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Chaperone-like activity of calnuc prevents amyloid aggregation Madhavi kanuru, and Gopala Krishna Aradhyam Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00660 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 10, 2016

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Biochemistry

Chaperone-like activity of calnuc prevents amyloid aggregation Madhavi Kanuru and Gopala Krishna Aradhyam* Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai – 600 036, INDIA *To whom correspondence should be addressed: Gopala Krishna Aradhyam. Tel.: 91-44-22574112; FAX: 9144-22574102; E-mail: [email protected]

Funding: Madhavi Kanuru acknowledges CSIR for a senior research fellowship. Work in the author’s lab is funded by IIT Madras, CSIR, DST, DBT and ICMR (all Govt. of India funding agencies).

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Biochemistry

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Abbreviations: MDH, malate dehydrogenase;ADH, alcohol dehydrogenase; CS, citrate

Synthase; GdmCl, guanudnium chloride; AD,Alzheimer’s disease; PD, Parkinson’s disease; Αβ, Amyloid β; UPR, Unfolded Protein Response; ThT, Thioflavin; DTNB,Dithio-2-nitrobenzoic acid; GST, Glutathione-S-transferase; SEM, Scanning Electron Microscopy; TEM. Transmission Electron Microscopy.MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4′,6-Diamidino-2-phenylindole dihydrochloride.

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Biochemistry

Key words: calnuc,chaperone, aggregation, enzyme activity, Ca2+-binding, amyloid fibril, cell toxicity Abstract Calnuc is a ubiquitously expressed protein of the EF-hand Ca2+-binding super family. Previous studies have implicated it in Ca2+-sensitive physiological processes, whereas details of its function and involvement in human diseases are lacking. Drawing upon the sequence homology of calnuc with calreticulin, we propose its function as a molecular chaperone-like protein. In cells under thermal, chemical (urea, guanidinium chloride; GdmCl) and acidic stress, calnuc exhibits properties similar to that of established chaperone-like proteins (GRP78, spectrin, and α-crystallin), effectively demonstrated by its ability to suppress aggregation of malate dehydrogenase (MDH), alcohol dehydrogenase (ADH) and catalase. Calnuc aids in refolding of MDH with retention of 80% of its enzymatic activity. In HEK293 cells subjected to heat shock, calnuc chaperones luciferase, protecting its activity. Our in vitro and cell culture results establish the ability of calnuc to inhibit fibrillation of insulin and lysozyme and validate its neuro-protective role in cells treated with amyloid fibrils. Calnuc also rescues cells from fibrillar toxicity (caused by misfolded or aggregated proteins), providing a plausible explanation for the previous observation of its low expression in brains affected by Alzheimer’s disease. We propose that calnuc is possibly involved in controlling protein unfolding diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), prion disease and type II diabetes.


Biochemistry

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Introduction Aetiology of known (and the so-called) “conformational” disorders is due to the folding problems of proteins e.g.,α-synuclein, Amyloid β (Αβ) peptides, transthyretin, lysozyme and prion proteins– all of them leading to aggregation or amyloid-like states1.Any disturbance, either due to stress or mutations, resulting in misfolding of proteins (leading to their aggregation or formation of amyloid fibrillary plaques) could become detrimental for the cell causing an array of neurodegenerative disorders, such as AD, PD, prion disease and also type II diabetes2-7. Under such conditions “molecular chaperone proteins” play a protective role by interacting with the partially folded intermediates of amyloidogenic proteins preventing their misfolding, subsequent fibril formation and rescue the cell8-10. Genes that code for molecular chaperones are induced and expressed in response to extreme stress conditions in order to “salvage” the cell by the “Unfolded Protein Response” (UPR) signalling pathway. Glucose-regulated protein 78, 94 (GRP78, GRP94) and enzymes like proteindisulfideisomerase (PDI) are some of the well-known chaperones, expressed by the functional activation of transcription transducers like Endoplasmic Reticulum (ER) transmembrane “activating transcription factor” (ATF6), PKR-like ER kinase (PERK) and inositol-requiring 1 (IRE1)10-11. The general thinking is that there are many, yet to be characterized, ER stress-inducible genes that have the ability to regulate UPR. A previous report alludes to the up-regulation of the calnuc gene during the ER-mediated unfolded protein response, although its function is not clear12. Cellular stress mediated increased expression of calnuc in ER results in ATF6 inactivation similar to that of GRP78 (a UPR target chaperone protein)12. Despite this observation, the involvement of calnuc, if any, in regulating stress is not known. Calnuc (also known as nucleobindin or Nucb1) exhibits 30% sequence similarity for the entire protein with ER resident Ca2+-binding protein calreticulin, a well-known molecular chaperone. Both the proteins possess similar structural features, an N-terminal Zn2+-binding site and C-terminal Ca2+binding motifs (evidence for calreticulin being a chaperone can be seen in references13-17). These similarities prompted us to investigate the possibility of similar chaperone activity and function for calnuc. Here, we demonstrate that calnuc attenuates the aggregation of unfolded proteins and protects enzymes against the loss of activity caused by thermal stress conditions. We have used MDH, lactate dehydrogenase, citrate synthase (CS), catalase and ADH for aggregation, refolding and reactivation studies. The presence of calnuc during refolding (from the completely unfolded state) significantly increases the biological activity of these enzymes. Calnuc prevents insulin and lysozyme from forming fibril structures. Cell viability, LDH leakage assay and DAPI staining of apoptotic nuclei indicate calnuc’s role in attenuating fibril-induced cell toxicity. Co-localisation of recombinant calnuc along with the Aβ(1-42)-GFP chimera in cells underscores their interaction in cell culture and points to calnuc’s posssible physiological function in preventing amyloid fibril-induced cell toxicity and that it could play an important role in obviating neurodegenerative disorders. Materials and Methods Cloning, expression and purification Cloning of calnuc gene into a mammalian vector. Human calnuc gene (1.4 Kb) was cloned between Hind III and SacII restriction sites in the dsRed-N1vector. The positive clone was confirmed by colony PCR, insert release upon restriction digestion and nucleotide sequencing. Protein expression in HEK293 cells was confirmed by performing western blot and fluorescence microscopy.

Over expression and purification of calnuc and its mutants: Calnuc and its fragments were cloned into pTYB12 vector which is expressed as a fusion to a selfcleavable chitin-intein tag upon incubation with dithiothreitol, releasing a “tag less” protein. Overexpression of calnuc and its fragments in BL21(DE3) cells was induced with 200 µM isopropylthio-β-d-galactoside (IPTG) at OD600 – 0.6 and the cells grown at 23 °C for 16 h. Purification of Chitin-Intein fused calnuc, its fragments, and the mutants was done from the cell pellets that were


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Biochemistry

resuspended in 20 mM Tris, 300 mM NaCl (pH 8.0) (buffer A) followed by sonication on ice using an ultrasonicator (Vibracell Sonics and Materials,Inc. Newtown, CT, USA). The supernatant was loaded on to a column containing chitin resin (New England Biolabs), equilibrated and washed with 20 column volumes of buffer A, flushed with wash buffer B (20 mM Tris, 300 mM NaCl, 50 mM DTT pH 8.0) and incubated at 4 °C for 24 h. Recombinant calnuc and its fragments were released from the chimera by the addition and incubation of DTT at 4 °C for 24 h to trigger the self-cleavage of the intein tag. Finally, the protein was eluted with 20 mM Tris, 50 mM NaCl (pH 8.0) and its purity checked on SDS-PAGE (12%) gel. Sub-cloning of Aβ(1-42) into pGEX6P1:The clone Aβ(1-42)-GFP in the pEGFP-C2 vector was a kind gift from Prof. Seongman Kang (School of Life Sciences and Biotechnology, Korea University, Seoul). The Aβ(1-42) region (120 bp) was sub-cloned into pGEX6P1 between EcoRI and NotI restriction sites and confirmed by DNA sequencing. Expression and purification of GST-Aβ fusion protein. For the expression of GST-Aβ(1-42) in BL21DE3, cells were inoculated in 4 ml LB broth containing 100 µg/ml ampicillin and grown at 37 °C for 16 h, further, they were sub cultured and grown at 37 °C until OD600 was 0.8 – 1. The expression was induced with 1 mM IPTG and the cells were harvested after culturing for 6 to7 h at 24 °C. GST-Aβ(1-42) was affinity purified by glutathione agarose column. The harvested cells were suspended in lysis buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA pH 8.0) and lysed using an ultrasonicator (Vibracell Sonics and Materials Inc. Newtown, CT); 10% (w/v) SDS was added to the lysate and centrifuged at 12000 rpm at 4 °C for 45 min. Triton X-100 0.8% (v/v) was added to the supernatant and loaded on to GST column and equilibrated with buffer (20 mM Tris, 100 mM NaCl, pH 8.0). GST-Aβ(1-42) was eluted using elution buffer (50 mM Tris-HCl and 10 mM reduced glutathione) and purity was assessed on 12% SDS-PAGE. Pure fractions were pooled, concentrated, set up for precision protease cleavage overnight at 4 °C and passed through GST column to remove the GST tag. The cleaved fraction of Aβ(1-42) was checked on 16% tricine PAGE. The peptide was buffer exchanged with 25 mM ammonium bicarbonate and the sample was lyophilized and stored at – 80 °C. Temperature-dependent aggregation of catalase, ADH and MDH. Aggregation of Catalase, ADH or MDH due to heat treatment, in 20 mM HEPES (pH 7.5) with 50 mM NaCl, was monitored in the absence or presence of calnuc by measuring light scattering at 360 nm using a Perkin Elmer UV-VIS spectrophotometer with a thermostatic cell holder assembly maintained at 50 °C through a circulating water bath. The concentration of catalase, ADH and MDH was 1 mg/mL in all the samples and calnuc was measured at 1 µM and 2 µM concentrations. No precipitation or turbidity was observed during these experiments. Samples were incubated for 5 min at room temperature in buffer containing 20 mM HEPES (pH 7.5) with 50 mM NaCl and monitored aggregation at 50 °C for a time scan of 20 min. Control experiments were performed with two other proteins [T. maritima acetyl esterase (TmAcE) and Anabaena aryl esterase(AnaEst)] instead of calnuc, to demonstrate specificity. Chaperone-like activity of calnuc was also monitored in the presence of ATP. ATP did not affect its chaperone-like activity. Assay of enzymatic activity: Enzyme activity was determined by taking aliquots at different times from the assay mixture incubated at 50 °C both in the presence and absence of calnuc (2 µM). Enzyme activity of MDH (0.2 µM) was determined by monitoring the decrease in absorbance at 340 nm due to the conversion of NADH (13 µM) to NAD+ using oxaloacetate (50 µM) as substrate18. In the case of citrate synthase (CS), the “thiol” group of CoA-SH, formed during the reaction, further reacts with DTNB and forms 5-thio-2-nitrobenzoic acid (TNB is the yellow product) which is monitored by measuring absorbance at 412 nm (19). The assay mixture consists of CS (0.5 µM), oxaloacetate (50 µM) and acetyl Co-A (10 µM) and DTNB (20 µM) in buffer containing 20 mM HEPES pH 7.5, 50 mM NaCl. Glutathione-S-transferase (GST) activity was monitored by an increase in the absorbance at 340 nm due to the formation of GS-DNB Conjugate during the reaction, wherein


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the GST catalyzes the conjugation of L-glutathione to CDNB through the thiol group of the glutathione. The reaction mixture was prepared with 4 mM reduced glutathione (GSH), 0.4 mM CDNB, 5 µg of GST, and 20 mM HEPES pH 7.5 buffer with 50 mM NaCl in a final volume of 1.0 mL. The reaction was started by addition of GST. The rate of increase in the absorption is directly proportional to the GST activity in the sample20. Refolding assay of MDH. All unfolding and refolding reactions were carried out at 25 °C. Malate dehydrogenase (6 µM) was unfolded in 6 M guanidine hydrochloride (GdmCl) in 20 mM HEPES buffer containing, pH 7.5, 40 mM NaCl and 20 mM DTT for 1 h. The refolding experiment was initiated by the 100-fold dilution of MDH in 20 mM HEPES, pH 7.5, 40 mM NaCl and 10 mM DTT. Enzyme activity was checked at various refolding times both in the presence and absence of calnuc (0.2 µM). The relative activity of the enzyme was calculated by considering the activity of the same amount of native enzyme as 100% and plotted at different time intervals. Generation of insulin, Aβ and lysozyme fibrils. Insulin (1 mg/ml) was dissolved in 25 mM HCl and 100 mM NaCl, pH 1.5 and its aggregation was achieved by incubating at 37 °C for 7 days or at 50 °C for 24 h 21. Lysozyme (2 mg/ml) fibrils were generated under acidic conditions (25 mM HCl, pH 1.6) by incubating at 50 °C for 10 days22. Fibrils of Aβ were generated by incubating Aβ(1–42) (10 µM) at 37 °C for 72 h. Formation of amyloid aggregates was confirmed by monitoring the structural changes infar UV-CD spectra followed by ThT fluorescence and visualized by HR-SEM. The effect of calnuc (ranging from 1 – 5 µM) on amyloid aggregate formation was also monitored similarly. Thioflavin T (ThT) assay. The formation of insulin amyloid fibrils was detected by monitoring the significant enhancement of the ThT fluorescence intensity21. The stock solution of ThT at a concentration of 1 mM was prepared in 95% (v/v) methanol and protected from light prior to use. The working solution of ThT at a concentration of 10 µM was diluted from stock and prepared in Phosphate buffered saline (136.7 mM NaCl, 2.68 mM KCl, 0.01 M Na2HPO4, 1.76 mM KH2PO4, and 1.54 mM NaN3, pH 7.4) for immediate use. The formation of aggregated insulin and lysozyme fibrils were triggered by incubating insulin (1 mg/mL, pH 1.6) at 60 °C for 1 hour or at 37 °C for 5 days and lysozyme (2 mg/mL, pH 1.6) at 37 °C for 10 days in 25 mM HCl and 50 mM NaCl respectively. The experiment at 60 °C was also repeated in the presence and absence of 10 mM H2O2. ThT (10 µM) was then added to these amyloid aggregates formed under different conditions in the presence and absence of calnuc. ThT fluorescence was observed by exciting the samples at 440 nm and recording the emission from 460 to 600 nm (emission maximum was observed at 485 nm). The excitation and emission slits were maintained at 5 nm each. All ThT fluorescence experiments were performed in triplicates. Far-UV circular dichroism (CD) spectroscopy. The secondary structural changes of amyloid fibrils sample solutions monitored by ThT fluorescence were further evaluated by far-UV CD spectroscopy. CD spectra of these samples were recorded after diluting 100-fold with buffer over the wavelength range of 195–260 nm with a 0.2 cm path length sample cell. All CD measurements were collected at room temperature using a bandwidth of 1.0 nm, and a scanning speed of 100 nm/min. Each CD spectrum was the average of four scans. Scanning Electron Microscopy (SEM) measurements. HR-Scanning electron microscopy (F E I Quanta FEG 200) was used to observe the morphology of the insulin (1 mg/ml) fibril formation both in the presence and absence of calnuc (0.5 mg/ml). The sample was dried on a glass slide under vacuum for all measurements. The sample was observed after sputtering with gold. Transmission electron microscopy (TEM) measurements. The morphology of the insulin fibril formation in the presence orabsence of calnuc was monitored under TEM. Samples were prepared by adding 2 µl of 100 fold diluted insulin fibril solution (1 mg/ml) and in presence of calnuc (0.5 mg/ml) to Formvar and carbon-coated nickel grids and were allowed to dry in sterile conditions. Grids loaded with samples were viewed on a Philips CM12 transmission electron microscope with EDAX attachment.


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Biochemistry

Cell culture based luciferase assay. HEK293 cells were co-transfected with pGL3-luciferase vector and pCDNA6V5 plasmid containing his-tagged calnuc. As a control, cells were transfected with pGL3-luciferase; 24 h post transfection, 20 µg/ml of cycloheximide (protein synthesis inhibitor; in order to prevent synthesis of heat shock proteins) was added and cells incubated for 30 min. The cells were then subjected to stress by increasing the temperature to 45 °C for 15 min (heat shock). One set of cells was used to check the loss of activity and the other was allowed to recover at room temperature to monitor the efficiency of refolding. Cells were lysed in lysis buffer (100 mM Phosphate pH 7.8, 10 µM DTT, 1% Triton X-100) and the luciferase activity measured with the ‘Luciferase Assay System’ (Promega)23. All experiments were carried out in triplicates. For studying the effect of calcium ions on the chaperone activity of calnuc, all the buffers, reagents and the proteins were prepared in Chelex-purified water. EDTA was removed by extensive dialysis against metal ion and EDTA-free buffer. Cell toxicity of aggregated insulin and Aβ(1-42) fibrils. IMR32 cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin (10 U/ml) and 10 µg/ml streptomycin. Cells were cultured in T25 flasksin an incubator with 95% air and 5% CO2 at 37 °C. The medium was changed every 2–3 days. A day prior to treatment, the cells were plated based on the experimental parameters. MTT Assay. The viability of cells was assessed by measuring their ability to reduce MTT dye (yellow color) to formazan (purple color). For MTT assay, 3000 to 5000 IMR32 cells in 100 µl of buffer were plated in each well of a 96-well plate. Cells were further grown for an additional 24 h at 37 °C in an incubator with 5% CO2 supply. The fibrils (at a concentration of 10 µM/well) were added to the medium both in the presence and absence of calnuc and its fragments (at various concentrations ranging from 1-5 µM/well) and followed by incubation for 24 h, after which the media was removed and 100 µl of serum-free media containing 10 µl of MTT (5 mg/ml prepared in PBS) was added to each well. The cells were further incubated for 4 h in dark at 37 °C and 200 µl of DMSO was added to each well to dissolve the reduced MTT (formazan) crystals formed by viable cells, giving them a characteristic purple color. Absorbance measurement was recorded on spectrophotometric plate reader at 570 and 650 nm for each well. Cell viability was calculated from the difference in readings from both wavelengths with respect to untreated cells as control.24 LDH leakage assay to check cytotoxicity. The estimation of leakage of cytosolic enzyme, lactate dehydrogenase (LDH), is a standard protocol to determine cell viability. After the treatment of cells with amyloid fibrils, both in the presence and absence of calnuc, their viability was determined by measuring the activity of LDH. Activity was measured spectrophotometrically by monitoring the conversion of NADH to NAD+ in the presence of sodium pyruvate (at 340 nm). LDH released to extracellular medium in the treated cells was measured and quantified as percentage of that observed in control cells (untreated and considered as 100%).24 DAPI staining. IMR32 cells were plated in 6-well culture dishes at a density of 2×105 cells per well, in full-serum medium and incubated for 24 h. The cells were then treated with native and fibrillar insulin (5 µM) and Aβ(1-42) fibrils (10 µM) for 48 h and one well was used as a positive control by maintaining cells under serum deprivation. After the incubation period, the media was removed by aspiration and the cells were washed three times with cold PBS (pH 7.4). Cells were then fixed with 4% formaldehyde for 5-10 min followed by PBS wash and permeabilized in 0.1% Triton-X 100 for 20-30 seconds, removed and washed with PBS. These cells were stained with DAPI (1x) and incubated for 20 min indark at room temperature followed by a PBS wash. Cells were then viewed througha fluorescence microscope (Optika B353, Italy).25 Co-localization of calnuc and Aβ(1-42). IMR32 cells were plated in 6-well culture dishes at a density of 2×105 cells per well, in full-serum medium and incubated for 24 h. Cells were cotransfected with both Aβ(1-42)-GFP and calnuc dsRed using lipofectamine as per “Invitrogen” protocol. Briefly, DNA (2 µg) and lipofectamine (4 µl) complex were prepared in serum-free medium and incubated for 10 min. The medium was removed from the wells and the serum-free


Biochemistry

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medium was added. After incubation, the complex was added to the wells and incubated at 37 °C in an incubator with 5% CO2. After 4 h, the serum-free medium was removed and the complete medium was added and placed back in the incubator for 24 h. Cells were viewed under a fluorescence microscope (Optika B353, Italy) and FRET was directly monitored to indicate co-localization. Results Calnuc chaperones proteins aggregating due to thermal stress. Chaperone-like activity of calnuc was assessed by monitoring prevention of temperature-dependent (at 50 °C) aggregation of proteins (Catalase, ADH, MDH and GST). We incubated these proteins in presence or absence of calnuc and monitored aggregation at 50 °C. At these temperatures and as a function of time, aggregation of the protein results in increased light scattering monitored in a fluorescence spectrometer (Fig. 1). Calnuc significantly suppressed the thermal aggregation of all substrate proteins as a function of time and in a dose-dependent manner. The scattering decreased, both in terms of rate of aggregation (slope of the plot) and the level of aggregation (saturation value) (Fig. 1). Calnuc guards the biological activity of the test enzymes. In addition to precluding aggregation, the effect of calnuc in preventing the loss of biological activity of the substrate proteins was also ascertained. Residual activity of the substrate enzymes (MDH, CS and GST) against thermal stress was assessed by measuring activity in the presence and absence of calnuc. These enzymes lose ~85% activity upon incubation at 50 °C for 10 min (Fig. 2). Calnuc restricts the loss in residual activity to 60 - 70%. Calnuc, by itself, neither exhibits any activity with the specific substrates of the enzymes nor does it interfere with the specific activity of these enzymes (Fig. 2A). Domain-independent chaperone-like function of calnuc. To pinpoint the minimal region of calnuc involved in chaperone-like activity, various fragments were designed and generated as illustrated in Fig 3. Initially, we inspected the prevention of stress-induced aggregation by both N- and C-fragments of calnuc. Both fragments exhibited 60–70% decrease in aggregation of malate dehydrogenase, similar to calnuc (Fig. 3B). Interestingly both the fragments were also able to protect the activity of malate dehydrogenase up to 60-80% under thermal stress conditions. Whereas MDH exhibited only 15-20% activity (by the end of 10 min incubation at 50 °C), in the presence of N-and C-fragments of calnuc, its activity was 80% (Fig. 3C). Calnuc restores the enzymatic activity of proteins upon refolding to their native structure. Monitored by reactivation of GdmCl-unfolded MDH. Successful refolding of an unfolded enzyme should restore its activity. The ability of calnuc to restore the activity of its target proteins was investigated by monitoring the recovery of residual activity during an enzyme’s refolding process (we selected MDH) and the ability of calnuc to speed it up. Calnuc significantly improves the recovery of MDH activity (Fig. 4A). While MDH recovered only 15% of its activity after 60 min, in the presence of calnuc, there was an enhancement in residual activity to 75% (loss of activity decreasing from 85% to 25%). We also monitored the ability of calnuc fragments in assisting the refolding of MDH to its native conformation. MDH exhibits 80% of recovery in its activity in the presence of N-fragment and 60% in the presence of C-fragment (Fig. 4B). These data imply that calnuc not only prevents aggregation but also helps in restoration of the enzymatic activity suggesting refolding to its native functional form (as assessed by its enzymatic activity). Effect of Ca2+ and Zn2+ on chaperone activity of calnuc. We have investigated the effect of Ca2+/Zn2+-binding on chaperone-like activity of calnuc by monitoring the thermal aggregation of catalase. Our results indicate a reduction in chaperone activity of calnuc in its Ca2+ bound form. Apo form exhibited 60-70% decrease in aggregation whereas in the presence of Ca2+, only 30–40% prevention of aggregation was observed (Fig. 5). On the other hand Zn2+didn’t exhibit any effect on chaperone activity and executed similar activity as that of apo form. This demonstrates that Ca2+ binding leads to a reduction in calnuc’s ability to protect proteins against thermal stress stimulated aggregation.


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Biochemistry

Chaperone-like activity of calnuc in cell culture. To assess the chaperone-like activity of calnuc a cell-based luciferase assay was performed. The luciferase activity was monitored under normal condition when subjected to thermal stress and also during the process of refolding. The expression of endogenous chaperones (e.g., heat shock proteins) was regulated by treating cells with protein synthesis inhibitor cycloheximide, prior to heat shock. Cells co-transfected with calnuc exhibited 50% activity, whereas in the case of luciferase only, the activity was only 30% immediately after the heat shock treatment (Fig. 6). This implies that calnuc plays a role in protecting luciferase from unfolding caused bythermal stress. It was also evident from luciferase activity read out (denoted as grey bars in Fig. 6) that calnuc enhances its overall refolding. In the presence of calnuc, the luciferase activity recovered was up to 97%, whereas only 70% activity was seen in the absence of calnuc.23 Prevention of fibril formation by calnuc. Calnuc inhibits fibrillation of insulin and lysozyme. Thioflavin T (ThT) is a fluorescent dye that binds to amyloid fibrils. A significant enhancement of its fluorescence signal is observed upon binding to amyloid fibrils; therefore it is used as a sensitive probe for monitoring amyloid fibrillation (21). Fluorescence emission spectra for the binding of ThT to insulin and lysozyme fibrils, both in the absence and presence of calnuc, are demonstrated in Fig. 7A & B. Insulin and lysozyme by themsleves cause an enhancement of fluorescence emission of ThT, implying the presence of aggregated insulin fibrils. On the other hand, the fluorescence signal of the probe was negligible in the presence of calnuc, reasserting calnuc’s ability to prevent fibrillation of insulin. Our results gain importance from the fact thata similar phenomenon was reported for human Islet Amyloid Polypeptide (hIAPP).26 Monitoring structural changes in insulin and lysozyme during fibril formation. Far-UV CD spectra were used to monitor fibril formation of insulin and lysozyme, at acidic pH and in a time-dependent manner. Changes in protein conformation during fibrillation processes were detected by observing the secondary structural signature, both in the presence and absence of calnuc. Insulin and lysozyme exhibit α-helical secondary structure with CD spectral local minima at 222 and 208 nm and a positive band below 200 nm. As a function of time during fibril formation, a conformational transition from αhelix to β-sheet was observed (Fig. 7C and D). This structural shift was not noticed in the presence of calnuc, which is in agreement with the reduction of fibril formation, as monitored by ThT fluorescence. Calnuc prevents the fibrillation of insulin. Insulin molecules unfold partially and interact with each other to form linear aggregates or fibrils under amyloidogenic conditions, like acidic pH and high temperature27. Insulin fibril formation was initiated by subjecting it to suchc onditions in the presence and absence of calnuc. Formation of linear aggregates or fibrils of insulin was prevented and a homogeneous distribution was observed in the presence of calnuc, as seen in the HR-SEM images (Fig. 8A and B). Furthermore, the fibril structures of insulin and lysozyme were studied by TEM (Fig. 8C and D). The effect of calnuc on the formation of fibril structures by insulin and lysozyme is visible in TEM images shown in Fig. 8E and F. Calnuc inhibits the development of fibril like structures in these proteins. These observations suggest the role of calnuc in the protection of proteins from the effect of amyloidogenic conditions. Calnuc protects cells from insulin and Aβ(1–42) fibril cytotoxicity. We further examined the ability of calnuc to prevent the fibril-induced cellular toxicity, which is mostly associated with amyloidogenic species and is relevant to neurological disorders.28-30 Here, we have analyzed the cellular toxicity induced by the fibrils in IMR32 cell lines, by applying pre-generated fibrils to the culture media in the presence and absence of calnuc and its fragments. Cell viability of treated and untreated cells was monitored by MTT assay. Cell survival rates improved from~50% to ~80%due to the presence of calnuc (Fig. 9A). These observations demonstrate the role of calnuc in preventing the amyloid fibril-induced cell toxicity. To probe the probable mode of action of calnuc, we used the well-known fact that amyloid fibrils cause cell death via apoptosis.31-33 To determine the effect of calnuc on the insulin-mediated cell death


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via the apoptosis pathway, we performed the LDH leakage assay and nuclear DAPI staining. The released cytosolic enzyme, LDH, was measured to determine the effect of calnuc in reducing the fibril-induced apoptosis. We found that there is a decrease in LDH release (activity) when cells were treated with calnuc as compared to those treated with fibrils (Fig. 9B). In accordance, DAPI staining of cells treated with insulin fibrils showed the enhanced chromatin condensation and fragmentation, whereas this process was reduced in the presence of calnuc (Fig. 9C). On the other hand, LDH leakage assay (which is a potential marker of membrane damage during apoptosis) has revealed almost 40% enhanced LDH release from the cells treated with fibrils as compared to control cells. This significant LDH release was lost in the presence of calnuc (Fig. 9B). These conclusive findings confirm the role of calnuc in preventing the fibril-induced apoptosis. Further confirmation was obtained from IMR32 cells treated with fibrils and stained with the DAPI, a DNA stain. Granular staining of condensed chromatin is indicative of cells undergoing apoptosis (Fig. 9C). As a positive control for apoptotic nuclei, cells were incubated for 24-48 h under serum deprivation, which caused a pattern similar to that induced by fibrils. Treating with calnuc led to a reduction in condensed chromatin appearance in nuclei compared to fibril-induced apoptosis (Fig. 9C). To further preliminarily probe the probable mechanism of action of calnuc, we have also monitored the presence of calnuc complex with the Aβ(1-42) peptide aggregates by fluorescence tags. Calnuc was tagged to dsRed and Aβ(1-42) with GFP tag. The merge image displaying yellow fluorescence confirms the existence of calnuc along with the amyloid Aβ(1-42) peptide, demonstrating their interaction (Fig. 9D). These results are in agreement with the previous results demonstrating the interaction of calnuc with amyloid precursor protein (APP) and affecting its biogenesis.34 These investigations prove the role of calnuc in preventing aggregation of misfolded proteins. Calnuc might play a role in neurodegenerative disorders even as scattered reports indicate its probable involvement in several physiological processes.35, 36 Discussion We demonstrate that calnuc prevents the temperature-induced aggregationof proteins such as ADH, catalase, MDH and GST. Additionally, calnuc prevents the loss of their functional (enzymatic) activity. These observations accentuate the fact that calnuc has the potential to act as a molecular chaperone and protects susceptible proteins from the loss of structure and biological activity. The recovery of activity of the target enzymes is apparently due to the assistance provided by calnuc in refolding the unfolded/misfolded proteins. The ability of calnuc in chaperoning proteins was reduced in Ca2+-bound form whereas the presence of Zn2+ didn’t exhibit any alteration in its activity. We have further confirmed calnuc’s ability to chaperone proteins to their functional structure by a cell culture based luciferase assay demonstrating that calnuc protects and enhances luciferase activity during thermal stress. By demonstrating (in vitro) the prevention of amyloid fibril formation of insulin and lysozyme, we propose involvement of calnuc in controlling neurodegenerative disorders through its effect on the formation of amyloid fibrils. These results are in line with what has been demonstrated for other molecular chaperones, like clusterin and α-crystallin, which play a vital role in repression of neurodegenerative diseases by preventing the formation of amyloid fibrils.37 We propose that calnuc prevents the onset of Alzheimer’s disease by inhibiting the formation of amyloid fibrils owing to its chaperone-like activity and has the capacity to play a protective role in neurodegenerative disorders. Amyloid aggregates have previously been shown to cause toxicity to cells.28-33 In this direction, our results demonstrate the effective role of calnuc in inhibiting amyloid aggregates and reducing the associated cell toxicity. Decrease in cell viability was observed in cells treated with insulin fibrils and was inhibited in the presence of calnuc. Co-localization studies demonstrate the interaction of calnuc with amyloid peptide, effectively underscoring the ability of calnuc in preventing amyloid aggregates. Our observations provide evidence that calnuc can attenuate cell toxicity induced by amyloid fibrils. Calnuc is localized invarious compartments/organelles of the cell, plays a key role in UPR and probably interacts with Aβ.26, 34 We present another dimension to its diverse functions, that calnuc chaperones proteins, enabling them to fold to their native structure, maybe while they are being transported and/or at their destination inhibiting their aggregation into amyloidogenic plaques. Such a


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Biochemistry

protective role of calnuc should aid in prevention of neurodegenerative diseases. We conclude that calnuc exhibits novel chaperone-like activity that helps the cells survive stress conditions. Acknowledgments: We thank Dr. A. Sai Krishna for helping in initial cloning experiments and Dr. Prashant Kumar for his help in cell culture experiments. We thank Ms. Jayashree Aradhyam for editing the manuscript. We thank Dr. N. Manoj (IIT Madras) for giving us TmAcE and AnaEst proteins. Conflict of interest: The authors declare that they have no conflict of interest with the contents of this article. Author contributions: GKA and MK conceived the idea for the project. MK conducted the experiments. GKA and MK analyzed the results and wrote the paper. References 1. Novitskaya, V., Bocharova, O. V., Bronstein, I., and Baskakov, I. V. (2006) Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. J. Biol. Chem.281, 13828–13836. 2. Caughey, B., and Lansbury, P. T. (2003) Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Ann. Rev. Neurosci.26, 267–298. 3. Stefani, M., and Dobson, C. M. (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J. Mol. Med.81, 678–699. 4. Chiti, F., and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human disease. Ann. Rev. Biochem.75, 333–366. 5. Roychaudhuri, R., Yang, M., Hoshi, M. M., and Teplow, D. B. (2009) Amyloid beta-protein assembly and Alzheimer disease. J. Biol. Chem.284, 4749–4753. 6. Yankner, B. A., and Lu, T. (2009) Amyloid beta-protein toxicity and the pathogenesis of Alzheimer disease. J. Biol. Chem.284, 4755–4759. 7. Bellotti, V., Mangione, P., andStoppini, M. (1999) Biological activity and pathological implications of misfolded proteins. Cell. Mol. Life Sci.55, 977–991. 8. Carver, J. A., Rekas, A., Thorn, D. C., and Wilson, M. R. (2003) Small heat-shock proteins and clusterin: intra- and extracellular molecular chaperones with a common mechanism of action and function? IUBMB Life55, 661–668. 9. Ecroyd, H., and Carver, J. A. (2009) Crystallinproteins and amyloid fibrils. Cell. Mol. Life Sci.66, 62–81. 10. Liu, C. Y., and Kaufman, R. J. (2003) The unfolded protein response. J. Cell Sci. 116, 18611862. 11. Schröder, M., and Kaufman, R.J. (2005) ER stress and the unfolded protein response. Mutat. Res. 569, 29-63. 12. Tsukumo, Y., Tomida, A., Kitahara, O., Nakamura, Y., Asada, S., Mori, K., and Tsuruo, T. (2007) Nucleobindin 1 controls the unfolded protein response by inhibiting ATF6 activation. J. Biol. Chem.282, 29264–29272. 13. Sværke, C., and Houen, G.(1998) Chaperone properties of calreticulin. Acta. ChemicaScandinavica52, 942–949. 14. Kanuru, M., Raman, R., and Aradhyam, G. K. (2013) Serineprotease activity of calnuc:regulation by Zn2+and G proteins. J. Biol. Chem. 288, 1762–1773. 15. Baksh, S., Spamer, C., Heilmann, C., and Michalak, M. 1995 Identification of the Zn2+binding region in calreticulin. FEBS Lett.376, 53-57. 16. Tan, Y., Chen, M., Li, Z., Mabuchi, K., and Bouvier, M. (2006) The calcium- and zincresponsive regions of calreticulin reside strictly in the N-/C-domain. Biochim.Biophys.Acta1760, 745-753. 17. Somogyi, E., Petersson, U., Sugars, R. V., Hultenby, K., and Wendel, M.(2004) Nucleobindin—a Ca2+-binding protein present in the cells and mineralized tissues of the tooth. Calcified Tissue International74, 366–376.


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18. Bergmeyer, H. U., Gawehn, K., and Grassl, M.(1974) Methods of Enzymatic Analysis. Academic Press. NewYork. 19. Morgunov, I., and Srere, P. A.(1998) Interaction between citrate synthase and malate dehydrogenase substrate channeling of oxaloacetate. J.Biol. Chem.273, 29540-29544. 20. Mozer, T. J., Tiemeier, D. C., and Jaworski, E. G. (1983) Purification and characterization of corn glutathione S-transferase. Biochemistry22, 1068-1072. 21. Sankhala, R. S., and Swamy, M. J. (2010) The Major Protein of Bovine Seminal Plasma, PDC-109, is a Molecular Chaperone. Biochemistry49, 3908-3918. 22. Kumita, J. R., Poon, S., Caddy, G. L., Hagan, C. L., Dumoulin, M., Yerbury, J. J., Stewart, E. M., Robinson, C. V., Wilson, M. R., and Dobson, C. M. (2007) The Extracellular Chaperone Clusterin Potently Inhibits Human Lysozyme Amyloid Formation by Interacting with Prefibrillar Species. J. Mol. Biol.369, 157–167. 23. Zhai, R. G., Zhang, F., Hiesinger, P. R., Cao, Y., Haueter, C. M., and Bellen, H. J. (2008) NADsynthaseNMNAT acts as a chaperone to protect against neurodegeneration. Nature452, 887-892. 24. Agostinho, P., Oliveira, C. R. (2003) Involvement of calcineurin in the neurotoxic effects induced by amyloid-beta and prion peptides. Eur. J. Neurosci.17: 1-8. 25. Dehle, F., Ecroyd, H., Musgrave, I., and Carver, J. (2010) αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide. Cell Stress Chaperones15, 1013-1026. 26. Gupta, R., Kapoor, N., Raleigh, D. P., and Sakmar, T. P. (2012) Nucleobindin 1 Caps Human Islet Amyloid Polypeptide Protofibrils to Prevent Amyloid Fibril Formation. J. Mol. Biol.421, 378-389. 27. Kurouski, D., Luo, H., Sereda, V., Robb, F. T., and Lednev, I. K. (2012) Rapid degradation kinetics of amyloid fibrils under mild conditions by an archaealchaperonin. Biochem. Biophy. Res. Commun.422, 97-102. 28. El-Agnaf, O. M., Jakes, R., Curran, M. D., Middleton, D., Ingenito, R., Bianchi, E., Pessi, A., Neill, D., and Wallace, A. (1998) Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments. FEBS Lett. 440,71-75. 29. Chimon S, Shaibat, M. A., Jones, C. R., Calero, D. C., Aizezi, B., and Ishii, Y. (2007) Evidence of fibril-like beta-sheet structures in a neurotoxic amyloid intermediate of Alzheimer's beta-amyloid. Nat. Struct. Mol. Biol.14, 1157-1164. 30. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature416. 507-511. 31. Troy, C. M., Rabacchi, S. A., Friedman, W. J., Frappier, T. F., Brown, K., Shelanski, M. L. (2000) Caspase-2 mediates neuronal cell death induced by beta-amyloid. J. Neurosci.20, 1386-1392. 32. O'Donovan, C. N., Tobin, D., and Cotter, T. G. (2001) Prion protein fragment PrP-(106-126) induces apoptosis via mitochondrial disruption in human neuronal SH-SY5Y cells. J. Biol. Chem.276, 43516-43523. 33. Onoue, S., Ohshima, K., Endo, K., Yajima, T., Kashimoto, K. (2002) PACAP protects neuronal PC12 cells from the cytotoxicity of human prion protein fragment 106-126. FEBS Lett.522, 65–70. 34. Lin, P., Li, F., Zhang, Y. W., Tong, G., Farquhar, M. G. and Xu, H.(2007) Calnuc binds to Alzheimer’s beta-amyloid precursor protein and affects its biogenesis.J. Neurochem.100, 1505-1514. 35. Aradhyam, G. K., Balivada, L. M., Kanuru, M., Vadivel, P., and Vidhya, B. S. (2010) Calnuc. Emerging roles in calcium signaling and human diseases. IUBMB Life62, 436-446. 36. Gorbatyuk, M. S., and Gorbatyuk, O. S. (2013) The Molecular Chaperone GRP78/BiP as a Therapeutic Target for Neurodegenerative Disorders. J. Genet. Syndr. Gene Ther.4, doi:10.4172/2157-7412.1000128.


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Biochemistry

37. Narayan, P., Meehan, S., Carver, J. A., Wilson, M. R., Dobson, C. M., and Klenerman, D. (2012) Amyloid‑β Oligomers are Sequestered by both Intracellular and Extracellular Chaperones. Biochemistry51, 9270-9276. Figure Legends. Figure 1. Calnuc prevents temperature-dependent aggregation of amyloidogenic proteins. The role of calnuc in rescuing ADH, catalase, MDH and GST from temperature-dependent aggregation was assessed by monitoring absorbance at 360 nm in a Perkin Elmer UV-VIS spectrophotometer attached to a thermostatic cell holder assembly maintained at 50 °C through a circulating water bath. Substrate and calnuc were incubated initially for 5 min at room temperature(25 °C), placed in cell holder temperature maintained at 50 °C and monitored aggregation for a time scan of 20 min. Concentrations of the substrates were maintained at 1 mg/ml whereas the substrate to calnuc ratio was maintained at 1:4 and 1:2 ratios. Experiments were carried out in 20 mM HEPES pH 7.5 buffer containing 50 mM NaCl. Panels A, B, C and D represent aggregation of ADH, Catalase, MDH and GST respectively, both in the presence and absence of calnuc. The figure is a representative of independent experiments repeated several times (n=6). Figure 2.Calnuc protects residual enzyme activity during thermal stress. The activity of the enzymes (MDH, CS and GST) were assessed after incubating samples at room temperature under native conditions and at 50 °C, both in the presence and absence of calnuc (2 µM). Activity was determined by taking aliquots at different times from the assay mixture incubated at 50 °C. Concentrations of the substrates were maintained at 1 mg/ml whereas the aggregation prevention was examined at 1 µM and 2 µM concentrations of calnuc. Experiments were carried out in 20 mM HEPES pH 7.5 buffer containing 50 mM NaCl. In all the panels, open circles (o) represent the activity of the enzyme and closed circles (●) represent the activity of the enzyme in the presence of calnuc. Panels A, B and C represent the activity of MDH, CS and GST in the presence and absence of calnuc respectively. The figure is a representative of independent experiments repeated several times. In all panels, closed square (▄) indicates the activity of enzymes in presence of calnuc (as a control) under native conditions. Figure 3. Fragments of calnuc prevent thermal aggregation and enzyme activity. Panel A is the schematic representation of calnuc and its fragments used in this study. Calnuc (461 amino acids), a multi-domain protein, has two Zn2+-binding sites (located near the Nterminus), a DNA binding basic rich region and the C-terminal region consisting of two calcium-binding EF-hand motifs, a Gαi binding region, a leucine zipper region (LZ) and poly glutamine rich region (pQ). Calnuc N-and C-fragments were generated to assess their role in chaperone activity. Panel B represents the role of calnuc and its fragments in rescuing the thermal aggregation of MDH (0.2 µM). Panel C depicts the activity of MDH in the presence of various fragments of calnuc (2 µM). The figure is a representative of independent experiments repeated several times. Figure 4. Calnuc enhances residual enzyme activity of MDH during refolding. Malate dehydrogenase (6 µM) was denatured in 6 M GdmHCl (guanidine hydrochloride) in a buffer containing 20 mM HEPES, pH 7.5, 40 mM NaCl and 20 mM DTT for 4 h. A refolding experiment was initiated by 100-fold dilution of MDH (0.06 µM) in 20 mM HEPES, pH 7.5, 40 mM NaCl and 10 mM DTT. Consequently, enzyme activity was determined in the absence and presence of calnuc (0.2 µM) or one of its fragments. Panel A depicts the refolding of MDH in the presence and absence of calnuc (0.2 µM) at the denoted time intervals. Closed squares and closed circles represent the recovery in residual activity of MDH and in the presence of calnuc respectively. Panel B represents the activity


Biochemistry

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of MDH refolded in the presence and absence of calnuc and its fragments at the end of 60 min. The relative activity of the enzyme was calculated by considering the activity of the same amount of native enzyme as 100%. Effect of Ca2+and Zn2+on chaperone activity of calnuc. The chaperone activity of calnuc was monitored in presence of Ca2+and Zn2+ to understand their effect upon binding. Catalase (1 mg/ml) and 3.5 µM of calnuc, both in the presence and absence of Ca2+ (100 µM) and Zn2+ (10 µM) were used for the aggregation experiments. Control experiments were carried out to determine the effect of Ca2+ (100 µM) and Zn2+ (10 µM) alone on catalase. All experimental conditions are same as that mentioned in legend of Fig. 1. The values are presented as percentage of aggregation compared with control ± SEM (n=3). Figure 6. Chaperone activity of calnuc in cell culture (IMR32 cells). Effect of transient expression of calnuc in IMR32 cells was monitored by thermal (45°C) inactivation and post-heat treatment reactivation of transiently transfected Luciferase. Activity was measured, both, in the presence and absence of calnuc. Samples without heat shock served as controls (dark gray bars). Light gray bars denote the activity of luciferase (in the presence and absence of calnuc) after heat shock at 45 °C for 15 min. Luciferase activity was also measured after 2 h recovery at room temperature (gray bars). Error bars, Standard error of mean. *P

Chaperone-like Activity of Calnuc Prevents ... - ACS Publications

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