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Secretagogin is a Ca2+-dependent stress-responsive chaperone that may also play a role in aggregation-based proteinopathies

Open AccessPublished:July 20, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102285
      Secretagogin (SCGN) is a three-domain hexa-EF-hand Ca2+-binding protein that plays a regulatory role in the release of several hormones. SCGN is expressed largely in pancreatic β-cells, certain parts of the brain, and also in neuroendocrine tissues. The expression of SCGN is altered in several diseases, such as diabetes, cancers, and neurodegenerative disorders; however, the precise associations that closely link SCGN expression to such pathophysiologies are not known. In this work, we report that SCGN is an early responder to cellular stress, and SCGN expression is temporally upregulated by oxidative stress and heat shock. We show the overexpression of SCGN efficiently prevents cells from heat shock and oxidative damage. We further demonstrate that in the presence of Ca2+, SCGN efficiently prevents the aggregation of a broad range of model proteins in vitro. Small-angle X-ray scattering (BioSAXS) studies further reveal that Ca2+ induces the conversion of a closed compact apo-SCGN conformation into an open extended holo-SCGN conformation via multistate intermediates, consistent with the augmentation of chaperone activity of SCGN. Furthermore, isothermal titration calorimetry establishes that Ca2+ enables SCGN to bind α-synuclein and insulin, two target proteins of SCGN. Altogether, our data not only demonstrate that SCGN is a Ca2+-dependent generic molecular chaperone involved in protein homeostasis with broad substrate specificity but also elucidate the origin of its altered expression in several cancers. We describe a plausible mechanism of how perturbations in Ca2+ homeostasis and/or deregulated SCGN expression would hasten the process of protein misfolding, which is a feature of many aggregation-based proteinopathies.

      Keywords

      Abbreviations:

      ADH (alcohol dehydrogenase), CS (citrate synthase), ΔCTD (C-terminal deleted), HSP70 (heat shock protein 70), ITC (isothermal titration calorimetry), MDH (malate dehydrogenase), ΔNTD (N-terminal deleted), SCGN (secretagogin), SAXS (small-angle X-ray scattering)
      Secretagogin (SCGN) is a 32 kDa Ca2+-binding protein of the hexa EF-hand family, which was first discovered in the cDNA library of human pancreatic β-cells as a facilitator of insulin secretion (
      • Wagner L.
      • Oliyarnyk O.
      • Gartner W.
      • Nowotny P.
      • Gröger M.
      • Kaserer K.
      • et al.
      Cloning and expression of secretagogin, a novel neuroendocrine and pancreatic islet of Langerhans-specific Ca2+-binding protein.
      ). It is highly enriched in pancreatic islets and well-characterized for its role in glucose metabolism by regulating insulin expression and release through SNAP25 interaction (
      • Wagner L.
      • Oliyarnyk O.
      • Gartner W.
      • Nowotny P.
      • Gröger M.
      • Kaserer K.
      • et al.
      Cloning and expression of secretagogin, a novel neuroendocrine and pancreatic islet of Langerhans-specific Ca2+-binding protein.
      ,
      • Rogstam A.
      • Linse S.
      • Lindqvist A.
      • James P.
      • Wagner L.
      • Berggård T.
      Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin.
      ,
      • Qin J.
      • Liu Q.
      • Liu Z.
      • Pan Y.-Z.
      • Sifuentes-Dominguez L.
      • Stepien K.
      • et al.
      Structural and mechanistic insights into secretagogin-mediated exocytosis.
      ). SCGN is also expressed in neuroendocrine cells of gastrointestinal tract and certain brain tissues. Deregulated SCGN expression is reported in a broad range of disorders that include diabetes (
      • Hansson S.
      • Vachet P.
      • Eriksson J.
      • Pereira M.
      • Skrtic S.
      • Wallin H.
      • et al.
      Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.
      ,
      • Malenczyk K.
      • Szodorai E.
      • Schnell R.
      • Lubec G.
      • Szabó G.
      • Hökfelt T.
      • et al.
      Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for β cell specification.
      ,
      • Sharma A.K.
      • Khandelwal R.
      • Kumar M.J.M.
      • Ram N.S.
      • Chidananda A.H.
      • Raj T.A.
      • et al.
      Secretagogin regulates insulin signaling by direct insulin binding.
      ), neurodegenerative disorders (
      • Attems J.
      • Ittner A.
      • Jellinger K.
      • Nitsch R.
      • Maj M.
      • Wagner L.
      • et al.
      Reduced secretagogin expression in the hippocampus of P301L tau transgenic mice.
      ,
      • Lachén-Montes M.
      • González Morales A.
      • Iloro I.
      • Elortza F.
      • Ferrer I.
      • Gveric D.
      • et al.
      Unveiling the olfactory proteostatic disarrangement in Parkinson’s disease by proteome-wide profiling.
      ,
      • Zahola P.
      • Hanics J.
      • Pintér A.
      • Máté Z.
      • Gáspárdy A.
      • Zsófia H.
      • et al.
      Secretagogin expression in the vertebrate brainstem with focus on the noradrenergic system and implications for Alzheimer’s disease.
      ), cancer (
      • Zhan X.
      • Evans C.O.
      • Oyesiku N.M.
      • Desiderio D.M.
      Proteomics and transcriptomics analyses of secretagogin down-regulation in human non-functional pituitary adenomas.
      ,
      • Xing X.
      • Lai M.
      • Gartner W.
      • Xu E.
      • Huang Q.
      • Li H.
      • et al.
      Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin.
      ,
      • Juhlin C.C.
      • Zedenius J.
      • Höög A.
      Clinical routine application of the second-generation neuroendocrine markers ISL1, INSM1, and secretagogin in neuroendocrine neoplasia: staining outcomes and potential clues for determining tumor origin.
      ), and ulcerative colitis (
      • Sifuentes-Dominguez L.
      • Li H.
      • Llano E.
      • Liu Z.
      • Singla A.
      • Patel A.
      • et al.
      SCGN deficiency results in colitis susceptibility.
      ). The consistent deregulated expression of SCGN leads to the proposition that SCGN could be considered a potential biomarker for certain cancers, such as prostate, large cell neuroendocrine, pituitary, colorectal, and renal cancers (
      • Zhan X.
      • Evans C.O.
      • Oyesiku N.M.
      • Desiderio D.M.
      Proteomics and transcriptomics analyses of secretagogin down-regulation in human non-functional pituitary adenomas.
      • Xing X.
      • Lai M.
      • Gartner W.
      • Xu E.
      • Huang Q.
      • Li H.
      • et al.
      Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin.
      ,
      • Birkenkamp-Demtröder K.
      • Wagner L.
      • Brandt Sørensen F.
      • Bording Astrup L.
      • Gartner W.
      • Scherübl H.
      • et al.
      Secretagogin is a novel marker for neuroendocrine differentiation.
      ,
      • Adolf K.
      • Wagner L.
      • Bergh A.
      • Stattin P.
      • Ottosen P.
      • Borre M.
      • et al.
      Secretagogin is a new neuroendocrine marker in the human prostate.
      ,
      • Kim D.S.
      • Choi Y.P.
      • Kang S.
      • Gao M.Q.
      • Kim B.
      • Park H.R.
      • et al.
      Panel of candidate biomarkers for renal cell carcinoma.
      ,
      • Dong Y.
      • Li Y.
      • Liu R.
      • Li Y.
      • Zhang H.
      • Liu H.
      • et al.
      Secretagogin, a marker for neuroendocrine cells, is more sensitive and specific in large cell neuroendocrine carcinoma compared with the markers CD56, CgA, Syn and Napsin A.
      ,
      • Yu L.
      • Suye S.
      • Huang R.
      • Liang Q.
      • Fu C.
      Expression and clinical significance of a new neuroendocrine marker secretagogin in cervical neuroendocrine carcinoma.
      ).
      Besides insulin, SCGN also regulates the secretion of stress-related corticotrophin-releasing hormone (
      • Romanov R.
      • Alpár A.
      • Zhang M.D.
      • Zeisel A.
      • Calas A.
      • Landry M.
      • et al.
      A secretagogin locus of the mammalian hypothalamus controls stress hormone release.
      ) and matrix metalloprotease-2 from selective neurons (
      • Hanics J.
      • Szodorai E.
      • Tortoriello G.
      • Malenczyk K.
      • Keimpema E.
      • Lubec G.
      • et al.
      Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration.
      ). Thus, SCGN is a multifunctional regulator of several hormones and is also emerging as a stress-related Ca2+ sensor (
      • Romanov R.
      • Alpár A.
      • Zhang M.D.
      • Zeisel A.
      • Calas A.
      • Landry M.
      • et al.
      A secretagogin locus of the mammalian hypothalamus controls stress hormone release.
      ,
      • Alpár A.
      • Zahola P.
      • Hanics J.
      • Hevesi Z.
      • Korchynska S.
      • Benevento M.
      • et al.
      Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress.
      ,
      • Maj M.
      • Wagner L.
      • Tretter V.
      20 Years of secretagogin: exocytosis and beyond.
      ). Despite such strong correlations, the functions and mechanistic details are not understood. Recent reports have also illustrated the role of SCGN in preventing insulin and α-synuclein aggregation (
      • Sharma A.K.
      • Khandelwal R.
      • Kumar M.J.M.
      • Ram N.S.
      • Chidananda A.H.
      • Raj T.A.
      • et al.
      Secretagogin regulates insulin signaling by direct insulin binding.
      ,
      • Chidananda A.H.
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Secretagogin binding prevents α-synuclein fibrillation.
      ). Protein misfolding leads to the onset of cellular stress, which, in various pathological conditions, leads to protein aggregation. These findings prompted us to explore the possibility that SCGN could be involved in managing cellular stress by preventing protein misfolding.
      In this study, we demonstrate that SCGN is a Ca2+-dependent molecular chaperone. We report a connection between SCGN expression and cellular stress; SCGN is temporally upregulated during heat and oxidative stress, suggestively alleviating the aggregation of target proteins and mitigating cell death. This finding underscores the strong association of SCGN with ER stress and the redox-responsive Ca2+ switch of SCGN (
      • Malenczyk K.
      • Szodorai E.
      • Schnell R.
      • Lubec G.
      • Szabó G.
      • Hökfelt T.
      • et al.
      Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for β cell specification.
      ,
      • Khandelwal R.
      • Sharma A.K.
      • Chadalawada S.
      • Sharma Y.
      Secretagogin is a redox-responsive Ca2+ sensor.
      ), which would aid in regulating SCGN function both in the ER (oxidizing) and the cytosol (reducing). Using the BioSAXS, we further demonstrate that the Ca2+-dependent chaperone activity of SCGN is associated with its transformation from closed to open conformation upon Ca2+ binding. Thus, we identify a new role of SCGN as a Ca2+-dependent general chaperone and suggest its larger, yet unappreciated, involvement in precluding protein misfolding disorders.

      Results

      SCGN expression is altered by heat and oxidative stress

      To discern a correlation of SCGN expression with protein misfolding diseases, we first looked for alterations in the temporal expression of endogenous SCGN under stress conditions. Change in cellular SCGN expression during stress was monitored by subjecting RIN-5F cells to heat and oxidative stresses. As a standard procedure, heat shock was induced by incubating cells at 42 °C (
      • Cates J.
      • Graham G.C.
      • Omattage N.
      • Pavesich E.
      • Setliff I.
      • Shaw J.
      • et al.
      Sensing the heat stress by mammalian cells.
      ,
      • Siddiqui M.A.
      • Ahmad J.
      • Farshori N.N.
      • Saquib Q.
      • Jahan S.
      • Kashyap M.P.
      • et al.
      Rotenone-induced oxidative stress and apoptosis in human liver HepG2 cells.
      ), while oxidative stress was induced chemically by treating the cells with rotenone, a mitochondria complex I inhibitor known to induce constitutive protein aggregation in vitro. The endogenous SCGN expression was monitored at both the transcript and protein levels, at different time points after exposure to stress.
      A maximum of a 4-fold increase in Scgn gene expression was observed 3 h postshock treatment (Fig. 1A). After steadily increasing for 3 h postheat shock treatment, a gradual decline in Scgn expression was observed till 6 h. The expression of inducible heat shock protein 70 (Hsp70), which was used as a positive control was found to be maximum at 6 h which coincides with the previous reports (
      • Ritossa F.
      Discovery of the heat shock response.
      ,
      • Abravaya K.
      • Phillips B.
      • Morimoto R.I.
      Attenuation of the heat response in HeLa cells is mediated by the release of bound heat shock transcription factor and is modulated by changes in growth and heat shock temperatures.
      ). This suggests that SCGN is an early responder to heat shock. A similar pattern was noted in rotenone-treated cells, where Scgn transcript remains significantly upregulated between 6 to 9 h followed by a retreat to below normal levels (Fig. 1B). This was compared with glucose-regulated protein (Grp78) levels, a known marker for oxidative stress (
      • Jing X.
      • Shi Q.
      • Bi W.
      • Zeng Z.
      • Liang Y.
      • Wu X.
      • et al.
      Rifampicin protects pc12 cells from rotenone-induced cytotoxicity by activating GRP78 via PERK-eIF2α-ATF4 pathway.
      ), whose expression was found elevated around 6 to 9 h as demonstrated by quantitative real-time PCR (qRT-PCR) analyses. These results demonstrate significant upregulation of Scgn transcripts by thermal or oxidative stress.
      Figure thumbnail gr1
      Figure 1SCGN expression is altered during heat and oxidative stress. A, transcript levels of SCGN in RIN-5F cells that were subjected to heat shock treatment at 42 °C for 45 min and then lysed after 0, 1, 2, 3, 4, 5, and 6 h of the recovery period. SCGN mRNA levels as analyzed by qRT-PCR. B, SCGN mRNA levels in RIN-5F cells after rotenone treatment (1 μM) for 3, 6, 9, 12, 18, and 24 h. C, Western blot depicting SCGN levels in RIN-5F cells subjected to heat shock treatment as in (A), E, Western blot depicting SCGN protein levels in RIN-5F cells treated with rotenone at 3, 6, 9, 12, 18, and 24 h of time points, and (D and F) quantitative analyses of immunoblots showing signal intensity performed using ImageJ. Signal intensity is normalized with β-actin. HSP70 and GRP78 were used as positive controls for qRT-PCR and western blots in heat and oxidative stress, respectively. ∗ Represents p-value <0.05 and ∗∗represents p-value <0.01 with a minimum of three biological replicates in a group. HSP70, heat shock protein 70; SCGN, secretagogin.
      To check if the change in transcript level also translates to a change in SCGN protein levels, we examined SCGN levels after heat shock and rotenone treatment. A noticeable increase in SCGN levels, similar to that seen in HSP70 expression, was seen until 6 h of recovery after heat shock treatment (Fig. 1, C and D). Oxidative stress induced by rotenone treatment resulted in an increase in SCGN protein levels for up to 9 h, followed by a significant reduction in the protein levels (Fig. 1, E and F). Consistent with the transcript level, GRP78 protein level was found to be upregulated 6 h post rotenone treatment. The transient increase in transcript levels of SCGN during the initial short-term recovery phase after heat shock or oxidative stress is followed by rapid transcript degradation (Fig. 1, A and B). Likewise, the SCGN protein levels elevate during the early response to both heat shock and oxidative stress, but during the late response to oxidative stress, SCGN is maintained at a lower steady-state level (Fig. 1, D and F). This indicates that SCGN is an early responder to stress.

      SCGN suppresses the aggregation of proteins in a Ca2+-dependent manner

      The stress-inducible expression of SCGN points toward a possible chaperone-like activity of SCGN. To establish this, we investigated if SCGN would act as an antiaggregant against various model proteins: alcohol dehydrogenase (ADH), citrate synthase (CS), lysozyme, and malate dehydrogenase (MDH). While the aggregation of the heat-labile enzymes ADH, CS, and MDH was induced by heating them at 41.5, 45, and 50 °C respectively, the aggregation of lysozyme was induced chemically by incubation with 20 mM DTT (
      • Shao F.
      • Bader M.W.
      • Jakob U.
      • Bardwell J.C.
      DsbG, a protein disulfide isomerase with chaperone activity.
      ,
      • Abgar S.
      • Vanhoudt J.
      • Aerts T.
      • Clauwaert J.
      Study of the chaperoning mechanism of bovine lens alpha-crystallin, a member of the alpha-small heat shock superfamily.
      ,
      • Raychaudhuri S.
      • Sinha M.
      • Mukhopadhyay D.
      • Bhattacharyya N.P.
      HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity.
      ,
      • Matukumalli S.R.
      • Tangirala R.
      • Rao C.M.
      Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation.
      ). These are standard conditions to yield maximum aggregation with given substrates and are described in Methods. The aggregation kinetics of the substrates was monitored either alone or in the presence of BSA (control) or SCGN. Since SCGN is a Ca2+ sensor protein, all the experiments described above were performed either in the presence of EDTA or Ca2+. As expected, the sample turbidity of the substrates alone (monitored at 465 nm, 500 nm, 450 nm, and 360 nm for ADH, CS, MDH, and lysozyme, respectively) rapidly increases with time and eventually reaches a maximum saturation represented as a plateau phase (Fig. 2, AH). BSA, used as a control, was merely effective or even displayed slightly enhanced aggregation (with ADH and MDH) but did not reduce aggregation, which is consistent with earlier reports (
      • Finn T.E.
      • Nunez A.C.
      • Sunde M.
      • Easterbrook-Smith S.B.
      Serum albumin prevents protein aggregation and amyloid formation and retains chaperone-like activity in the presence of physiological ligands.
      ). However, the incubation of substrates with SCGN significantly decreases aggregation, more effectively in the presence of Ca2+ (Fig. 2, AD). In the presence of Ca2+, the aggregation of substrates was suppressed substantially as compared to that observed in the absence of Ca2+ (Fig. 2, EH). While SCGN could reduce the aggregation in the absence of Ca2+, the efficacy of chaperone action of SCGN was increased significantly in the presence of Ca2+ (80–90%) (Fig. 2,IL). In the case of ADH, about 50% protection was observed in the presence of EDTA, which increased to 90% in the presence of Ca2+ (Fig. 2I). At a 1:2 M ratio of SCGN and substrates, SCGN could reduce scattering (absorbance value) to ∼40%, while at a ratio of 2:1, the inhibition of aggregation was more than 80%, suggesting dose-dependent inhibition of aggregation by SCGN (Fig. 2,IL). These results suggest that Ca2+ drastically increases the anti-aggregation activity of SCGN and might be a prerequisite for efficient chaperone action of SCGN in the cellular milieu.
      Figure thumbnail gr2
      Figure 2SCGN prevents aggregation of various model substrates. Aggregation assay of substrates: alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), citrate synthase (CS), and lysozyme in the presence of SCGN at different stoichiometric ratios. Bovine serum albumin (BSA) was used as a control. The aggregation kinetics was monitored in the presence of either 2 mM Ca2+: (A) ADH, (B) MDH, (C) CS, (D) lysozyme, or in the presence of 10 μM EDTA: (E) ADH, (F) MDH, (G) CS, and (H) lysozyme. Percentage change in chaperone activity of SCGN in the presence of 10 μM EDTA or 2 mM Ca2+ with: (I) ADH, (J) MDH, (K) CS, and (L) lysozyme, at different molar ratios. Each experiment has been performed in three replicates. Red and blue lines represent the percent decrease in absorbance upon incubation with apo- and holo-SCGN at different molar ratios, respectively. SCGN, secretagogin.

      SCGN stabilizes luciferase activity against heat shock

      Having established that SCGN possesses strong anti-aggregation properties toward general protein substrates, we explored the chaperone activity of SCGN in the cellular milieu by luciferase assay. Taking advantage of the fact that luciferase loses its activity when exposed to heat shock, we checked if SCGN overexpression would rescue luciferase from heat-induced loss of activity. HEK-293T cells were cotransfected with a pGL3 vector containing luciferase construct and eGFP(N3)-SCGN construct. SCGN overexpression was validated in HEK-293T cell line before performing the experiment using RT-PCR and Western blot (Fig. S1). To rule out the possibility of GFP demonstrating any activity, cells transfected with eGFP(N3) plasmid that express GFP alone were used as controls. The expression of other heat-inducible chaperones was suppressed by pretreating the cells with cycloheximide. Under our experimental conditions of heat shock in luciferase-transfected HEK-293T cells (control), we observed about 60% loss of luciferase activity, whereas in SCGN-overexpressing cells, the loss of luciferase activity was ∼35% (Fig. 3A). Moreover, SCGN overexpression further increased luciferase activity up to 80% after 4 h of recovery, suggesting the role of SCGN in refolding and regaining luciferase activity. The control and GFP-transfected cells did not show any increase in luciferase activity.
      Figure thumbnail gr3
      Figure 3SCGN overexpression rescues heat shock–induced misfolding of luciferase and prevents cell death against oxidative stress. A, percent luciferase activity of control or SCGN-overexpressing HEK-293T cells treated with heat shock (HS), without heat shock (WHS), and after heat shock recovery for 4 h (HSR). Untransfected cells and GFP transfected cells were kept as control. ∗ Represents p-value <0.05 and ∗∗ represents p-value <0.01 with a minimum of three biological replicates in a group. B, MTT assay of rotenone-treated HEK-293T cells with or without SCGN overexpression incubated with 100 nM, 500 nM, 1 μM, 10 μM, and 100 μM rotenone for 24 h. Cell viability was measured as a measure of absorbance at 540 nm. ∗ Represents p-value <0.05 and ∗∗ represents p-value <0.01 with a minimum of eight biological replicates in a group. SCGN, secretagogin. ## denotes significance of difference between control and 100 nM rotenone treatments, p-value <0.01.

      SCGN protects cells during oxidative stress

      Under stress, the expression of HSPs is increased to alleviate protein misfolding and cell death. As SCGN expression was temporally altered by heat and oxidative stress and SCGN exhibits chaperone activity both in vitro and ex vivo, we assessed if SCGN action is similar to that of an HSP aiding cell survival during cellular stress. Cell death was induced by treating HEK-293T cells with increasing concentrations of rotenone. Cell viability was examined by MTT assay in the rotenone-treated cells with or without SCGN overexpression (Fig. 3). A substantial decrease (up to 40%) in cell viability was observed with increasing concentrations of rotenone (100 nM, 500 nM, 1 μM, 10 μM, and 100 μM) (Fig. 3B). In contrast, SCGN overexpressing cells were up to 35% more viable than the control cells, demonstrating that SCGN plays a role in rescuing from cell death.

      Closed to an open conformational transition of SCGN by Ca2+

      Since SCGN is a Ca2+ sensor protein that exhibits Ca2+-dependent molecular chaperone activity, we next analyzed the effect of Ca2+ on the structural rearrangement of SCGN using small-angle X-ray scattering (SAXS). To ensure sample purity and homogeneity, a quality check of purified samples was performed before SAXS experiments by SDS-PAGE and gel filtration analysis (Fig. S2, A and B). The homodispersity of the SCGN samples was also ensured by dynamic light scattering analyses prior to SAXS (Fig. S2C). SAXS data of protein solutions in the range of 5 to 17 mg/ml concentrations and of the respective matched buffers as blank controls were collected. Data obtained with increasing concentrations of SCGN exhibit a profile characteristic of a compact globular molecule as depicted from the log I(q) versus log q plot (Fig. S3A). The Kratky plot confirms a well-folded conformation and exhibits only subtle differences between apo, Ca2+-, and Mg2+-bound SCGN (Fig. S3B). At low q values, a plot of ln(I(q)) versus q2 is linear and independent of protein concentration indicating the homogeneity of the protein sample as observed in the Guinier plot (Fig. S4A). The Guinier Rg calculated from the Guinier plot correlates well with distance distribution function (P(r)) Rg and CRYSOL Rg calculated from the crystal structures (PDB ID: 2be4 and 6jlh) of Danio rerio SCGN (Fig. S4B). The protein samples remained unaffected post-X-ray exposure as confirmed with SDS-PAGE analyses of post-SAXS samples (Fig. S5A). This was further corroborated with MALDI analysis of SCGN samples before and after X-ray exposure. The m/z peak corresponding to a molecular weight of 33 kDa (monomeric SCGN) did not alter (Fig. S5, B and C).
      At protein concentrations of ∼5 mg/ml, the radius of gyration (Rg) of apo-protein was found to be 23.4 Å, which increases drastically to 37.7 Å upon the addition of Ca2+. Interestingly, at a higher protein concentration (13.5 mg/ml), the radius of gyration in the presence of Ca2+ increased to 47.5 Å. On the other hand, even the saturated level of Mg2+ (8 mM) did not induce any change in Rg value (23.7 Å) with increasing concentrations, suggesting a Ca2+-specific structural rearrangement (Table S1). Structural models prepared using ab initio programs GASBOR and DAMMIF suggest that both the apo- and Mg2+-bound forms of SCGN exist as a V-shaped globular molecule. This conformation is transformed to an open, elongated structure by Ca2+, thus forming a cleft plausibly for substrate binding (Fig. 4A).
      Figure thumbnail gr4
      Figure 4Ca2+ binding leads to elongation of SCGN. SAXS-derived GASBOR (right) and DAMMIF ab initio models (left) of SCGN. The SAXS data were collected with ∼10 mg/ml SCGN for (A) Apo, Ca2+- (8 mM) and Mg2+- (8 mM) bound SCGN, and (B) SCGN in reducing (5 mM DTT) and oxidizing conditions (100 μM H202). SAXS, small-angle X-ray scattering; SCGN, secretagogin.
      To rule out the possibility of the Ca2+-dependent increase in Rg value being a result of dimerization or higher oligomer formation, we recorded structural changes in SCGN in reducing and oxidizing milieu using SAXS analysis. In reducing conditions (in the presence of 5 mM DTT), the Rg value of apo-SCGN was 22.8 Å, which was slightly increased to 25.6 Å in oxidizing conditions (in the presence of 100 μM H2O2). SCGN exists as a monomer in reducing conditions, while in oxidizing conditions, the protein tends to oligomerize. However, the effect of Ca2+ was more pronounced in oxidizing conditions. At low protein concentrations of holo-SCGN under oxidizing conditions, the Rg value was increased to 37.4 Å, and at higher protein concentrations, the Rg value was further increased to 46.9 Å. However, the Rg value of holo-SCGN in reducing conditions was increased only up to 33.3 Å, suggesting that the increase of Rg is not due to disulfide-mediated dimerization but is a consequence of Ca2+ binding (Table 1). This further confirms that dimerization of the Ca2+-bound form in oxidizing conditions is accentuated at higher protein concentrations, as reported earlier (
      • Khandelwal R.
      • Sharma A.K.
      • Chadalawada S.
      • Sharma Y.
      Secretagogin is a redox-responsive Ca2+ sensor.
      ,
      • Lee J.-J.
      • Yang S.-Y.
      • Park J.
      • Ferrell J.E.
      • Shin D.-H.
      • Lee K.-J.
      Calcium ion induced structural changes promote dimerization of secretagogin, which is required for its insulin secretory function.
      ).
      Table 1SAXS-derived structural parameters of SCGN at various concentrations in oxidizing (100 μM H2O2) and reducing conditions (5 mM DTT) in the presence and absence of Ca2+ using ATSAS software analysis
      SCGN (mg/ml)I0/cRgGuinier (Å)RgP(r) (Å)Dmax (Å)Guinier rangeMW porod (kDa)
      DTT8.70.3222.8± 1.223.075116232.3
      11.510.3222.9± 1.022.974126131.9
      16.650.3121.5± 1.722.06776631.6
      DTT + Ca2+8.750.3928.8± 1.028.692154735.3
      11.340.429.2± 0.928.995114534.9
      18.50.5533.3± 2.937.5167154044.1
      H2O25.390.4325.6± 0.626.610695434.6
      9.180.424.2± 1.123.97665732.1
      10.560.3823.5± 0.423.476115932
      H2O2 + Ca2+4.90.6937.4± 1.341.618442950.7
      7.340.6435.5± 1.540.8177103751.7
      15.30.9546.9± 2.852.521682684.5
      It is also important to note that the Dmax for Ca2+-bound protein samples (∼8 mg/ml) in reducing conditions is increased to 92 Å (from 75 Å for apo-SCGN), while in oxidizing conditions, Dmax increased considerably to 184 Å (from 106 Å for apo-SCGN). This supports our earlier data which showed that SCGN forms oligomers in a concentration-dependent manner under oxidizing conditions, while the addition of DTT abrogates oligomerization (
      • Khandelwal R.
      • Sharma A.K.
      • Chadalawada S.
      • Sharma Y.
      Secretagogin is a redox-responsive Ca2+ sensor.
      ,
      • Sharma A.
      • Khandelwal R.
      • Sharma Y.
      • Rajanikanth V.
      Secretagogin, a hexa EF-hand calcium-binding protein: high level bacterial overexpression, one-step purification and properties.
      ). While the GASBOR and DAMMIF models for apo SCGN with DTT and H2O2 are fairly similar, the model of holo-SCGN in oxidizing condition appears more elongated, in accordance with increased Rg values (Fig. 4B). On the other hand, Ca2+-bound SCGN in the presence of DTT remains a monomer that adopts a partially open conformation with the two terminal domains flanking away from the central region. Thus, intracellular Ca2+ concentration and cellular redox status would enable SCGN to adopt multiple conformations resulting in diverse substrate binding, a characteristic of a molecular chaperone.

      Structural and mechanistic insights into Ca2+-dependent enhancement of chaperone action of SCGN

      Having identified the closed (apo-SCGN) and open (holo-SCGN) conformations of SCGN, we decided to dissect the dynamics of these conformational changes. As shown above, Ca2+ binding causes major rearrangements with the relocation of both terminal domains and the formation of an open elongated structure, which is largely an extended arrangement of the domains. To reveal the progression of intermediate events, structural changes in SCGN (7 mg/ml) were monitored by SAXS by the sequential addition of Ca2+. The Rg value of the apo-SCGN (23.2 Å) did not change significantly when titrated with Ca2+ up to 100 μM Ca2+ but reduced marginally to 22.7 Å at 150 μM Ca2+ (Table 2). At 200 μM Ca2+ concentration, the Rg and Dmax values were restored to that of the apo-protein. At 250 μM Ca2+, an expansion of both Rg (26.8 Å) and Dmax (93 Å) was noted, signaling the initiation of a conformational transition. At this Ca2+ concentration, the V-shape geometry of apo-SCGN turns toward a U-shape, as seen in the GASBOR model, whereas one of the terminal domains is already flanked away as seen in the DAMMIF model (Figs. 5, and S7). At intermediate Ca2+ concentrations (250 μM [Ca2+] for DAMMIF model and 400 μM [Ca2+] for GASBOR model), the protein begins to open up, as displayed by the movement of one domain being pushed away from the center of the protein. Finally, at saturated Ca2+ concentrations (1 mM Ca2+), the Rg (31 Å) and Dmax (123 Å) values increased, indicating the opening of the molecule into an extended conformation (Table 2). In other words, the sequential addition of Ca2+ induces a structural rearrangement that initiates the movement of the N- and C-domains away from the middle domain, creating a crevice in the central domain leading to the creation of a site ready and available for substrate binding. The global structural changes induced by Ca2+ display step by step transformation of SCGN into a molecular chaperone.
      Table 2Rg values obtained from the Guinier approximation of the SAXS intensity profiles
      Ratio Ca2+/SCGN[Ca2+]I0/cRgGuinier (Å)RgP(r) (Å)Dmax (Å)Guinier rangeMW Porod (kDa)
      -0 μM0.3222.8± 1.223.075116232.3
      0.2350 μM0.323.2± 0.323.779176032.8
      0.46100 μM0.3123.4± 0.323.67976031.7
      0.69150 μM0.2722.7± 0.222.87386232
      0.92200 μM0.3223.3± 0.223.37786032.6
      1.15250 μM0.3826.8± 0.32793135134.6
      1.38300 μM0.3728± 2.227.28694534.7
      1.84400 μM0.429.2± 0.631.613274639.1
      2.3500 μM0.4430.1± 1.131.2107104538.9
      4.61 mM0.3931.2± 0.732.612354337.1
      SCGN (7 mg/ml) was titrated with increasing Ca2+ concentration ranging from 50 μM to 1 mM.
      Figure thumbnail gr5
      Figure 5Closed to open conformational transition of SCGN by Ca2+ as shown by SAXS modeling analysis. A, the GASBOR ab initio model and (B) DAMMIF molecular models of SCGN constructed upon Ca2+ titrations ranging from 50 μM to 1 mM. The BioSAXS data of SCGN were recorded at 7 mg/ml concentration. SAXS, small-angle X-ray scattering; SCGN, secretagogin.
      To understand the multiple states of SCGN conformation during Ca2+ binding, we analyzed the SAXS data using the MULTI-FOXS program, which provides population-weighted ensembles fitting to a SAXS profile of the protein in solution (
      • Schneidman-Duhovny D.
      • Hammel M.
      • Tainer J.A.
      • Sali A.
      FoXS, FoXSDock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles.
      ). Ensembles of models obtained were based on the crystal structure of apo-SCGN (PDB ID: 2be4 (
      • Bitto E.
      • Bingman C.A.
      • Bittova L.
      • Frederick R.O.
      • Fox B.G.
      • Phillips Jr., G.N.
      X-Ray structure of Danio rerio secretagogin: a hexa-EF-hand calcium sensor.
      )) and Ca2+-bound SCGN (PDB ID: 6jlh (
      • Qin J.
      • Liu Q.
      • Liu Z.
      • Pan Y.-Z.
      • Sifuentes-Dominguez L.
      • Stepien K.
      • et al.
      Structural and mechanistic insights into secretagogin-mediated exocytosis.
      )) from D. rerio. The MULTI-FOXS analysis reveals that the SAXS-based ensembles of the apo-state of mouse SCGN correlate well with the crystal structure of D. rerio apo-SCGN (PDB ID: 2be4) (Fig. 6A). The model built from the SAXS data of the SCGN at saturated Ca2+ concentration fits well with the crystal structure of Ca2+-bound D. rerio SCGN (PDB ID: 6jlh) (Fig. 6B). A substantial expansion of SCGN molecule from about 65 Å in the apo form to 82 Å in Ca2+-bound form is observed indicating the adoption of an open conformation exerted by Ca2+ binding to the molecule (Fig. 6, A and B). The models of apo- and Ca2+-bound SCGN overlap only in the C-terminal domain, whereas Ca2+ causes a large shift of the N-terminal domain along with the central region (Fig. 6C). The intermediate state during this transition captured at 250 μM [Ca2+] is a mixed ensemble of three randomly selected ensembles with several different orientations that do not overlap at all, demonstrating the high flexibility of this intermediate state (Fig. 6D). This is further corroborated by a sharp increase in the Rg value (Fig. 6D and Table 2). A structural comparison of the SAXS-derived model of Ca2+-bound SCGN with the Ca2+-bound crystal structure of a related Ca2+ sensor, calbindin-D28K, highlights the distinctive structural transformation of SCGN (Fig. S8). Unlike SCGN, Ca2+ does not induce a drastic conformational change in calbindin D28K (Fig. S8). We thus demonstrate how Ca2+ may activate SCGN by transforming the molecule from closed to open conformation to be geared for performing Ca2+-dependent functions.
      Figure thumbnail gr6
      Figure 6Structural analysis of apo to holo transitional states of SCGN. Multi-FOXS models showing best-fit ensembles of (A) apo and (B) Ca2+-bound SCGN. The overlay of two randomly selected conformational ensembles is represented in blue and orange. C, Apo (blue) and Ca2+-bound SCGN (orange) models overlap only in the C-terminal domain, while the N-terminal domain completely shifts by 180°. D, superimposition of three randomly selected ensembles of SCGN at 250 μM Ca2+ (transition state) (represented in blue, gray, and orange). They do not overlap demonstrating flexible states of mixed ensembles. SCGN, secretagogin.

      Ca2+ enables the binding of client proteins to SCGN

      It is seen above that Ca2+ transforms SCGN from closed to open conformation. To investigate how this process impacts the interaction of SCGN with client proteins, we selected α-synuclein, an intrinsically unstructured protein prone to aggregation and implicated in proteinopathy, and insulin known to interact with SCGN (
      • Sharma A.K.
      • Khandelwal R.
      • Kumar M.J.M.
      • Ram N.S.
      • Chidananda A.H.
      • Raj T.A.
      • et al.
      Secretagogin regulates insulin signaling by direct insulin binding.
      ,
      • Chidananda A.H.
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Secretagogin binding prevents α-synuclein fibrillation.
      ). The binding of α-synuclein to SCGN was examined by isothermal titration calorimetry (ITC). The thermogram of SCGN binding to α-synuclein in the presence of Ca2+ follows an endothermic process and data best fit to one-set of binding site model (Fig. 7A). The binding of SCGN to α-synuclein is an enthalpically unfavorable but entropically favorable process with enthalpy change (ΔH) being 1.24 cal/mol and entropy change (ΔS) being 435 cal/mol/deg. The dissociation constant (Kd) for binding of α-synuclein to SCGN is 110 μM, indicating a weak binding. We further confirmed it by monitoring the stability of SCGN–α-synuclein complex using analytical gel filtration. We found that SCGN does not form a stable complex with α-synuclein suggesting a weak interaction with the substrate (Fig. S9). The interaction of insulin with SCGN was also studied by ITC. As per analysis, the Ca2+-free SCGN binds insulin with an affinity of (Kd) ∼213 μM which, in the presence of Ca2+, increases up to ∼73 μM (Fig. 7, C and D). These results demonstrate that Ca2+ modulates the binding positively. Our results align with the finding that the binding of target proteins to chaperones is weak which enables target proteins to dissociate easily from the complex to be available to form stronger interactions of the folded state (
      • Taylor A.
      • Shkedi A.
      • Nadel C.M.
      • Gestwicki J.E.
      The interactions of molecular chaperones with client proteins: why are they so weak?.
      ). On the other hand, in the absence of Ca2+, we did not observe any measurable heat change (Fig. 7B), suggesting that binding of SCGN and α-synuclein takes place only when Ca2+ is present. This highlights the indispensable role of Ca2+ in mediating structural changes in SCGN, which allows substrate binding.
      Figure thumbnail gr7
      Figure 7Client proteins binding to SCGN. A and B, ITC thermogram depicting heat change per injection upon titration of α-synuclein (600 μM) with SCGN (60 μM): (A) in the presence of 2 mM Ca2+ and (B) in the absence of Ca2+. C and D, ITC thermogram of SCGN-insulin binding. ITC experiments were carried out with SCGN (30 μM) and research grade recombinant insulin (1 mM) (C) in the presence of 3 mM Ca2+, and (D) absence of Ca2+. Appropriate blanks were subtracted from the data. All the experiments were repeated at least thrice. The presented thermograms are from the representative set. ITC, isothermal titration calorimetry; SCGN, secretagogin.

      The substrate selectivity for chaperone action is determined by the distinct domains of SCGN

      The movement of the terminal domains by Ca2+ leading to the creation of a binding site for substrates led us to assign the role of each domain, if any, in imparting chaperone activity. To this end, we prepared two deletion constructs of SCGN: C-terminal deleted (ΔCTD) and N-terminal deleted (ΔNTD) constructs. The recombinant ΔCTD (20.4 kDa) and ΔNTD (19.1 kDa) proteins having the central domain in common were tested for chaperone activity. The choice of selection of two domains out of the three was to have stable protein preparations. While both proteins possess chaperone activity, there was a variation in the ability of ΔCTD (20.4 kDa) and ΔNTD (19.1 kDa) to suppress aggregation at identical protein to substrate ratios, depending on the model substrate. For example, in the case of CS, ΔCTD was more efficient than ΔNTD in precluding aggregation, while in the case of lysozyme, ADH, and MDH, ΔNTD was more efficient (Fig. 8). Apart from these results, our previous studies also displayed variations in the efficiency of suppressing aggregation. In the case of insulin, ΔCTD efficiently halts its aggregation (
      • Malenczyk K.
      • Szodorai E.
      • Schnell R.
      • Lubec G.
      • Szabó G.
      • Hökfelt T.
      • et al.
      Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for β cell specification.
      ), while for α-synuclein, ΔNTD of SCGN is more effective in preventing fibrillation (
      • Chidananda A.H.
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Secretagogin binding prevents α-synuclein fibrillation.
      ). We speculate that the size, shape, and charge of the substrate play an important role in deciding its interaction with either the N-terminal or C-terminal domain upon binding at the central domain. Thus, distinct regions of SCGN play a role in deciding how it interacts with its substrate for chaperone action.
      Figure thumbnail gr8
      Figure 8Central domain of SCGN is indispensable for chaperone activity. Aggregation kinetics of model substrates: (A) CS, (B) MDH, (C) ADH, and (D) lysozyme was monitored in the presence of varying concentrations of ΔNTD-SCGN and ΔCTD-SCGN in the presence of either 10 μM EDTA (red) or 2 mM Ca2+ (blue). The percentage decrease in absorbance was calculated and plotted at the saturation point. ADH, alcohol dehydrogenase, CS, citrate synthase; MDH, malate dehydrogenase; SCGN, secretagogin; ΔCTD, C-terminal deleted; ΔNTD, N-terminal deleted.

      Discussion

      Dysregulation of SCGN homeostasis is implicated in the progression of several pathophysiologies like diabetes, neurodegeneration, and cancer (
      • Hansson S.
      • Vachet P.
      • Eriksson J.
      • Pereira M.
      • Skrtic S.
      • Wallin H.
      • et al.
      Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.
      ,
      • Attems J.
      • Ittner A.
      • Jellinger K.
      • Nitsch R.
      • Maj M.
      • Wagner L.
      • et al.
      Reduced secretagogin expression in the hippocampus of P301L tau transgenic mice.
      ,
      • Zahola P.
      • Hanics J.
      • Pintér A.
      • Máté Z.
      • Gáspárdy A.
      • Zsófia H.
      • et al.
      Secretagogin expression in the vertebrate brainstem with focus on the noradrenergic system and implications for Alzheimer’s disease.
      ,
      • Zhan X.
      • Evans C.O.
      • Oyesiku N.M.
      • Desiderio D.M.
      Proteomics and transcriptomics analyses of secretagogin down-regulation in human non-functional pituitary adenomas.
      ,
      • Xing X.
      • Lai M.
      • Gartner W.
      • Xu E.
      • Huang Q.
      • Li H.
      • et al.
      Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin.
      ,
      • Juhlin C.C.
      • Zedenius J.
      • Höög A.
      Clinical routine application of the second-generation neuroendocrine markers ISL1, INSM1, and secretagogin in neuroendocrine neoplasia: staining outcomes and potential clues for determining tumor origin.
      ,
      • Sifuentes-Dominguez L.
      • Li H.
      • Llano E.
      • Liu Z.
      • Singla A.
      • Patel A.
      • et al.
      SCGN deficiency results in colitis susceptibility.
      ,
      • Yu L.
      • Suye S.
      • Huang R.
      • Liang Q.
      • Fu C.
      Expression and clinical significance of a new neuroendocrine marker secretagogin in cervical neuroendocrine carcinoma.
      ). A common causative factor in the onset and advancement of these disorders is cellular stress. There can be various factors that are responsible for cellular stress, namely heat shock, redox imbalance resulting in oxidative stress, heavy metal exposure, and deregulation of Ca2+ homeostasis (
      • Morimoto R.I.
      Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators.
      ,
      • Hagen T.M.
      Oxidative stress, redox imbalance, and the aging process.
      ,
      • Park C.
      • Jeong J.
      Synergistic cellular responses to heavy metal exposure: a minireview.
      ,
      • Preissler S.
      • Rato C.
      • Yan Y.
      • Perera L.,A.
      • Czako A.
      • Ron D.
      Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes.
      ,
      • Lebeau P.F.
      • Platko K.
      • Byun J.H.
      • Austin R.C.
      Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells.
      ). Multiple studies have reported a change in serum/plasma levels of SCGN during diabetes, neurodegeneration, and cancer, suggesting it to be a potential biomarker for these diseases (
      • Hansson S.
      • Vachet P.
      • Eriksson J.
      • Pereira M.
      • Skrtic S.
      • Wallin H.
      • et al.
      Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.
      ,
      • Zahola P.
      • Hanics J.
      • Pintér A.
      • Máté Z.
      • Gáspárdy A.
      • Zsófia H.
      • et al.
      Secretagogin expression in the vertebrate brainstem with focus on the noradrenergic system and implications for Alzheimer’s disease.
      ,
      • Xing X.
      • Lai M.
      • Gartner W.
      • Xu E.
      • Huang Q.
      • Li H.
      • et al.
      Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin.
      ,
      • Gartner W.
      • Lang W.
      • Leutmetzer F.
      • Domanovits H.
      • Waldhäusl W.
      • Wagner L.
      Cerebral expression and serum detectability of secretagogin, a recently cloned EF-hand Ca2+-binding protein.
      ). Further, overexpression of SCGN has been shown to rescue neurodegeneration and insulin resistance (
      • Malenczyk K.
      • Szodorai E.
      • Schnell R.
      • Lubec G.
      • Szabó G.
      • Hökfelt T.
      • et al.
      Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for β cell specification.
      ,
      • Attems J.
      • Ittner A.
      • Jellinger K.
      • Nitsch R.
      • Maj M.
      • Wagner L.
      • et al.
      Reduced secretagogin expression in the hippocampus of P301L tau transgenic mice.
      ). SCGN expression is known to be regulated by the activation of Ca2+ permeable TRPV1 channels (
      • Bromberg Z.
      • Weiss Y.
      The role of the membrane-initiated heat shock response in cancer.
      ), and TRPV1 agonists also upregulate Hsp70 (
      • Malenczyk K.
      • Girach F.
      • Szodorai E.
      • Storm P.
      • Segerstolpe A.
      • Tortoriello G.
      • et al.
      A TRPV1-to-secretagogin regulatory axis controls pancreatic β-cell survival by modulating protein turnover.
      ,
      • Bromberg Z.
      • Goloubinoff P.
      • Saidi Y.
      • Weiss Y.G.
      The membrane-associated transient receptor potential vanilloid channel is the central heat shock receptor controlling the cellular heat shock response in epithelial cells.
      ) that suggests a relation between SCGN expression and heat shock response. During stress, the transcript and protein expression dynamics of most of the stress proteins is inconsistent (
      • Vogel C.
      • Silva G.M.
      • Marcotte E.M.
      Protein expression regulation under oxidative stress.
      ,
      • Cheng Z.
      • Teo G.
      • Krueger S.
      • Rock T.M.
      • Koh H.W.
      • Choi H.
      • et al.
      Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress.
      ). Upon cellular stress, the mRNAs of stress proteins exhibit transient spike and decline, while their protein levels either elevate and maintain a new steady-state (GRP78 and HSP90B1) or remain constant throughout response (HSP90AA1) (
      • Vogel C.
      • Silva G.M.
      • Marcotte E.M.
      Protein expression regulation under oxidative stress.
      ,
      • Cheng Z.
      • Teo G.
      • Krueger S.
      • Rock T.M.
      • Koh H.W.
      • Choi H.
      • et al.
      Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress.
      ). The Scgn transcript dynamics concurs with those of a few stress-responsive proteins like GRP78, HSP90B1, and P58IPK (
      • Vogel C.
      • Silva G.M.
      • Marcotte E.M.
      Protein expression regulation under oxidative stress.
      ). The increased Scgn transcript levels during the early response, however, do not correlate with the increase in intracellular protein levels. Further, during the late response to oxidative stress, SCGN protein is maintained at a lower steady-state level, unlike that of constitutively-expressed GRP78 or HSP90B1 chaperones (
      • Vogel C.
      • Silva G.M.
      • Marcotte E.M.
      Protein expression regulation under oxidative stress.
      ). We speculate that this could be either due to yet unknown posttranscriptional regulation or due to secretion of SCGN into the media poststress. Despite these intriguing correlations, the precise role and regulation of SCGN expression during cellular stress remain unknown.
      We further demonstrate that SCGN effectively prevents both the heat-inducible as well as the chemically inducible (DTT) aggregation of a wide range of proteins in vitro in a Ca2+-dependent manner. This finding, along with the interaction of SCGN with several bona fide molecular chaperones and 26S proteasome subunits, indicates that SCGN could be a part of the protein folding assembly to aid in the protein homeostasis and/or folding process (
      • Maj M.
      • Wagner L.
      • Tretter V.
      20 Years of secretagogin: exocytosis and beyond.
      ,
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Veiled potential of secretagogin in diabetes: correlation or coincidence?.
      ). Another layer of regulation of SCGN activity could stem from the localization of SCGN and the corresponding organelle milieu that dictates the Ca2+ and the redox signal/stimulus needed to activate SCGN. We have reported earlier that in an oxidizing environment (like in ER), SCGN could act as a Ca2+ buffer, while in the cytosol’s reducing environment, it might act as a Ca2+ sensor (
      • Sharma A.K.
      • Khandelwal R.
      • Kumar M.J.M.
      • Ram N.S.
      • Chidananda A.H.
      • Raj T.A.
      • et al.
      Secretagogin regulates insulin signaling by direct insulin binding.
      ,
      • Lee J.-J.
      • Yang S.-Y.
      • Park J.
      • Ferrell J.E.
      • Shin D.-H.
      • Lee K.-J.
      Calcium ion induced structural changes promote dimerization of secretagogin, which is required for its insulin secretory function.
      ,
      • Sharma A.
      • Khandelwal R.
      • Sharma Y.
      • Rajanikanth V.
      Secretagogin, a hexa EF-hand calcium-binding protein: high level bacterial overexpression, one-step purification and properties.
      ,
      • Sanagavarapu K.
      • Weiffert T.
      • Ní Mhurchú N.
      • O'Connell D.
      • Linse S.
      Calcium binding and disulfide bonds regulate the stability of secretagogin towards thermal and urea denaturation.
      ). Thus, we speculate that SCGN would largely function as a chaperone in the ER’s oxidizing environment with high Ca2+ concentration. However, in the cytosol, an increase in cytosolic Ca2+ concentration upon release from intracellular stores could lead to the activation of SCGN.
      We further checked if SCGN could mediate its chaperone functions in a cellular scenario. We found that SCGN overexpression prevents the loss of luciferase activity in HEK-293T cells. Our results further bolster the earlier published results of SCGN-mediated inhibition of fibrillogenesis of insulin and α-synuclein (
      • Malenczyk K.
      • Szodorai E.
      • Schnell R.
      • Lubec G.
      • Szabó G.
      • Hökfelt T.
      • et al.
      Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for β cell specification.
      ,
      • Chidananda A.H.
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Secretagogin binding prevents α-synuclein fibrillation.
      ). Based on these results, it is reasonable to anticipate that SCGN would indeed protect other aggregation-prone proteins, specifically those implicated in neurodegenerative diseases, such as Aβ-42 and Tau from undergoing fibrillogenesis. We also demonstrated that SCGN overexpression could protect cells from oxidative stress. This is in line with the earlier reports that demonstrated a correlation of SCGN expression with ER stress, protein folding, quality control, and cell survival (
      • Bromberg Z.
      • Weiss Y.
      The role of the membrane-initiated heat shock response in cancer.
      ,
      • Khandelwal R.
      • Sharma A.K.
      • Biswa B.B.
      • Sharma Y.
      Extracellular secretagogin is internalized into the cells through endocytosis.
      ). This is possibly mediated by maintaining the level and activity of proteins, such as ubiquitin carboxyl-terminal hydrolases USP9X and USP7 that boost β-cell proliferation, implying a direct role in protein folding (
      • Malenczyk K.
      • Szodorai E.
      • Schnell R.
      • Lubec G.
      • Szabó G.
      • Hökfelt T.
      • et al.
      Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for β cell specification.
      ).
      We now identify chaperone activity as one of the crucial functions of SCGN vis-a-vis Ca2+ and elucidate why deregulated SCGN levels accelerate protein misfolding disorders. Reduced levels of SCGN in certain tissues, as noted in protein folding diseases (
      • Attems J.
      • Ittner A.
      • Jellinger K.
      • Nitsch R.
      • Maj M.
      • Wagner L.
      • et al.
      Reduced secretagogin expression in the hippocampus of P301L tau transgenic mice.
      ,
      • Zahola P.
      • Hanics J.
      • Pintér A.
      • Máté Z.
      • Gáspárdy A.
      • Zsófia H.
      • et al.
      Secretagogin expression in the vertebrate brainstem with focus on the noradrenergic system and implications for Alzheimer’s disease.
      ,
      • Khandelwal R.
      • Sharma A.K.
      • Chadalawada S.
      • Sharma Y.
      Secretagogin is a redox-responsive Ca2+ sensor.
      ), and increased release of SCGN in response to endoplasmic reticulum stress (
      • Hansson S.
      • Vachet P.
      • Eriksson J.
      • Pereira M.
      • Skrtic S.
      • Wallin H.
      • et al.
      Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.
      ) is consistent with our findings. We provide a rationale for a direct interplay among the three: (i) cellular stress, (ii) deregulation of Ca2+ and/or SCGN expression levels, and (iii) protein misfolding diseases, such as Parkinson’s and Alzheimer’s disease. A reduction in SCGN expression, or even a perturbation in Ca2+ homeostasis, would consequently affect SCGN’s propensity to function as a chaperone, which would hasten the aggregation of the concerned proteins, accelerating the onset of disease.
      We next checked the alterations in structural features of SCGN by Ca2+ in the augmentation of SCGN’s chaperone activity. BioSAXS has provided us with an innovative perspective on the Ca2+-dependent folding mechanism of SCGN in unprecedented detail. The pair distribution curve (the P(r) function), the Guinier plot, Io/C value, and concentration-dependent monitoring of Rg values are good quality control checks, and the current work appropriately accounted for them. Monitoring the ensembles of structures during the sequential addition of Ca2+ has allowed us to display the presence of a conformational switch of SCGN. Ca2+ binding triggers the drifting of both terminal domains away from the central domain, creating an open groove, enough to accommodate the binding of a broad range of substrates. The ends of both terminal domains appear to move away from each other, with the connecting residues—between NTD and the central domain (residues 94–100) and between the central domain and CTD (residues 173–178)—acting like flexible hinges (computed using the HingeProt program) (
      • Emekli U.
      • Schneidman-Duhovny D.
      • Wolfson H.J.
      • Nussinov R.
      • Haliloglu T.
      HingeProt: automated prediction of hinges in protein structures.
      ). This drifting movement of both terminal domains serves as a mechanistic model for the transition from the closed (apo form) to open and activated conformation (Ca2+ bound). It is to be noted that a similar Ca2+-dependent elongation of the structure is reported in gelsolin, a Ca2+-binding protein (
      • Ashish
      • Paine M.S.
      • Perryman P.B.
      • Yang L.
      • Yin H.L.
      • Krueger J.K.
      Global structure changes associated with Ca2+ activation of full-length human plasma gelsolin.
      ), which is not known to act as a chaperone. Calbindin D28K is another hexa EF-hand protein that does not show such structural change upon Ca2+ binding (
      • Noble J.W.
      • Almalki R.
      • Roe S.M.
      • Wagner A.
      • Duman R.
      • Atack J.R.
      The X-ray structure of human calbindin-D28K: an improved model.
      ). Thus, our study opens a new avenue for monitoring structural transitions in Ca2+-dependent chaperones to better understand their mechanism of action.
      To summarize, the present study describes a previously unidentified function of SCGN wherein it not only efficiently chaperones various protein substrates but also helps in maintaining a healthy proteome in cells during conditions of stress. The interplay between stress, neurodegenerative diseases, and SCGN expression was unclear for a long time. The importance of SCGN as a Ca2+-dependent chaperone and its role in stress physiology has helped resolve this enigma. This study enables the extraction of important insights into the multifunctional nature of SCGN and lays a foundation for future therapeutic discoveries for neurodegenerative diseases exploiting the SCGN model.

      Experimental procedures

      Chemicals and antibodies

      Alcohol dehydrogenase lyophilized powder sourced from baker’s yeast was obtained from SRL. CS and MDH sourced from the porcine heart (Sigma), and lysozyme from chicken egg white (Sigma) were used for chaperone assays. The antibodies used in these experiments are anti-SCGN bs-(11744R; Bioss), anti-Hsp70 (MA3-007; Invitrogen), anti-GRP78(C50B12; CST), anti-β-Actin (bs0061R; Bioss), and HRP conjugated secondary antibody (bs0295G; Bioss).

      Cloning and protein purification

      The overexpression of mouse SCGN cloned in pET21b expression vector in E. coli BL21-DE3 cells was induced with 1 mM IPTG at A600. of 0.6 and incubated at 25 °C for 10 h postinduction as reported previously (
      • Sharma A.
      • Khandelwal R.
      • Sharma Y.
      • Rajanikanth V.
      Secretagogin, a hexa EF-hand calcium-binding protein: high level bacterial overexpression, one-step purification and properties.
      ,
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Secretagogin purification and quality control strategies for biophysical and cell biological studies.
      ). Briefly, the soluble fraction of the bacterial pellet was loaded on a Ni-NTA-Sepharose (GE Healthcare) column, which was pre-equilibrated with 50 mM Tris, pH 7.5, 100 mM KCl (equilibration buffer), and washed with wash buffer (50 mM Tris, pH 7.5, 100 mM KCl, and 2% Triton X-100) followed by a wash with equilibration buffer. The protein was eluted with a gradient of 0 to 250 mM imidazole in 50 mM Tris, pH 7.5, and 100 mM KCl buffer.

      ΔNTD-SCGN and ΔCTD-SCGN

      The overexpression of ΔNTD-SCGN or ΔCTD-SCGN in E. coli BL21-DE3 cultures was induced with 1 mM IPTG at 37 °C after the A600 reached about 0.6 and incubated at 18 °C for 16 h postinduction (
      • Chidananda A.H.
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Secretagogin binding prevents α-synuclein fibrillation.
      ). The ΔNTD-SCGN was purified by employing a two-step procedure. The soluble fraction from the bacterial pellet was first loaded onto a phenyl-Sepharose column (GE Healthcare). After one wash with lysis buffer, the protein was eluted in an elution buffer of 50 mM Tris, pH 7.5 and 100 µM EDTA. In the second step of purification, the protein eluted in the previous step was loaded on a Q-Sepharose column in the same buffer (50 mM Tris, pH 7.5 and 0.5 mM EDTA). The nonspecific proteins were removed first by a wash buffer containing 2% Triton X-100 followed by a wash buffer without Triton X-100. The protein was eluted using a gradient of 200 mM to 1 M NaCl. The purification of ΔCTD-SCGN was performed on an anion exchange chromatography on a Q-Sepharose resin using the same protocol that is described for ΔNTD-SCGN (
      • Chidananda A.H.
      • Sharma A.K.
      • Khandelwal R.
      • Sharma Y.
      Secretagogin binding prevents α-synuclein fibrillation.
      ). All the proteins were further purified using size-exclusion chromatography on a Sephadex 75pg column coupled to an FPLC machine (Bio-Rad). Purified proteins were decalcified by incubation with 100 μM EDTA followed by buffer exchange against Chelex-purified buffer till the added EDTA is removed.

      Chaperone activity assay

      Aggregation kinetics was monitored in time-dependent mode on a Lambda 35 UV spectrophotometer (PerkinElmer) equipped with a water bath or on a Hitachi Fluorescence Spectrophotometer F-7000 connected to a temperature controller (TCC100). Thermal aggregation of ADH (1.3 μM), MDH (1.5 μM), or CS (2 μM) solutions in 50 mM Hepes, 100 mM KCl, pH 7.5, was induced at 41.5 °C, 50 °C, and 45 °C respectively, and monitored at 465 nm, 450 nm, and 500 nm, respectively (
      • Shao F.
      • Bader M.W.
      • Jakob U.
      • Bardwell J.C.
      DsbG, a protein disulfide isomerase with chaperone activity.
      ,
      • Abgar S.
      • Vanhoudt J.
      • Aerts T.
      • Clauwaert J.
      Study of the chaperoning mechanism of bovine lens alpha-crystallin, a member of the alpha-small heat shock superfamily.
      ,
      • Raychaudhuri S.
      • Sinha M.
      • Mukhopadhyay D.
      • Bhattacharyya N.P.
      HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity.
      ,
      • Matukumalli S.R.
      • Tangirala R.
      • Rao C.M.
      Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation.
      ). Chemical aggregation of lysozyme (10 μM) was initiated by the addition of 20 mM DTT in 50 mM Hepes, 100 mM KCl, pH 7.5 buffer. Change in turbidity of ADH, CS, and lysozyme was monitored by measuring the increase/decrease in absorbance units. Aggregation of MDH was monitored by recording 90° scattering at 465 nm on a fluorescence spectrophotometer. The aggregation kinetics of all the substrates was recorded either with 2 mM Ca2+ or with 10 μM EDTA. The reaction mixtures of each substrate with or without SCGN (or BSA as a control) were incubated at respective temperatures with constant stirring and monitored at the abovementioned wavelengths. The reaction mixtures of each substrate with the deletion constructs of SCGN (ΔNTD and ΔCTD) were also incubated at temperatures as mentioned above, and absorbance was monitored at 500 nm, 450 nm, 465 nm, and 360 nm wavelengths for CS, MDH, ADH, and lysozyme, respectively. Experiments were performed with varying stoichiometric ratios of SCGN to substrates (1:1, 1:2, and 2:1). All the experiments were performed in triplicates, and the obtained data were analyzed and plotted using Origin Lab (2019b version) and GraphPad Prism 8. The percent change in chaperone activity of SCGN is calculated using,
      %chaperoneactivityofSCGN=100(ASubstrateASCGN+SubstrateASubstrate) ∗ 100


      where ASubstrate is the absorbance at a respective wavelength for the substrates used (ADH, MDH, CS, and lysozyme), and ASCGN + Substrate is the highest absorbance value obtained with substrate and SCGN.

      Cell culture

      RIN-5F and HEK-293T cells were cultured in RPMI and DMEM media, respectively, supplemented with 10% fetal bovine serum (containing penicillin, streptomycin, and kanamycin) and maintained at 37 °C in a 5% CO2 incubator. Luciferase construct in basic pGL3 vector [procured from Promega luciferase assay system kit (Catalog No. E4030)], pEGFP(N3)-SCGN plasmid, and SCGN-eGFPN3 constructs were used for transient overexpression in HEK-293T cells. Transfection of cells was performed with Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer's protocol.

      Heat shock treatment

      RIN-5F cells (0.2 million) were seeded in a T-25 flask. After 24 h, the cells were subjected to heat shock at 42 °C for 45 min. Cells without heat shock were used as control. Samples in triplicate were collected at 0, 1, 2, 3, 4, 5, and 6 h of recovery from heat shock. The cells were either lysed in RIPA buffer for Western blotting or in Trizol reagent for qRT-PCR analysis.

      Rotenone treatment

      RIN-5F cells were seeded in a 6-well plate at a density of 2 × 105 cells/well. The next day, the cells were incubated with 1 μM rotenone (Sigma), and the samples were collected at 3, 6, 9, 12, 18, and 24 h. Untreated cells were maintained as control. Cells were lysed in RIPA buffer for Western blotting and in Trizol reagent for RNA preparation for qRT-PCR. Each experiment was performed in triplicate.

      Western blotting

      Protein in cell lysate was quantified using a Takara BCA protein estimation kit. For each sample, 35 μg total protein was loaded on 12% SDS PAGE gel and transferred onto a nitrocellulose membrane at 100 V for 2 h. After blocking with 5% BSA for 2 h, blots were incubated overnight at 4 °C with either anti-SCGN antibody (1:4000) or anti-HSP70 antibody (1:2000) or anti-GRP78 (1:2000) or β-Actin (1:20,000). The next day, the blots were washed with TBST (50 mM Tris, pH 7.5, 100 mM KCl, 0.1% Tween-20) four times at 10 min intervals. Subsequently, the blots were incubated with anti-mouse or anti-rabbit secondary antibody conjugated with HRP (1:10,000) for an hour. Post incubation, the blots were washed four times every 10 min with TBST and developed using the chemiluminescence method with a Bio-Rad ECL substrate kit. The obtained bands were analyzed using Image J software and normalized with β-actin.

      Quantitative real-time PCR

      Total RNA from the heat shock and oxidative stress samples was extracted by Trizol-chloroform method followed by DNase treatment. After assessing RNA quality, 1 μg of total RNA from each sample was used for cDNA synthesis (Takara PrimeScript first strand cDNA synthesis kit). mRNA expression levels were quantified by qRT-PCR on an ABI Prism 7900 HT (Applied Biosystems) using the SYBR Green detection system (Applied Biosystems). The reactions were performed according to the following conditions: initial hold at 95 °C for 30 s, followed by 40 cycles of amplification at 95 °C for 10 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. At least three technical replicates were prepared in each group of samples. β-actin/RPL11 or TBP was used as a reference control. The relative mRNA expression was calculated using the 2-ΔΔct as a mean of at least three technical replicates. Primers for the target genes used for qRT-PCR are listed in Supplementary Information (Table S2).

      MTT assay

      HEK-293T cells transfected with either pEFGP(N3) or pEGFP(N3)-SCGN constructs were seeded at a cell density of 15,000 cells/well in a 96-well plate. After 24 h, cells were incubated with increasing concentrations of rotenone (Sigma) (0, 100 nM, 500 nM, 1 μM, 10 μM, and 100 μM) for 24 h. Cells without any treatment and cells treated with 5% Triton X-100 were employed as controls. After 24 h of incubation, the cells were washed twice with PBS. Thereafter, 0.5 mg/ml of MTT was added to serum-free DMEM and incubated for 4 h at 37 °C in a CO2 incubator. Subsequently, MTT was removed, 100 μl of DMSO was added to dissolve MTT crystals and incubated for 30 min at 37 °C. Post incubation, the absorbance was recorded at 540 nm in an EnSpire Multimode plate reader and for% cell viability was calculated in the percentage of untreated control cells. Each group consisted of eight biological replicates.

      Cell culture–based luciferase assay

      HEK-293T cells (0.2 × 106) seeded in 6-well plates were transfected using lipofectamine LTX reagent (Invitrogen) by following the manufacturer’s protocol. Transfection was performed in incomplete media with firefly luciferase pGL3 vector (Promega luciferase assay system; Catalog No. E4030). Either only luciferase or luciferase with pEGFP(N3)-SCGN construct were cotransfected into cells. Luciferase and pEGFP(N3) vector cotransfection were used as controls. The transfection medium was replaced by DMEM complete media after 6 h. After 36 h of transfection, cells were incubated with 20 μg/ml cycloheximide for 30 min to suppress the expression of other chaperones. Subsequently, cells were subjected to heat shock at 42 °C for 45 min. Untreated cells were used as control. The luciferase activity for one set of cells was measured immediately after heat shock treatment, while for another set of cells after 4 h of recovery using EnSpire Multimode Plate Reader for reading time of 5 s with an initial delay time of 2 s in luminescence mode. All experiments were performed in triplicates. The percentage luciferase activity is calculated considering without heat shock (WHS) as a control for each group using the formula.
      % Luciferase Activity=(Test/Control)100


      Small-angle X-ray scattering

      SAXS data were collected as mentioned earlier on a Rigaku BioSAXS 2000 system at a wavelength of 1.54178 Å with 2D Kratky collimation, equipped with Rigaku HyPix-3000 hybrid pixel array detector attached to MicroMax 007 HF generator, operated at 40 kV and 30 mA (
      • Srivastava S.S.
      • Jamkhindikar A.A.
      • Raman R.
      • Jobby M.K.
      • Chadalawada S.
      • Sankaranarayanan R.
      • et al.
      A transition metal-binding, trimeric βγ-crystallin from methane-producing thermophilic archaea, Methanosaeta thermophila.
      ). SAXS experiments were performed in either reducing (5 mM DTT) or oxidizing conditions (100 μM H2O2) with multiple concentrations of apo-SCGN ranging from 5 mg/ml to 15 mg/ml and in the presence of either 8 mM Ca2+ or 8 mM Mg2+. The protein solutions were prepared in 50 mM Tris, pH 7.5, 100 mM KCl buffer. The matched buffer for each sample was collected from flow through and used as a buffer blank.
      Second set of experiments included Ca2+ titration (from 50 μM to 1 mM) with SCGN concentration at 7 mg/ml (with 5 mM DTT). It was an appropriate concentration at which the quality of data was good (signal/noise ratio). The sample and its matched buffer were exposed to X-ray for 60 min. The data were processed with the Automatic Data Analysis Pipeline (AAP), which is based on the ATSAS suite (version 2.7) and ATSAS online (Version 3.7) (
      • Franke D.
      • Petoukhov M.V.
      • Konarev P.V.
      • Panjkovich A.
      • Tuukkanen A.
      • Mertens H.
      • et al.
      Atsas 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions.
      ). The Guinier analysis was performed using RAW software (
      • Hopkins J.B.
      • Gillilan R.E.
      • Skou S.
      BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis.
      ).
      All SAXS models were constructed using DAMMIN and GASBOR online tools (
      • Svergun D.I.
      • Petoukhov M.V.
      • Koch M.H.J.
      Determination of domain structure of proteins from X-ray solution scattering.
      ,
      • Franke D.
      • Svergun D.I.
      DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering.
      ). Multi-FOXS analysis was carried out with apo crystal structure (PDB ID: 2be4) as well as with Ca2+-bound structure (PDB ID: 6jlh) of D. rerio SCGN.

      Isothermal titration calorimetry

      The energetics of SCGN binding to substrates (α-synuclein and insulin) were studied at 30 °C on an ITC-200 instrument. SCGN samples (60 μM in the cell) and 600 μM α-synuclein (in the syringe) were prepared in Chelex-purified 50 mM Tris buffer, pH 7.5, and 100 mM KCl in the presence of 2 mM Ca2+. Insulin binding to SCGN in the absence/presence of Ca2+ were performed on a Microcal VP-ITC instrument at 30 °C in Chelex-purified 50 mM Tris, pH 7.5, 100 mM KCl. All experiments were performed using 1 mM insulin in the syringe and 30 μM SCGN. For Ca2+ free titrations, 100 μM EGTA was added both to SCGN and insulin, while for Ca2+ saturated titrations, 3 mM Ca2+ was added both to SCGN and insulin. The graphs obtained were buffer subtracted by titrating into α-synuclein or insulin with buffer under similar conditions. Data fittings were performed with the help of Origin 8 software.

      Dynamic light scattering

      Dynamic light scattering measurements of SCGN (13 mg/ml) samples prepared in buffer (50 mM Tris, pH 7.5, and 100 mM KCl) were performed on an SZ-100 Horiba instrument SCGN (13 mg/ml) at room temperature with 20 accumulations each time. A buffer blank reading was used for buffer subtraction. Prior to analyses, the protein samples were centrifuged at 15,000 rpm for 15 min.

      MALDI analysis

      SCGN samples (7 mg/ml) to be analyzed were mixed with equal volumes of a sinapinic acid matrix (1:1). The 2 μl mixed sample was loaded onto a MALDI plate and allowed to air dry. The MALDI plate was placed in a mass spectrometer to acquire the spectra. The m/z values were analyzed using the software MASCOT.

      Statistical analysis

      All data were analyzed using GraphPad Prism 8 and expressed as mean ± standard deviation. A p-value of <0.05 was considered statistically significant between different test conditions determined by a two-sided Student’s t test for western and qRT-PCR experiments. Data from luciferase and MTT assay were analyzed, and p-value (<0.05 significance) was calculated by two-way ANOVA with Bonferroni multiple comparison test.

      Data availability

      All data generated and analyzed during this study are included in this article.

      Supporting information

      This article contains supporting information. Supporting Figures S1-S9 and Supporting Tables S1-S2.

      Conflict of interest

      The authors declare that they have no conflict of interest with the content of this article.

      Acknowledgments

      We acknowledge Dr Ghanshyam Swarup for providing the GRP78 antibody and Dr Venu Sankeshi for technical help. We are grateful to Shanti Swaroop Srivastava for and providing valuable inputs to the manuscript. We thank Syed Sayeed Abdul for providing excellent laboratory assistance. We also acknowledge the support of the Fine Biochemicals facility and Tissue Culture facility at CCMB.

      Author contributions

      A. H. C., A. K. S., and Y. S. conceptualization; A. H. C. and R. K. investigation; A. H. C. data curation; A. H. C., R. K., and A. K. S. writing-original draft; A. H. C., R. K., A. D. P., and A. K. S. formal analysis; A. H. C. visualization; R. K., A. D. P., and Y. S. writing-reviewing and editing; R. K. and A. J. resources; A. J. and A. K. S. methodology; A. K. S. validation; Y. S. supervision; Y. S. project administration; Y. S. funding acquisition.

      Funding and additional information

      This work is supported by the J. C. Bose National Fellowship (SERB) granted to Y. S., A. H. C., and R. K. were the recipients of the ICMR and CSIR-GATE research fellowship, respectively. The current affiliation of A. K. S. and R. K. is Translational Nutrition Biology Laboratory, ETH Zurich ( [email protected] ).

      Supporting information

      References

        • Wagner L.
        • Oliyarnyk O.
        • Gartner W.
        • Nowotny P.
        • Gröger M.
        • Kaserer K.
        • et al.
        Cloning and expression of secretagogin, a novel neuroendocrine and pancreatic islet of Langerhans-specific Ca2+-binding protein.
        J. Biol. Chem. 2000; 275: 24740-24751
        • Rogstam A.
        • Linse S.
        • Lindqvist A.
        • James P.
        • Wagner L.
        • Berggård T.
        Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin.
        Biochem. J. 2007; 401: 353-363
        • Qin J.
        • Liu Q.
        • Liu Z.
        • Pan Y.-Z.
        • Sifuentes-Dominguez L.
        • Stepien K.
        • et al.
        Structural and mechanistic insights into secretagogin-mediated exocytosis.
        Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 6559-6570
        • Hansson S.
        • Vachet P.
        • Eriksson J.
        • Pereira M.
        • Skrtic S.
        • Wallin H.
        • et al.
        Secretagogin is increased in plasma from type 2 diabetes patients and potentially reflects stress and islet dysfunction.
        PLoS One. 2018; 13e0196601
        • Malenczyk K.
        • Szodorai E.
        • Schnell R.
        • Lubec G.
        • Szabó G.
        • Hökfelt T.
        • et al.
        Secretagogin protects Pdx1 from proteasomal degradation to control a transcriptional program required for β cell specification.
        Mol. Metab. 2018; 14: 108-120
        • Sharma A.K.
        • Khandelwal R.
        • Kumar M.J.M.
        • Ram N.S.
        • Chidananda A.H.
        • Raj T.A.
        • et al.
        Secretagogin regulates insulin signaling by direct insulin binding.
        iScience. 2019; 21: 736-753
        • Attems J.
        • Ittner A.
        • Jellinger K.
        • Nitsch R.
        • Maj M.
        • Wagner L.
        • et al.
        Reduced secretagogin expression in the hippocampus of P301L tau transgenic mice.
        J. Neural Transm. 2011; 118: 737-745
        • Lachén-Montes M.
        • González Morales A.
        • Iloro I.
        • Elortza F.
        • Ferrer I.
        • Gveric D.
        • et al.
        Unveiling the olfactory proteostatic disarrangement in Parkinson’s disease by proteome-wide profiling.
        Neurobiol. Aging. 2018; 73: 123-134
        • Zahola P.
        • Hanics J.
        • Pintér A.
        • Máté Z.
        • Gáspárdy A.
        • Zsófia H.
        • et al.
        Secretagogin expression in the vertebrate brainstem with focus on the noradrenergic system and implications for Alzheimer’s disease.
        Brain Struct. Funct. 2019; 224: 2061-2078
        • Zhan X.
        • Evans C.O.
        • Oyesiku N.M.
        • Desiderio D.M.
        Proteomics and transcriptomics analyses of secretagogin down-regulation in human non-functional pituitary adenomas.
        Pituitary. 2003; 6: 189-202
        • Xing X.
        • Lai M.
        • Gartner W.
        • Xu E.
        • Huang Q.
        • Li H.
        • et al.
        Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin.
        Proteomics. 2006; 6: 2916-2923
        • Juhlin C.C.
        • Zedenius J.
        • Höög A.
        Clinical routine application of the second-generation neuroendocrine markers ISL1, INSM1, and secretagogin in neuroendocrine neoplasia: staining outcomes and potential clues for determining tumor origin.
        Endocr. Pathol. 2020; 31: 401-410
        • Sifuentes-Dominguez L.
        • Li H.
        • Llano E.
        • Liu Z.
        • Singla A.
        • Patel A.
        • et al.
        SCGN deficiency results in colitis susceptibility.
        Elife. 2019; 8e49910
        • Birkenkamp-Demtröder K.
        • Wagner L.
        • Brandt Sørensen F.
        • Bording Astrup L.
        • Gartner W.
        • Scherübl H.
        • et al.
        Secretagogin is a novel marker for neuroendocrine differentiation.
        Neuroendocrinology. 2005; 82: 121-138
        • Adolf K.
        • Wagner L.
        • Bergh A.
        • Stattin P.
        • Ottosen P.
        • Borre M.
        • et al.
        Secretagogin is a new neuroendocrine marker in the human prostate.
        Prostate. 2007; 67: 472-484
        • Kim D.S.
        • Choi Y.P.
        • Kang S.
        • Gao M.Q.
        • Kim B.
        • Park H.R.
        • et al.
        Panel of candidate biomarkers for renal cell carcinoma.
        J. Proteome Res. 2010; 9: 3710-3719
        • Dong Y.
        • Li Y.
        • Liu R.
        • Li Y.
        • Zhang H.
        • Liu H.
        • et al.
        Secretagogin, a marker for neuroendocrine cells, is more sensitive and specific in large cell neuroendocrine carcinoma compared with the markers CD56, CgA, Syn and Napsin A.
        Oncol. Lett. 2020; 19: 2223-2230
        • Yu L.
        • Suye S.
        • Huang R.
        • Liang Q.
        • Fu C.
        Expression and clinical significance of a new neuroendocrine marker secretagogin in cervical neuroendocrine carcinoma.
        J. Clin. Pathol. 2021; 74: 787-795
        • Romanov R.
        • Alpár A.
        • Zhang M.D.
        • Zeisel A.
        • Calas A.
        • Landry M.
        • et al.
        A secretagogin locus of the mammalian hypothalamus controls stress hormone release.
        EMBO J. 2015; 34: 36-54
        • Hanics J.
        • Szodorai E.
        • Tortoriello G.
        • Malenczyk K.
        • Keimpema E.
        • Lubec G.
        • et al.
        Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration.
        Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E2006-E2015
        • Alpár A.
        • Zahola P.
        • Hanics J.
        • Hevesi Z.
        • Korchynska S.
        • Benevento M.
        • et al.
        Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress.
        EMBO J. 2018; 37e100087
        • Maj M.
        • Wagner L.
        • Tretter V.
        20 Years of secretagogin: exocytosis and beyond.
        Front. Mol. Neurosci. 2019; 12: 29
        • Chidananda A.H.
        • Sharma A.K.
        • Khandelwal R.
        • Sharma Y.
        Secretagogin binding prevents α-synuclein fibrillation.
        Biochemistry. 2019; 58: 4585-4589
        • Khandelwal R.
        • Sharma A.K.
        • Chadalawada S.
        • Sharma Y.
        Secretagogin is a redox-responsive Ca2+ sensor.
        Biochemistry. 2017; 56: 411-420
        • Cates J.
        • Graham G.C.
        • Omattage N.
        • Pavesich E.
        • Setliff I.
        • Shaw J.
        • et al.
        Sensing the heat stress by mammalian cells.
        BMC Biophys. 2011; 4: 16
        • Siddiqui M.A.
        • Ahmad J.
        • Farshori N.N.
        • Saquib Q.
        • Jahan S.
        • Kashyap M.P.
        • et al.
        Rotenone-induced oxidative stress and apoptosis in human liver HepG2 cells.
        Mol. Cell. Biochem. 2013; 384: 59-69
        • Ritossa F.
        Discovery of the heat shock response.
        Cell Stress and Chaperones. 1996; 1: 97-98
        • Abravaya K.
        • Phillips B.
        • Morimoto R.I.
        Attenuation of the heat response in HeLa cells is mediated by the release of bound heat shock transcription factor and is modulated by changes in growth and heat shock temperatures.
        Genes Dev. 1991; 5: 2117-2127
        • Jing X.
        • Shi Q.
        • Bi W.
        • Zeng Z.
        • Liang Y.
        • Wu X.
        • et al.
        Rifampicin protects pc12 cells from rotenone-induced cytotoxicity by activating GRP78 via PERK-eIF2α-ATF4 pathway.
        PLoS One. 2014; 9e92110
        • Shao F.
        • Bader M.W.
        • Jakob U.
        • Bardwell J.C.
        DsbG, a protein disulfide isomerase with chaperone activity.
        J. Biol. Chem. 2000; 275: 13349-13352
        • Abgar S.
        • Vanhoudt J.
        • Aerts T.
        • Clauwaert J.
        Study of the chaperoning mechanism of bovine lens alpha-crystallin, a member of the alpha-small heat shock superfamily.
        Biophys. J. 2001; 80: 1986-1995
        • Raychaudhuri S.
        • Sinha M.
        • Mukhopadhyay D.
        • Bhattacharyya N.P.
        HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity.
        Hum. Mol. Genet. 2008; 17: 240-255
        • Matukumalli S.R.
        • Tangirala R.
        • Rao C.M.
        Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation.
        Sci. Rep. 2017; 741235
        • Finn T.E.
        • Nunez A.C.
        • Sunde M.
        • Easterbrook-Smith S.B.
        Serum albumin prevents protein aggregation and amyloid formation and retains chaperone-like activity in the presence of physiological ligands.
        J. Biol. Chem. 2012; 287: 21530-21540
        • Lee J.-J.
        • Yang S.-Y.
        • Park J.
        • Ferrell J.E.
        • Shin D.-H.
        • Lee K.-J.
        Calcium ion induced structural changes promote dimerization of secretagogin, which is required for its insulin secretory function.
        Sci. Rep. 2017; 7: 6976
        • Sharma A.
        • Khandelwal R.
        • Sharma Y.
        • Rajanikanth V.
        Secretagogin, a hexa EF-hand calcium-binding protein: high level bacterial overexpression, one-step purification and properties.
        Protein Expr. Purif. 2015; 109: 113-119
        • Schneidman-Duhovny D.
        • Hammel M.
        • Tainer J.A.
        • Sali A.
        FoXS, FoXSDock and MultiFoXS: Single-state and multi-state structural modeling of proteins and their complexes based on SAXS profiles.
        Nucl. Acids Res. 2016; 44: W424-429
        • Bitto E.
        • Bingman C.A.
        • Bittova L.
        • Frederick R.O.
        • Fox B.G.
        • Phillips Jr., G.N.
        X-Ray structure of Danio rerio secretagogin: a hexa-EF-hand calcium sensor.
        Proteins. 2009; 76: 477-483
        • Taylor A.
        • Shkedi A.
        • Nadel C.M.
        • Gestwicki J.E.
        The interactions of molecular chaperones with client proteins: why are they so weak?.
        J. Biol. Chem. 2021; 297101282
        • Morimoto R.I.
        Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators.
        Genes Dev. 1998; 12: 3788-3796
        • Hagen T.M.
        Oxidative stress, redox imbalance, and the aging process.
        Antioxid. Redox Signal. 2003; 5: 503-506
        • Park C.
        • Jeong J.
        Synergistic cellular responses to heavy metal exposure: a minireview.
        Biochim. Biophys. Acta (Gen. Subj.). 2018; 1862: 1584-1591
        • Preissler S.
        • Rato C.
        • Yan Y.
        • Perera L.,A.
        • Czako A.
        • Ron D.
        Calcium depletion challenges endoplasmic reticulum proteostasis by destabilising BiP-substrate complexes.
        Elife. 2020; 9e62601
        • Lebeau P.F.
        • Platko K.
        • Byun J.H.
        • Austin R.C.
        Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells.
        J. Biol. Chem. 2021; 296100779
        • Gartner W.
        • Lang W.
        • Leutmetzer F.
        • Domanovits H.
        • Waldhäusl W.
        • Wagner L.
        Cerebral expression and serum detectability of secretagogin, a recently cloned EF-hand Ca2+-binding protein.
        Cereb. Cortex. 2002; 11: 1161-1169
        • Bromberg Z.
        • Weiss Y.
        The role of the membrane-initiated heat shock response in cancer.
        Front. Mol. Biosci. 2016; 3: 12
        • Malenczyk K.
        • Girach F.
        • Szodorai E.
        • Storm P.
        • Segerstolpe A.
        • Tortoriello G.
        • et al.
        A TRPV1-to-secretagogin regulatory axis controls pancreatic β-cell survival by modulating protein turnover.
        EMBO J. 2017; 36: 2107-2125
        • Bromberg Z.
        • Goloubinoff P.
        • Saidi Y.
        • Weiss Y.G.
        The membrane-associated transient receptor potential vanilloid channel is the central heat shock receptor controlling the cellular heat shock response in epithelial cells.
        PLoS One. 2013; 8e57149
        • Vogel C.
        • Silva G.M.
        • Marcotte E.M.
        Protein expression regulation under oxidative stress.
        Mol. Cell. Proteomics. 2011; 10https://doi.org/10.1074/mcp.M111.009217
        • Cheng Z.
        • Teo G.
        • Krueger S.
        • Rock T.M.
        • Koh H.W.
        • Choi H.
        • et al.
        Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress.
        Mol. Sys. Biol. 2016; 12: 855
        • Sharma A.K.
        • Khandelwal R.
        • Sharma Y.
        Veiled potential of secretagogin in diabetes: correlation or coincidence?.
        Trends Endocrinol. Metabol. 2019; 30: 234-243https://doi.org/10.1016/j.tem.2019.01.007
        • Sanagavarapu K.
        • Weiffert T.
        • Ní Mhurchú N.
        • O'Connell D.
        • Linse S.
        Calcium binding and disulfide bonds regulate the stability of secretagogin towards thermal and urea denaturation.
        PLoS One. 2016; 11e0165709
        • Khandelwal R.
        • Sharma A.K.
        • Biswa B.B.
        • Sharma Y.
        Extracellular secretagogin is internalized into the cells through endocytosis.
        FEBS J. 2021; 289: 3183-3204
        • Emekli U.
        • Schneidman-Duhovny D.
        • Wolfson H.J.
        • Nussinov R.
        • Haliloglu T.
        HingeProt: automated prediction of hinges in protein structures.
        Proteins. 2008; 70: 1219-1227
        • Ashish
        • Paine M.S.
        • Perryman P.B.
        • Yang L.
        • Yin H.L.
        • Krueger J.K.
        Global structure changes associated with Ca2+ activation of full-length human plasma gelsolin.
        J. Biol. Chem. 2007; 282: 25884-25892
        • Noble J.W.
        • Almalki R.
        • Roe S.M.
        • Wagner A.
        • Duman R.
        • Atack J.R.
        The X-ray structure of human calbindin-D28K: an improved model.
        Acta Crystallogr. D Struct. Biol. 2018; 74: 1008-1014
        • Sharma A.K.
        • Khandelwal R.
        • Sharma Y.
        Secretagogin purification and quality control strategies for biophysical and cell biological studies.
        in: Calcium-Binding Proteins of the EF-Hand Superfamily. Methods in Molecular Biology. 1929. Humana Press, New York, NY2019
        • Srivastava S.S.
        • Jamkhindikar A.A.
        • Raman R.
        • Jobby M.K.
        • Chadalawada S.
        • Sankaranarayanan R.
        • et al.
        A transition metal-binding, trimeric βγ-crystallin from methane-producing thermophilic archaea, Methanosaeta thermophila.
        Biochemistry. 2017; 56: 1299-1310
        • Franke D.
        • Petoukhov M.V.
        • Konarev P.V.
        • Panjkovich A.
        • Tuukkanen A.
        • Mertens H.
        • et al.
        Atsas 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions.
        J. Appl. Crystallogr. 2017; 50: 1212-1225
        • Hopkins J.B.
        • Gillilan R.E.
        • Skou S.
        BioXTAS RAW: improvements to a free open-source program for small-angle X-ray scattering data reduction and analysis.
        J. Appl. Cryst. 2017; 50: 1545-1553
        • Svergun D.I.
        • Petoukhov M.V.
        • Koch M.H.J.
        Determination of domain structure of proteins from X-ray solution scattering.
        Biophys. J. 2001; 80: 2946-2953
        • Franke D.
        • Svergun D.I.
        DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering.
        J. Appl. Crystallogr. 2009; 42: 342-346