Heterologous Overexpression of Human NEFA and Studies on the Two EF-Hand Calcium-Binding Sites

Kroll, Katja; Otte, Stefan; Hirschfeld, Gregor; Barnikol-Watanabe, Shitsu; Götz, H.; Sternbach, Hans; Kratzin, Hartmut; Barnikol, Heinz Ulrich; Hilschmann, Norbert

doi:10.1006/bbrc.1999.0867

Cited by 19 publications

(11 citation statements)

References 34 publications

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“…The identification of multiple functionally unrelated interaction partners and their implication in various processes such as Ca 2+ homeostasis (Kroll et al 1999;Morel-Huaux et al 2002;Taniguchi et al 2000;Lin et al 1999), Gα protein mediated signalling (Lin et al 2000), unfolded protein response (Tsukumo et al 2007), TNFR1 shedding (Islam et al 2006) and hypothalamic feeding regulation (Oh et al 2006;Nonogaki et al 2008) suggests that nucleobindins are multifunctional proteins having various tissue-specific functions. So far, nothing is known about their function in stomach.…”

Section: Discussionmentioning

confidence: 99%

“…Within the cell both are located in the cytoplasmic and Golgi luminal pools, both are secreted into extracellular space and interact with other proteins in a calciumdependent manner (Lavoie et al 2002;Lin et al 2000;Morel-Huaux et al 2002;Taniguchi et al 2000;Islam et al 2006). Both nucleobindins were reported to interact with the postmitotic growth suppressor Necdin in neuronal cytoplasm (Taniguchi et al 2000) and have been shown to constitute a Ca 2+ storage pool in Golgi (Lin et al 1999;Kroll et al 1999;Morel-Huaux et al 2002). NUCB1 interacts with cyclooxygenases in COS-1 cells (Ballif et al 1996) and with G protein Gαi3 in the cytoplasm of rat kidney cells (Lin et al 2000), and acts as a negative regulator in ATF6-mediated unfolded protein response (Tsukumo et al 2007).…”

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confidence: 99%

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Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer

Kalniņa¹,

Siliņa²,

Bruvere³

et al. 2009

Eur J Histochem

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NUCB2 is an EF-hand Ca 2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues. The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform, generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca 2+ store in ER or Golgi apparatus, signalling via heterotrimeric Gα proteins and/or mediating the exocytosis of the secretory granules.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer

Kalniņa¹,

Siliņa²,

Bruvere³

et al. 2009

Eur J Histochem

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“…NUCB2 binds calcium via its two EF-hand domains, with a stoichiometry of 2 mol of Ca 2ϩ /mol of NUCB2 (31). In addition, exosome release from K562 human erythroleukemic cells can be regulated in a calcium-dependent fashion (52).…”

Section: Discussionmentioning

confidence: 99%

Extracellular TNFR1 Release Requires the Calcium-dependent Formation of a Nucleobindin 2-ARTS-1 Complex

Islam

Adamik²,

Hawari³

et al. 2006

Journal of Biological Chemistry

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Extracellular tumor necrosis factor (TNF) receptors function as TNF-binding proteins that modulate TNF activity. In human vascular endothelial cells (HUVEC), extracellular TNFR1 (type I TNF receptor, TNFRSF1A) is generated by two mechanisms, proteolytic cleavage of soluble TNFR1 ectodomains and the release of full-length 55-kDa TNFR1 in the membranes of exosome-like vesicles. TNFR1 release from HUVEC is known to involve the association between ARTS-1 (aminopeptidase regulator of TNFR1 shedding), an integral membrane aminopeptidase, and TNFR1. The goal of this study was to identify ARTS-1 binding partners that modulate TNFR1 release to the extracellular space. A yeast two-hybrid screen of a human placenta cDNA library showed that NUCB2 (nucleobindin 2), via its helix-loophelix domains, binds the ARTS-1 extracellular domain. The association between endogenous ARTS-1 and NUCB2 in HUVEC was demonstrated by co-immunoprecipitation experiments, which showed the formation of a calcium-dependent NUCB2⅐ARTS-1 complex that associated with a subset of total cellular TNFR1. Confocal microscopy experiments demonstrated that this association involved a distinct population of NUCB2-containing intracytoplasmic vesicles. RNA interference was utilized to specifically knock down NUCB2 and ARTS-1 expression, which demonstrated that both are required for the constitutive release of a full-length 55-kDa TNFR1 within exosomelike vesicles as well as the inducible proteolytic cleavage of soluble TNFR1 ectodomains. We propose that calcium-dependent NUCB2⅐ARTS-1 complexes, which associate with TNFR1 prior to its commitment to pathways that result in either the constitutive release of TNFR1 exosome-like vesicles or the inducible proteolytic cleavage of TNFR1 ectodomains, play an important role in mediating TNFR1 release to the extracellular compartment.

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“…Moreover, comparison of mRNA levels between cancerous and adjacent non-cancerous tissues by RT -PCR showed that the expression of NUCB2 is downregulated in 50% of gastric tumours. NUCB2 is a Ca 2+ binding protein, which also contains putative DNA binding domain, two helix -loop -helix motifs, EF-hands and leucine zipper (Barnikol-Watanabe et al, 1994;Kroll et al, 1999). It interacts with the postmitotic growth suppressor necdin and is proposed to be involved in the regulation of survival and death of postmitotic cells by controlling Ca 2+ homeostasis in the cytoplasm (Taniguchi et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Serological identification and expression analysis of gastric cancer-associated genes

et al. 2002

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Serological identification of tumour antigens by recombinant expression cloning has proved to be an effective strategy for the identification of cancer-associated genes having a relevance to cancer aetiology and progression, and for defining possible targets for immunotherapeutic intervention. In the present study we applied this technique to identify immunogenic proteins for gastric cancer that resulted in isolation of 14 distinct serum-reactive antigens. In order to evaluate their role in tumourigenesis and assess the immunogenicity of the identified antigens, we characterised each cDNA clone by DNA sequence analysis, mRNA tissue distribution, comparison of mRNA levels in cancerous and adjacent non-cancerous tissues and the frequency of antibody responses in allogeneic patient and control sera. Previously unknown splice variants of TACC1 and an uncharacterised gene Ga50 were identified. The expression of a newly identified TACC1 isoform is restricted to brain and gastric cancer tissues. Comparison of mRNA levels by semi-quantitative RT -PCR revealed a relative overexpression of three genes in cancer tissues, including growth factor granulin and Tbdn-1 -an orthologue of the mouse acetyltransferase gene which is associated with blood vessel development. An unusual DNA polymorphism -a three-nucleotide deletion was found in NUCB2 cDNA but its mRNA level was consistently decreased in gastric tumours compared with that in the adjacent noncancerous tissues. This study has revealed several new gastric cancer candidate genes; additional studies are required to gain a deeper insight into their role in the tumorigenesis and their potential as therapeutic targets.

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Heterologous Overexpression of Human NEFA and Studies on the Two EF-Hand Calcium-Binding Sites

Cited by 19 publications

References 34 publications

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer

Extracellular TNFR1 Release Requires the Calcium-dependent Formation of a Nucleobindin 2-ARTS-1 Complex

Serological identification and expression analysis of gastric cancer-associated genes

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