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Secretagogin is a Redox-Responsive Ca2+ Sensor Radhika Khandelwal, Anand Kumar Sharma, Swathi Chadalawada, and Yogendra Sharma Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00761 • Publication Date (Web): 20 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016
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Biochemistry
Secretagogin is a Redox-Responsive Ca2+ Sensor Radhika Khandelwal≠, Anand Kumar Sharma≠, Swathi Chadalawada and Yogendra Sharma* CSIR-Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad-500 007, India;
Running title: Secretagogin and redox sensitivity
*Corresponding author: Yogendra Sharma, PhD, Centre for Cellular and Molecular Biology (CCMB), Uppal Road, Hyderabad-500007, India. Phone: +91-40-27192561, Fax: +91-40-27160591, E-mail:
[email protected] ≠
RK and AKS are joint first authors
Keywords: Secretagogin, Redox sensitivity, Conformational stability
Financial disclosure (Funding Source Statement) This work was supported by the CSIR BioAge BSC0208 grant to YS. RK is the recipient of a CSIR-GATE fellowship from the CSIR, India, and AKS the recipient of a senior research fellowship from the UGC.
Conflict of Interest The authors declare no conflict of interest.
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Abbreviations: SCGN, Secretagogin, CaBP, Ca2+-binding protein, UPR, Unfolded protein response,
ER,
endoplasmic
reticulum,
DTT,
dithiothreitol,
carboxyethyl)phosphine, ITC, isothermal titration calorimetry
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TCEP,
Tris(2-
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Abstract Secretagogin (SCGN), a multifunctional, Ca2+ binding, regulatory protein, known to regulate insulin release, has recently been implicated in the control of stress-related corticotropinreleasing hormone (CRH) secretion. Localization of SCGN to multiple intracellular (such as cytosol, nucleus, and ER) and extracellular sites appears to provide multifunctional capabilities, however, the structural elements conferring such a wide-spread cellular distribution to SCGN remain unidentified. We report that the spatial and functional attributes of SCGN plausibly originate from the interplay between Ca2+ and its redox state. The mutation of selective Cys residues provides further insights into the origin and mode of redox-responsiveness. In the reducing milieu, SCGN exhibits a higher affinity for Ca2+, and more stability than in the oxidizing environment, suggesting it to be a redox-responsive Ca2+-sensor protein, which is further supported by its response to DTT (reducing stress) in MIN6 cells. Our data provide a biophysical/biochemical explanation for the diverse localization of SCGN in the cellular scenario and beyond the cell.
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Secretagogin (SCGN), a Ca2+-binding protein belonging to hexa EF-hand group, is predominantly expressed in pancreatic β-cells where it is known to regulate insulin release (1). The limited literature available suggests that SCGN plays a role in glucose-stimulated insulin secretion (2, 3). To facilitate insulin secretion, SCGN localizes to the cytoplasm and vesicular membrane, and interacts with SNAP-25, a protein involved in Ca2+-induced exocytosis (1, 4). In addition to its high expression in pancreatic β-cells, SCGN is also expressed in neurons and neuroendocrine cells in the brain and in endocrine glands (5-8). In the hypothalamus, SCGN has been shown to regulate the release of corticotropin-releasing hormone (CRH) (9), the first hormone of the stress response. This also lends credence to the fact that SCGN expression is disturbed in certain brain tumors or carcinomas and thus is considered a strong candidate biomarker (10-12). These anomalies are also related to a redox imbalance. This suggests SCGN be among the primary sensors of stress, however, confirmatory work remains due. SCGN localizes to multiple intracellular sites, including the cytosol, endoplasmic reticulum (ER), plasma membrane and nucleus (1, 9, 13) exposing it to the reducing as well as to oxidizing conditions. The structural elements involved in this spatially diverse localization (and function) remain unexplored. This is specifically important since SCGN has four Cys residues. We report here that both, the SCGN protein and its gene are redox-responsive elements, supporting the hypothesis that SCGN is a primary sensor of stress in the cell. SCGN tends to form redox-sensitive oligomer, which can be as high as 50% to the total protein concentration. Nevertheless, the functional implication of redox sensitivity remains unexplored. We here describe the effect of redox status on ligand binding and stability of SCGN. Our data support a logical hypothesis that the combination of redox status of the protein and availability of Ca2+ regulates the protein function and stability.
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Experimental Procedures Modeling mouse SCGN (mSCGN) structure: A 3-dimensional structure model of mouse SCGN was generated by using Phyre2 (Protein Homology/analogY Recognition Engine V 2.0) (15). Mouse SCGN protein sequence (Accession: NP_663374.1, GI: 21703798) was input for the model generation. Modeling was done in the intensive mode. From the template suggestion of Phyre2, best homologue was found to be zebrafish SCGN (structure PDB id: 2Q4U; PDB Title: ensemble refinement of the crystal structure of an EF-hand protein2 from Danio rerio dr.36843) which was selected as template (16). Total identity between query and the template was ~70% at homology confidence interval of 100%. Recombinant SCGN Purification: Mouse SCGN gene was cloned into the pET21b vector at NdeI and XhoI restriction sites and transformed into BL21 (DE3) cells for overexpression. The protein was purified as described earlier (14) with a modification of an on-column wash step. Briefly, the protein was purified from inclusion bodies by Ni-NTA affinity chromatography. Resin with bound protein was first washed with 50 mM Tris, pH 7.5, 100 mM KCl (wash buffer 1 or WB1) followed by 50 mM Tris, pH 7.5, 100 mM KCl, 2% Triton X-100 (WB2) and finally by WB1, 5 column volume each. Gel filtration was employed as a final step of purification. The purified protein was pooled and incubated with 0.1 mM EDTA to decalcify and buffer exchanged with Chelex-purified buffer (50 mM Tris, pH 7.5; 100 mM KCl) to remove EDTA. Alternatively, to circumvent refolding SCGN from inclusion bodies, we optimized the expression of SCGN in the minimal media (induction with 0.5 mM IPTG followed by incubation at 25 οC for 10 hours) in which SCGN was found to be expressed in the soluble fraction. The protein was purified by employing Ni-NTA chromatography. Cys mutations (C253S and C131S) were created into the parent plasmid by site-directed mutagenesis approach. Both the mutant
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proteins were purified by on-column refolding approach using protocol similar to wt SCGN as described above. Analytical gel filtration chromatography: The peak corresponding to the dimer of SCGN from the preparative gel filtration was divided into two fractions. One was loaded on a Superdex 75 column (analytical grade) without any modification while the second fraction was pretreated with 3 mM DTT before performing the chromatography. Native PAGE: For reducing conditions, protein samples were incubated with DTT (5 mM) for 10 min at room temperature. Non-reducing protein samples were incubated at room temperature for same time without addition of DTT. Native poly-acrylamide gels were prepared essentially as SDS-PAGE while omitting SDS in acrylamide gels and in running buffer. Reducing agent (βmercaptoethanol) was also avoided in sample buffer (loading dye). Spectral measurements: The surface hydrophobicity of the samples was measured using ANS (an external fluorophore) by exciting the sample at 365 nm and recording the emission spectra from 400-600 nm. Far-UV and near-UV CD spectral measurements were performed on a Jasco J-815 spectropolarimeter using 0.2 mm and 1 cm path length cuvettes respectively. All CD spectra were corrected for the buffer contribution as a blank. Equilibrium unfolding: Chemical unfolding was performed in increasing concentration of GdmCl. A stock solution of ultrapure Invitrogen GdmCl (8 M) was prepared in the Chelex purified water and filtered. The concentration was checked on a digital refractometer. Samples were prepared by addition of either 100 µM EDTA or 2 mM Ca2+ in increasing concentrations of GdmCl from 0-6.7 M. To perform unfolding in the reducing environment, 1 mM TCEP was included in one set of samples. Trp emission fluorescence spectra were recorded on an F-7000 Fluorescence Spectrophotometer (Hitachi Inc, Japan) by exciting the samples at 295 nm. The
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spectra were recorded from 300-450 nm at 5 nm excitation and emission slits. The unfolding transition data were fit as described earlier (17). Isothermal titration calorimetry (ITC): The energetics of Ca2+ binding to SCGN or mutants in the oxidizing and reducing environments were carried out by ITC at 30 °C on a Microcal VPITC instrument. Protein samples (30 µM in the cell) and 1 mM Ca2+ (in the syringe) was prepared in Chelex-purified 50 mM Tris, pH 7.5, 100 mM KCl (either in the presence or absence of 1 mM TCEP). The respective blanks were obtained by titrating buffer with 1 mM Ca2+ under identical parameter setting. Data were fit in the available equations using Origin 8, supplied by the manufacturer. The ITC experiments were repeated at least twice or more to check the reproducibility. The reported thermogram is a representative data set. MIN6 Cell culture and DTT treatment: MIN6 cells were maintained in DMEM supplemented with 10% serum. Cells were seeded at the density of 0.2 x 106 cells per well in the six-well cell culture plates. After 24 hours, cells were washed with PBS thrice and treated with different concentrations of DTT as indicated in the respective figures. After one hour of incubation, cells were washed with PBS and lysed in 0.5 ml of Trizol reagent. RNA isolation and quantitation using qRT PCR: Total RNA was extracted from MIN6 cells by Trizol reagent, according to the manufacturer’s protocol. cDNA was synthesized by a First Strand cDNA synthesis kit (Invitrogen) using random hexamers as per recommendation from the vendor. Primers were designed to span exon-exon junction for most of the genes. Primer sequences are available on request. The quantification of gene expression was performed by realtime PCR on an ABI viiA7 RT-PCR system via SYBR detection system. HPRT was used as endogenous control to normalize the expression of individual genes. Each sample was run in triplicate and data were analyzed using the 2-∆∆CT method.
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Statistical analysis: All the analyses were performed in the MS-Excel program using student’s ttest. * corresponds to p=
