Magdalena Maj

Expression of TAU in insulin-secreting cells and its interaction with the calcium-binding protein secretagogin

Tauopathies have been associated with Alzheimers disease (AD), which frequently manifests together with diabetes mellitus type 2. Calcium-binding proteins such as the recently identified secretagogin (SCGN) might exert protective effects. As pancreatic beta-cells and neurons share common electrophysiological properties, we investigated the appearance of TAU (listed as MAPT in the HUGO and MGI Databases) protein at the islets of Langerhans and beta-cell-derived cell lines which highly express the neuroendocrine-specific protein SCGN. Six predominant TAU isoforms could be identified by immunoblotting, which formed TAU deposits detectable by immunofluorescence and sarkosyl-insoluble pellets. Using GST-SCGN pull-down assays, a calcium-dependent SCGN-TAU interaction was found. In this line, sucrose density gradient fractionation and differential ultracentrifugation studies of TAU and SCGN revealed co-appearance of both proteins. Co-localization of TAU and SCGN within insulinoma cells and islets of Langerhans mainly restricted to insulin-positive beta-cells was demonstrated by confocal microscopy. Motivated by these findings, we looked if SCGN overexpression could exert protective function on Rin-5F cells, which showed differences in TAU levels. Testing the vulnerability of Rin-5F clones by MTT assay, we revealed that high TAU levels going along with highest TAU aggregates could not be antagonized by high levels of SCGN protein. Our findings demonstrated for the first time the association of TAU and the calcium-binding protein SCGN and support earlier results implicating that beta-cells might represent an extra cerebral site of tauopathy.

Cloning and molecular characterization of Dashurin encoded by C20orf116, a PCI-domain containing protein

BACKGROUND Characterization of gene products originating from undefined open reading frames and delineation of biological functions has become the task after the human genome has been decoded. METHODS We cloned the human C20orf 116 and defined its transcript in liver, kidney and various brain regions by Northern analysis. Antibodies against recombinant protein used for immunofluorescence and immunoblots confirmed its expression in these tissues. With the focus on kidney, its tubular expression and presence in glomerula were shown. RESULTS A 28 aa long signal peptide predicted by in silico analysis is reflected by visualization of size variants of approximately 3kDa difference suggesting a signal peptidase cleavage of the proform. Cell compartment separation confirmed the presence of Dashurin in peroxisomes/mitochondria, microsomes, cytosol and nucleus. This is in line with green fluorescent protein (GFP)-Dashurin fusion protein shuttling between cytosol and nucleus. Luciferase reporter studies revealed a 2-3 fold increase of promoter activities upon over-expression. Bioinformatic analysis identified a PCI-domain at the C-terminus providing protein-protein interaction capabilities. CONCLUSION Our present findings suggest the involvement of Dashurin in gene transcription or mRNA translation. GENERAL SIGNIFICANCE Dashurin shares the PCI-domain with three multisubunit protein complexes (26S proteasome, COP9 signalosome and eIF3 translation initiation factor).

Expression of secretagogin in clear-cell renal cell carcinomas is associated with a high metastasis rate.

Renal cell carcinomas are divided into several subgroups according to their histopathologic characteristics. The outcome, therapy responses, and the applicability of molecular-targeted therapies depend on the tumor classification and on the tumor stage. Recent advances within the biomarker research facilitated the exact classification of the molecular character of the renal tumor. For example, the calcium-binding proteins parvalbumin and S-100A1 are characteristically expressed in renal cell carcinoma subgroups. This led us to investigate the expression of the novel calcium-binding protein secretagogin in renal cell carcinomas. Tissue microarray cylinders including 94 clear-cell renal cell carcinomas, 61 non-clear-cell renal cell carcinomas (37 papillary renal cell and 24 chromophobe carcinomas), and 30 oncocytomas were analyzed by immunohistochemistry. This showed remarkable secretagogin expression in 37% of the clear-cell renal cell carcinomas. Non-clear-cell renal cell carcinomas and oncocytomas were completely negative. Consequently performed immunoblotting analyses confirmed this expression profile. Because publicly available data direct toward a formation of a hierarchical cluster of secretagogin overexpressing clear-cell renal cell carcinomas, we conducted a clinical follow-up of the patients with clear-cell renal cell carcinoma. This revealed significantly more metastasis within the secretagogin-positive clear-cell renal cell carcinoma subgroup (49% versus 28%; P < .05). In conclusion, we report on detection of the novel calcium-binding protein secretagogin within a subgroup of clear-cell renal cell carcinomas. The increased metastasis rates within the secretagogin-positive subgroup of clear-cell renal cell carcinomas direct toward a clinical impact of our findings.

The Microtubule-Associated Protein Tau and Its Relevance for Pancreatic Beta Cells

Structural and biochemical alterations of the microtubule-associated protein tau (MAPT) are associated with degenerative disorders referred to as tauopathies. We have previously shown that MAPT is present in human islets of Langerhans, human insulinomas, and pancreatic beta-cell line models, with biophysical similarities to the pathological MAPT in the brain. Here, we further studied MAPT in pancreatic endocrine tissue to better understand the mechanisms that lead to functional dysregulation of pancreatic beta cells. We found upregulation of MAPT protein expression in human insulinomas when compared to human pancreatic islets of Langerhans and an imbalance between MAPT isoforms in insulinomas tissue. We cloned one 3-repeat domain MAPT and transduced this into a beta-cell derived rodent cell line Rin-5F. Proliferation experiments showed higher growth rates and metabolic activities of cells overexpressing MAPT protein. We observed that a MAPT overexpressing cell line demonstrates altered insulin transcription, translation, and insulin secretion rates. We found the relative insulin secretion rates were significantly decreased in a MAPT overexpressing cell line and these findings could be confirmed using partial MAPT knock-down cell lines. Our findings support that MAPT may play an important role in insulin granule trafficking and indicate the importance of balanced MAPT phosphorylation and dephosphorylation for adequate insulin release.

Protein Networks Involved in Vesicle Fusion, Transport, and Storage Revealed by Array-Based Proteomics

Secretagogin is a calcium-binding protein whose expression is characterised in neuroendocrine, pancreatic, and retinal cells. We have used an array-based proteomic approach with the prokaryotically expressed human protein array (hEx1) and the eukaryotically expressed human protein array (Protoarray) to identify novel calcium-regulated interaction networks of secretagogin. Screening of these arrays with fluorophore-labelled secretagogin in the presence of Ca(2+) ions led to the identification of 12 (hEx1) and 6 (Protoarray) putative targets. A number of targets were identified in both array screens. The putative targets from the hEx1 array were expressed, purified, and subjected to binding analysis using surface plasmon resonance. This identified binding affinities for nine novel secretagogin targets with equilibrium dissociation constants in the 100 pM to 10 nM range. Six of the novel target proteins have important roles in vesicle trafficking; SNAP-23, ARFGAP2, and DOC2alpha are involved in regulating fusion of vesicles to membranes, kinesin 5B and tubulin are essential for transport of vesicles in the cell, and rootletin builds up the rootlet, which is believed to function as scaffold for vesicles. Among the targets are two enzymes, DDAH-2 and ATP-synthase, and one oncoprotein, myeloid leukaemia factor 2. This screening method identifies a role for secretagogin in secretion and vesicle trafficking interacting with several proteins integral to these processes.

Age related changes in pancreatic beta cells: A putative extra-cerebral site of Alzheimer's pathology

Frequent concomitant manifestation of type 2 diabetes mellitus (T2DM) and Alzheimers disease (AD) has been recently demonstrated by epidemiological studies. This might be due to functional similarities between β-cells and neurons, such as secretion on demand of highly specific molecules in a tightly controlled fashion. An additional similarity represents the age-related alteration of hyperphosphorylated tau in AD patients. Similarly, alterations have been identified in β-cells of T2DM patients. The islet amyloid polypeptide has been associated with β-cell apoptosis. As a consequence of increasing age, the accumulation of highly modified proteins together with decreased regenerative potential might lead to increasing rates of apoptosis. Moreover, reduction of β-cell replication capabilities results in reduction of β-cell mass in mammals, simultaneously with impaired glucose tolerance. The new challenge is to learn much more about age-related protein modifications. This can lead to new treatment strategies for reducing the incidence of T2DM and AD.

Molecular approaches to classify adult renal epithelial neoplasms.

Evaluation of: Powers MP, Alvarez K, Kim HJ, Monzon FA. Molecular classification of adult renal epithelial neoplasms using microRNA expression and virtual karyotyping. Diagn. Mol. Pathol. 20(2), 63–70 (2011). Novel reliable techniques for the exact classification of renal epithelial neoplasms, especially in cases with morphological ambiguity, are of high importance for diagnosis and patient treatment. Evaluation of gene-expression alterations in various malignancies by array screens and quantitative PCR is a well-known procedure. In this direction, the use of molecular methods, such as virtual karyotyping and miRNA profiling, represents a rather novel approach. In particular, great promise for reliable and specific characterization of renal tumor subtypes, such as clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC and oncoytoma, is offered – according to the recently presented work by Powers et al. – by a miRNA-profiling method. This technique might be used in the form of a clinically applicable biomarker diagnostic tool. In addition, researchers suggest that miRNAs might have a functional role and might therefore contribute to the progress towards personalized cancer therapy. In this article, a broader overview of recent approaches to classify renal epithelial neoplasms is presented, with a focus on promising miRNA gene-expression profiling. The latter needs further elaboration and testing with a larger sample number.

CXCL12/SDF-1 over-expression in human insulinomas and its biological relevance

This study was performed on the basis of previously obtained investigative gene array data concerning the over-expression of CXCL12/SDF-1 in human insulinomas versus human pancreatic islet preparations. The presence of CXCL12/SDF-1 was studied by RT-qPCR in human insulinomas (n=8) versus pancreatic islets (n=3), and was found to be significantly up-regulated in the former (p<0.012). The mRNA data were confirmed by immunostaining and confocal microscopy of human normal pancreatic islets, which showed the absence of CXCL12 protein and high expression in insulinoma tissue. Individual human insulinoma cells at cytospins stained positive for CXCL12 in the paranuclear region. These morphological data were extended by consecutive immunoblotting for cell-compartment-specific marker proteins of fractions obtained by sucrose gradient fractionation using Rin-5F insulinoma cells. CXCL12-containing fractions were positive for the membrane marker NSF but negative for SNAP-25 and appeared at a lighter density in the gradient than heavy insulin granules, suggesting packaging in specific granules different from insulin. In order to determine the biological relevance of the protein in insulinomas, we investigated the colony-forming potential of human CXCL12 stable-transfected rat Rin-5F insulinoma cells. These clones secreted human CXCL12 and contained 50-1000-fold higher copy numbers compared to its endogenous rat homologue. In colony-forming assays, these transfectant clones developed greater colony numbers, which were larger than wild-type and sham transfectants. To elucidate the mechanism of action, we identified a CXCL12 transfectant-specific increase in the pro-survival factor Mn-SOD, which is considered important for the inactivation of reactive oxygen species, thereby prolonging cell survival. These data demonstrate the importance of CXCL12 in the tumor biology of insulinoma.

Calcium binding protein Secretagogin expression in wild type and transgenic tau (P301L) mice

conditions of both diseases, and characterized their phenotypes longitudinally. Methods: We cross-bred amyloid precursor protein transgenic mice (APP-Tg) with two types of diabetes model mice, ob/ob and NSY mouse. APP-Tg crossed with ob/ob mouse was fed normal chow. APP-Tg crossed with NSY mouse was divided into three diet groups, and fed high-fat diet, high-sucrose diet and normal chow. We characterized metabolic phenotypes (body weight, blood glucose and insulin levels, glucose and insulin tolerance) and AD-pathology (cognitive function and brain Ab load) of these model animals longitudinally. Results: Two types of transgenic mice (APP-ob/ob and APP-NSY mouse) with pathological conditions of both diabetes and AD were successfully generated. Compared with APP-Tg, APPob/ob mice gained more body weight and developed marked hyperglycemia, hyperinsulinemia and glucose intolerance. These metabolic changes were associated with the very early (at the age of 2 months) manifestation of memory impairment. Surprisingly, this exacerbation of memory impairment was not accompanied by an increase in the Ab protein levels in the brain compared with APP-Tg. Furthermore, APP-ob/ob mice showed more prominent metabolic dysregulation compared with ob/ob mice. APP-NSY mice also showed early manifestation of cognitive dysfunction at the age of 3-month, without detectable amyloid plaques in the brain. Moreover, 5-month high-calorie feeding induced further exacerbation of memory impairment. Conclusions: We successfully generated two types of novel transgenic mouse models which show pathological phenotypes both of diabetes and AD. Exacerbation of memory impairment without an increase in Ab load in the brain of these diabetic APP-Tg implies the role of diabetic conditions on Ab-mediated neuronal dysfunction. Early manifestation of AD-like cognitive dysfunction of these animals provides a valuable model for drug development or screening for AD. Our findings also suggest the possibility that AD pathology could exacerbate metabolic phenotypes of diabetes.