scholarly journals Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

2006 ◽  
Vol 281 (43) ◽  
pp. 32619-32629 ◽  
Author(s):  
Vasanthy Vigneswara ◽  
Jonathan D. Lowenson ◽  
Claire D. Powell ◽  
Matthew Thakur ◽  
Kevin Bailey ◽  
...  
Keyword(s):  
Repair Enzyme ◽  
Knock Out ◽  
Methyl Donor ◽  
Brain Extracts ◽  
Knock Out Mice ◽  
First Time ◽  
The Brain ◽  
Phosphatidylethanolamine Binding Protein ◽  
Mammalian Protein

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein l-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1-/-) accumulate more isoaspartyl residues than their wild-type littermates, with the most “damaged” residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1-/- mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and two-dimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1-/- animals compared with Pcmt1+/+ littermates. Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxyl-terminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, β-synuclein, and α-synuclein, are all substrates for the l-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1-/- brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

scholarly journals Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

2018 ◽  
Author(s):  
Yuhki Saito ◽  
Yuan Yuan ◽  
Ilana Zucker-Scharff ◽  
John J. Fak ◽  
Yoko Tajima ◽  
...  
Keyword(s):  
Motor Coordination ◽  
Rna Binding ◽  
Synapse Formation ◽  
Rna Binding Proteins ◽  
Cell Types ◽  
Knock Out ◽  
Knock Out Mice ◽  
Cerebellar Purkinje Cells ◽  
The Brain

SUMMARYRNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell-types in vivo is unknown. We employed NOVA2 cTag-CLIP to generate functional RBP-RNA maps from single neuronal populations in the mouse brain. Combining cell-type specific data from Nova2-cTag and Nova2 conditional knock-out mice revealed differential NOVA2 regulatory actions (e.g. alternative splicing) on the same transcripts in different neurons, including in cerebellar Purkinje cells, where NOVA2 acts as an essential factor for proper motor coordination and synapse formation. This also led to the discovery of a mechanism by which NOVA2 action leads to different outcomes in different cells on the same transcripts: NOVA2 is able to regulate retained introns, which subsequently serve as scaffolds for another trans-acting splicing factor, PTBP2. Our results describe differential roles and mechanisms by which RBPs mediate RNA diversity in different neurons and consequent functional outcomes within the brain.

scholarly journals Lack of WWC2 Protein Leads to Aberrant Angiogenesis in Postnatal Mice

2021 ◽  
Vol 22 (10) ◽  
pp. 5321
Author(s):  
Viktoria Constanze Brücher ◽  
Charlotte Egbring ◽  
Tanja Plagemann ◽  
Pavel I. Nedvetsky ◽  
Verena Höffken ◽  
...  
Keyword(s):  
Signalling Pathway ◽  
Control Group ◽  
Knock Out ◽  
Ocular Angiogenesis ◽  
Sprouting Angiogenesis ◽  
Adult Mice ◽  
Upstream Regulator ◽  
Knock Out Mice ◽  
Hippo Signalling

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 3997-4002 ◽  
Author(s):  
Dirk Meyer ◽  
Carsten Schiller ◽  
Jürgen Westermann ◽  
Shozo Izui ◽  
Wouter L. W. Hazenbos ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Autoimmune Hemolytic Anemia ◽  
Knock Out ◽  
Effector Cells ◽  
Igg Isotypes ◽  
Knock Out Mice ◽  
Deficient Mice ◽  
Experimental Autoimmune

Abstract In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

scholarly journals Pannexin-1 Deficient Mice Have an Increased Susceptibility for Atrial Fibrillation and Show a QT-Prolongation Phenotype

2016 ◽  
Vol 38 (2) ◽  
pp. 487-501 ◽  
Author(s):  
Stella Petric ◽  
Sofia Klein ◽  
Lisa Dannenberg ◽  
Tillman Lahres ◽  
Lukas Clasen ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Cardiac Electrophysiology ◽  
Electrophysiological Study ◽  
Atp Release ◽  
Release Channel ◽  
Knock Out ◽  
Pannexin 1 ◽  
Significant Prolongation ◽  
Knock Out Mice

Background/Aims: Pannexin-1 (Panx1) is an ATP release channel that is ubiquitously expressed and coupled to several ligand-gated receptors. In isolated cardiac myocytes, Panx1 forms large conductance channels that can be activated by Ca2+ release from the sarcoplasmic reticulum. Here we characterized the electrophysiological function of these channels in the heart in vivo, taking recourse to mice with Panx1 ablation. Methods: Cardiac phenotyping of Panx1 knock-out mice (Panx1-/-) was performed by employing a molecular, cellular and functional approach, including echocardiography, surface and telemetric ECG recordings with QT analysis, physical stress testing and quantification of heart rate variability. In addition, an in vivo electrophysiological study entailed programmed electrical stimulation using an intracardiac octapolar catheter. Results: Panx1 deficiency results in a higher incidence of AV-block, delayed ventricular depolarisation, significant prolongation of QT- and rate corrected QT-interval and a higher incidence of atrial fibrillation after intraatrial burst stimulation. Conclusion: Panx1 seems to play an important role in murine cardiac electrophysiology and warrants further consideration in the context of hereditary forms of atrial fibrillation.

In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Peter L. Turecek ◽  
Jürgen Siekmann ◽  
Herbert Gritsch ◽  
Katalin Váradi ◽  
Rafi-Uddin Ahmad ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Full Length ◽  
Thrombin Inhibitor ◽  
Knock Out ◽  
Knock Out Mice

Abstract Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.

Recombinant Human Coagulation Factor IX Expressed in HEK293 Cells: Influence of Serin Phosphorylation and Tyrosine Sulfation on Pharmacokinetic Properties in FIX-Knock-out Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4098-4098
Author(s):  
Ernst Boehm ◽  
Michael Dockal ◽  
Meinhard Hasslacher ◽  
Artur Mitterer ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hek293 Cells ◽  
High Yield ◽  
High Similarity ◽  
Tyrosine Sulfation ◽  
Knock Out ◽  
Pharmacokinetic Properties ◽  
Knock Out Mice ◽  
In Vivo Recovery

Abstract Recombinant factor IX (rFIX) expressed in Chinese hamster ovary (CHO) cells has been shown to be safe and effective in clinical studies, but differs in pharmacokinetics from plasma-derived FIX (pdFIX). In clinical studies, CHO-derived rFIX had a 30–50 % lower in-vivo recovery when compared to pdFIX, whereas mean residence time and terminal half-life did not differ between preparations. Although rFIX shows high similarity to pdFIX in structure and function, differences in glycosylation and gamma-carboxylation degree can be detected. Moreover, although experimental proof has yet to be published, the lower degree of phosphorylation of amino acid serine 155, and the lower degree of sulfation of tyrosine 158 have been hypothesized to be causative for the lower in-vivo recovery of rFIX. These two modifications occur at less than 20 % for the tyrosine-sulfation and at less than 1 % for the serine phosphorylation in rFIX, whereas pdFIX has both modifications to more than 90 % completed. We identified human HEK293 cells to perform rFIX phosphorylation and sulfation to a higher extent than CHO cells. A rFIX-producing cell line derived from HEK293 cells was generated by stable transfection, and was adapted to suspension culture conditions to allow lab-scale fermentation. rFIX was produced and purified from a single fermentation run using two different down-stream process schemes: the first was able to enrich high-phosphorylated and -sulfated rFIX; the second to purify total rFIX from the supernatant at high yield. For pharmacokinetic comparison, these HEK293 materials, CHO-derived rFIX, and a pdFIX preparation were formulated in the same buffer. Determination of phosphorylation and sulfation by mass spectrometry showed a phosphorylation and sulfation degree of 50 % plus a 20 % single modification (phosphorylation or sulfation) for the HEK293-material purified by the modification enrichment method versus 15 % for both modifications plus a 15 % single modification for the material purified by the high-yield protocol. The values for CHO-derived rFIX and pdFIX were similar to those in the literature. Oligosaccharide mapping revealed glycosylation differences among CHO-, HEK293-, and pdFIX preparations, but high similarity between both HEK293-derived materials. We compared the pharmacokinetics of the various FIX preparations in FIX-knock-out mice. In-vivo recovery and area under the curve were statistically significantly higher for the high phosphorylated and sulfated HEK293-material than for total rFIX derived from HEK293 cells. However, these two parameters were lower for both HEK293-derived rFIX preparations than for CHO-derived rFIX, and lower for CHO-derived rFIX than for pdFIX. This may be due to glycosylation differences between these FIX preparations. Mean residence times and terminal half-lives were similar for all preparations. In summary, these findings emphasize that the degree of rFIX-sulfation and -phosphorylation influences the pharmacokinetic properties of rFIX.

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 3997-4002 ◽  
Author(s):  
Dirk Meyer ◽  
Carsten Schiller ◽  
Jürgen Westermann ◽  
Shozo Izui ◽  
Wouter L. W. Hazenbos ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Autoimmune Hemolytic Anemia ◽  
Knock Out ◽  
Effector Cells ◽  
Igg Isotypes ◽  
Knock Out Mice ◽  
Deficient Mice ◽  
Experimental Autoimmune

In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

scholarly journals Identification of a developmental switch in information transfer between whisker S1 and S2 cortex in mice

2021 ◽  
Author(s):  
Theofanis Karayannis ◽  
Linbi Cai ◽  
Jenq-Wei Yang ◽  
Shen-Ju Chou ◽  
Chia-Fang Wang ◽  
...  
Keyword(s):  
Information Transfer ◽  
Barrel Cortex ◽  
Wide Field ◽  
Sensory Stimuli ◽  
Knock Out ◽  
Electrophysiological Recordings ◽  
Primary Sensory Cortex ◽  
Animal Navigation ◽  
Knock Out Mice

The whiskers of rodents are a key sensory organ that provides critical tactile information for animal navigation and object exploration throughout life. Previous work has explored the developmental sensory-driven activation of the primary sensory cortex processing whisker information (wS1), also called barrel cortex. This body of work has shown that the barrel cortex is already activated by sensory stimuli during the first post-natal week. However, it is currently unknown when over the course of development these stimuli begin being processed by higher order cortical areas, such as secondary whisker somatosensory area (wS2). Here we investigate for the first time the developmental engagement of wS2 by sensory stimuli and the emergence of cortico-cortical communication from wS1 to wS2. Using in vivo wide-field imaging and electrophysiological recordings in control and conditional knock-out mice we find that wS1 and wS2 are able to process bottom-up information coming from the thalamus already right after birth. We identify that it is only at the end of the first post-natal week that wS1 begins to provide excitation into wS2, a connection which begins to acquire feed-forward inhibition characteristics after the second post-natal week. Therefore, we have uncovered a developmental window during which excitatory versus inhibitory functional connectivity between wS1 and wS2 takes place.

scholarly journals Cholesterol Regulates Exosome Release in Cultured Astrocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohammad Abdullah ◽  
Tomohisa Nakamura ◽  
Taslima Ferdous ◽  
Yuan Gao ◽  
Yuxin Chen ◽  
...  
Keyword(s):  
High Fat Diet ◽  
Normal Diet ◽  
Cellular Cholesterol ◽  
Knock Out ◽  
Pi3k Inhibitor Ly294002 ◽  
Akt Phosphorylation ◽  
Cholesterol Levels ◽  
Cultured Astrocytes ◽  
Knock Out Mice

Exosomes are vesicles secreted by various kinds of cells, and they are rich in cholesterol, sphingomyelin (SM), phosphatidylcholine, and phosphatidylserine. Although cellular sphingolipid-mediated exosome release has been reported, the involvement of other lipid components of cell membranes in the regulation of exosome release is poorly understood. Here, we show that the level of exosome release into conditioned media is significantly reduced in cultured astrocytes prepared from apolipoprotein E (ApoE) knock-out mice when compared to those prepared from wild-type (WT) mice. The reduced level of exosome release was accompanied by elevated levels of cellular cholesterol. The addition of cholesterol to WT astrocytes significantly increased the cellular cholesterol levels and reduced exosome release. PI3K/Akt phosphorylation was enhanced in ApoE-deficient and cholesterol-treated WT astrocytes. In contrast, the depletion of cholesterol in ApoE-deficient astrocytes due to treatment with β-cyclodextrin recovered the exosome release level to a level similar to that in WT astrocytes. In addition, the reduced levels of exosome release due to the addition of cholesterol recovered to the control levels after treatment with a PI3K inhibitor (LY294002). The cholesterol-dependent regulation of exosome release was also confirmed by in vivo experiments; that is, exosome levels were significantly reduced in the CSF and blood serum of WT mice that were fed a high-fat diet and had increased cholesterol levels when compared to those in WT mice that were fed a normal diet. These results suggest that exosome release is regulated by cellular cholesterol via stimulation of the PI3K/Akt signal pathway.

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