Monoclonal Antibodies to Discriminate the EF Hand Containing Calcium Binding Adaptor Proteins EFhd1 and EFhd2

Brachs, Sebastian; Lang, Caroline; Buslei, Rolf; Purohit, Pavitra; Fürnrohr, Barbara G.; Kalbacher, Hubert; Jäck, Hans‐Martin; Mielenz, Dirk

doi:10.1089/mab.2013.0014

Cited by 9 publications

(16 citation statements)

References 31 publications

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“…A dot-like neuropil staining (see arrowheads in Fig. 3B) of EFhd2 was observed in the hippocampus of EFhd2 +/+ mice but not of EFhd2 −/− mice using anti-EFhd2 mAb A4.18.18 [16]. Similar results were obtained with the anti-EFhd2 mAb A4.15.18 [16] (data not shown).…”

Section: Resultssupporting

confidence: 79%

The Ca2+ Sensor Protein Swiprosin-1/EFhd2 Is Present in Neurites and Involved in Kinesin-Mediated Transport in Neurons

et al. 2014

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Swiprosin-1/EFhd2 (EFhd2) is a cytoskeletal Ca2+ sensor protein strongly expressed in the brain. It has been shown to interact with mutant tau, which can promote neurodegeneration, but nothing is known about the physiological function of EFhd2 in the nervous system. To elucidate this question, we analyzed EFhd2−/−/lacZ reporter mice and showed that lacZ was strongly expressed in the cortex, the dentate gyrus, the CA1 and CA2 regions of the hippocampus, the thalamus, and the olfactory bulb. Immunohistochemistry and western blotting confirmed this pattern and revealed expression of EFhd2 during neuronal maturation. In cortical neurons, EFhd2 was detected in neurites marked by MAP2 and co-localized with pre- and post-synaptic markers. Approximately one third of EFhd2 associated with a biochemically isolated synaptosome preparation. There, EFhd2 was mostly confined to the cytosolic and plasma membrane fractions. Both synaptic endocytosis and exocytosis in primary hippocampal EFhd2−/− neurons were unaltered but transport of synaptophysin-GFP containing vesicles was enhanced in EFhd2−/− primary hippocampal neurons, and notably, EFhd2 inhibited kinesin mediated microtubule gliding. Therefore, we found that EFhd2 is a neuronal protein that interferes with kinesin-mediated transport.

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Section: Resultssupporting

confidence: 79%

The Ca2+ Sensor Protein Swiprosin-1/EFhd2 Is Present in Neurites and Involved in Kinesin-Mediated Transport in Neurons

et al. 2014

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“…We had detected its expression only in the pro B-cell line 38B9. 26 RT-PCR analysis of sorted BM B cells 34 revealed the strongest efhd1 mRNA expression in pro B cells ( Figure 3a). Western blot analysis of pro B cells from Rag1 − / − mice confirmed EFhd1 protein expression in pro B cells.…”

Section: Resultsmentioning

confidence: 99%

“…Western blot. Western blot analyses were performed according to standard procedures as previously described using rabbit anti EFhd1, 26 rabbit anti Actin (Sigma) and mouse anti Transferrin receptor antibodies (Thermo Scientific, Dreieich, Germany), followed by horseradish-peroxidase coupled secondary antibodies and development by enhanced chemiluminescence. Goat anti μ HC coupled to horseradish-peroxidase was purchased from Southern Biotech.…”

Section: Methodsmentioning

confidence: 99%

“…[22][23][24] We revealed its expression only in the Bcr-Abl expressing pro B-cell line 38B9, 25 but not in B-cell lines expressing a pre BCR or a BCR. 26 EFhd1 is a target gene of two transcription factors involved in B-cell identity and control of early B-cell development, Foxo1 and Brg1, in pro B cells. 27,28 In very aggressive human melanomas, EFhd1 is co-expressed with PGC-1α, a co-activator of PPAR that is involved in mitochondrial biogenesis.…”

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A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1

et al. 2017

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B-cell development in the bone marrow comprises proliferative and resting phases in different niches. We asked whether B-cell metabolism relates to these changes. Compared to pro B and small pre B cells, large pre B cells revealed the highest glucose uptake and ROS but not mitochondrial mass, whereas small pre B cells exhibited the lowest mitochondrial membrane potential. Small pre B cells from Rag1;33.C9 μ heavy chain knock-in mice revealed decreased glycolysis (ECAR) and mitochondrial spare capacity compared to pro B cells from Rag1 mice. We were interested in the step regulating this metabolic switch from pro to pre B cells and uncovered that Swiprosin-2/EFhd1, a Ca-binding protein of the inner mitochondrial membrane involved in Ca-induced mitoflashes, is expressed in pro B cells, but downregulated by surface pre B-cell receptor expression. Knockdown and knockout of EFhd1 in 38B9 pro B cells decreased the oxidative phosphorylation/glycolysis (OCR/ECAR) ratio by increasing glycolysis, glycolytic capacity and reserve. Prolonged expression of EFhd1 in EFhd1 transgenic mice beyond the pro B cell stage increased expression of the mitochondrial co-activator PGC-1α in primary pre B cells, but reduced mitochondrial ATP production at the pro to pre B cell transition in IL-7 cultures. Transgenic EFhd1 expression caused a B-cell intrinsic developmental disadvantage for pro and pre B cells. Hence, coordinated expression of EFhd1 in pro B cells and by the pre BCR regulates metabolic changes and pro/pre B-cell development.

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“…Western blot of epididymal WAT (eWAT) using Total OXPHOS WB-Antibody-Cocktail (ab110413; Abcam), rabbitanti-mouse NNMT (ARP42281_T100; Aviva Systems Biology), rabbit-anti-mouse a-tubulin (2144; NEB), and horseradish peroxidase-anti-rabbit IgG (7074; Cell Signaling Technology) for detection was conducted as described previously (18,19). Optical density (OD) was quantified by ImageJ (20).…”

Section: Biochemical Analysismentioning

confidence: 99%

Genetic Nicotinamide N-Methyltransferase (Nnmt) Deficiency in Male Mice Improves Insulin Sensitivity in Diet-Induced Obesity but Does Not Affect Glucose Tolerance

et al. 2018

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Antisense oligonucleotide knockdown (ASO-KD) of nicotinamide N-methyltransferase (NNMT) in high-fat diet (HFD)–fed mice has been reported to reduce weight gain and plasma insulin levels and to improve glucose tolerance. Using NNMT-ASO-KD or NNMT knockout mice (NNMT−/−), we tested the hypothesis that Nnmt deletion protects against diet-induced obesity and its metabolic consequences in males and females on obesity-inducing diets. We also examined samples from a human weight reduction (WR) study for adipose NNMT (aNNMT) expression and plasma 1-methylnicotinamide (MNAM) levels. In Western diet (WD)–fed female mice, NNMT-ASO-KD reduced body weight, fat mass, and insulin level and improved glucose tolerance. Although NNMT−/− mice fed a standard diet had no obvious phenotype, NNMT−/− males fed an HFD showed strongly improved insulin sensitivity (IS). Furthermore, NNMT−/− females fed a WD showed reduced weight gain, less fat, and lower insulin levels. However, no improved glucose tolerance was observed in NNMT−/− mice. Although NNMT expression in human fat biopsy samples increased during WR, corresponding plasma MNAM levels significantly declined, suggesting that other mechanisms besides aNNMT expression modulate circulating MNAM levels during WR. In summary, upon NNMT deletion or knockdown in males and females fed different obesity-inducing diets, we observed sex- and diet-specific differences in body composition, weight, and glucose tolerance and estimates of IS.

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Monoclonal Antibodies to Discriminate the EF Hand Containing Calcium Binding Adaptor Proteins EFhd1 and EFhd2

Cited by 9 publications

References 31 publications

The Ca2+ Sensor Protein Swiprosin-1/EFhd2 Is Present in Neurites and Involved in Kinesin-Mediated Transport in Neurons

The Ca2+ Sensor Protein Swiprosin-1/EFhd2 Is Present in Neurites and Involved in Kinesin-Mediated Transport in Neurons

A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1

Genetic Nicotinamide N-Methyltransferase (Nnmt) Deficiency in Male Mice Improves Insulin Sensitivity in Diet-Induced Obesity but Does Not Affect Glucose Tolerance

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