α-synuclein in sh-sy5y human neuroblastoma cells

2004 ◽  
Vol 27 (8) ◽  
pp. 850-856 ◽  
Author(s):  
Myung-Duk Suh ◽  
Chang-Ha Park ◽  
Sung-Soo Kim ◽  
Myeng-Og Kil ◽  
Geon-Hee Lee ◽  
...  
Keyword(s):  
Tissue Transglutaminase ◽  
Neuroblastoma Cells ◽  
Aggregate Formation ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma

scholarly journals Farnesyl Diphosphate Synthase Is a Cytosolic Enzyme in Leishmania major Promastigotes and Its Overexpression Confers Resistance to Risedronate

2006 ◽  
Vol 5 (7) ◽  
pp. 1057-1064 ◽  
Author(s):  
Aurora Ortiz-Gómez ◽  
Carmen Jiménez ◽  
Antonio M. Estévez ◽  
Juana Carrero-Lérida ◽  
Luis M. Ruiz-Pérez ◽  
...  
Keyword(s):  
Drug Target ◽  
Leishmania Major ◽  
Intracellular Localization ◽  
Initial Step ◽  
Isoprenoid Biosynthesis ◽  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type

ABSTRACT Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.

scholarly journals TFIIA Plays a Role in the Response to Oxidative Stress

2006 ◽  
Vol 5 (7) ◽  
pp. 1081-1090 ◽  
Author(s):  
Susan M. Kraemer ◽  
David A. Goldstrohm ◽  
Ann Berger ◽  
Susan Hankey ◽  
Sherry A. Rovinsky ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Expression Profiles ◽  
Intracellular Localization ◽  
Regulation Of Gene Expression ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT To characterize the role of the general transcription factor TFIIA in the regulation of gene expression by RNA polymerase II, we examined the transcriptional profiles of TFIIA mutants of Saccharomyces cerevisiae using DNA microarrays. Whole-genome expression profiles were determined for three different mutants with mutations in the gene coding for the small subunit of TFIIA, TOA2. Depending on the particular mutant strain, approximately 11 to 27% of the expressed genes exhibit altered message levels. A search for common motifs in the upstream regions of the pool of genes decreased in all three mutants yielded the binding site for Yap1, the transcription factor that regulates the response to oxidative stress. Consistent with a TFIIA-Yap1 connection, the TFIIA mutants are unable to grow under conditions that require the oxidative stress response. Underexpression of Yap1-regulated genes in the TFIIA mutant strains is not the result of decreased expression of Yap1 protein, since immunoblot analysis indicates similar amounts of Yap1 in the wild-type and mutant strains. In addition, intracellular localization studies indicate that both the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress. As such, the decrease in transcription of Yap1-dependent genes in the TFIIA mutant strains appears to reflect a compromised interaction between Yap1 and TFIIA. This hypothesis is supported by the observations that Yap1 and TFIIA interact both in vivo and in vitro. Taken together, these studies demonstrate a dependence of Yap1 on TFIIA function and highlight a new role for TFIIA in the cellular mechanism of defense against reactive oxygen species.

scholarly journals Probing tissue transglutaminase mediated vascular smooth muscle cell aging using a novel transamidation-deficient Tgm2-C277S mouse model

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Huilei Wang ◽  
James Chen ◽  
Sandeep Jandu ◽  
Sean Melucci ◽  
William Savage ◽  
...  
Keyword(s):  
Tissue Transglutaminase ◽  
Significant Loss ◽  
In Vivo Studies ◽  
Cell Aging ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Independent Functions ◽  
Vascular Stiffening

AbstractTissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

scholarly journals Rat wild-type parathyroid hormone receptor (PTH-R) and mutant PTH-RP132L show the different intracellular localization in vitro

2008 ◽  
Vol 29 (2) ◽  
pp. 61-69 ◽  
Author(s):  
Junko SHIMOMURA-KUROKI ◽  
Sobhan UBAIDUS ◽  
Paulo HL FREITAS ◽  
Minqi LI ◽  
Yoko ISHIDA ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Hormone Receptor ◽  
Intracellular Localization ◽  
Wild Type ◽  
Parathyroid Hormone Receptor

scholarly journals Binding of Calmodulin to the Carboxy-Terminal Region of p21 Induces Nuclear Accumulation via Inhibition of Protein Kinase C-Mediated Phosphorylation of Ser153

2005 ◽  
Vol 25 (16) ◽  
pp. 7364-7374 ◽  
Author(s):  
Aina Rodríguez-Vilarrupla ◽  
Montserrat Jaumot ◽  
Neus Abella ◽  
Núria Canela ◽  
Sonia Brun ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Kinase C ◽  
Carboxy Terminal

ABSTRACT Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261227
Author(s):  
Marcos Gouveia ◽  
Tjaša Sorčan ◽  
Špela Zemljič-Jokhadar ◽  
Rui D. M. Travasso ◽  
Mirjana Liović
Keyword(s):  
Mathematical Model ◽  
Reaction Network ◽  
Cell Stiffness ◽  
Cell Cortex ◽  
Complex Reaction ◽  
Aggregate Formation ◽  
Wild Type ◽  
Linear Ordinary Differential Equations ◽  
Insight Into

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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