Research Article Curcumin, a Natural Antioxidant, Acts as ...

Research ArticleCurcumin a Natural Antioxidant Acts asa Noncompetitive Inhibitor of Human RNase Lin Presence of Its Cofactor 2-5A In Vitro

Ankush Gupta and Pramod C Rath

Molecular Biology Laboratory School of Life Sciences Jawaharlal Nehru University New Delhi 110067 India

Correspondence should be addressed to Pramod C Rath pramodrathgmailcom

Received 5 February 2014 Revised 8 June 2014 Accepted 10 June 2014 Published 2 September 2014

Academic Editor Christophe N Peyrefitte

Copyright copy 2014 A Gupta and P C Rath This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Ribonuclease L (RNase L) is an antiviral endoribonuclease of the innate immune system which is induced and activated by viralinfections interferons and double stranded RNA (dsRNA) in mammalian cells Although RNase L is generally protective againstviral infections abnormal RNase L expression and activity have been associated with a number of diseases Here we show thatcurcumin a natural plant-derived anti-inflammatory active principle inhibits RNase L activity hence it may be exploited fortherapeutic interventions in case of pathological situations associated with excess activation of RNase L

1 Introduction

The 2101584051015840-oligoadenylate- (2-5A-) dependent ribonuclease L(RNase L) is one of the key antiviral enzymes of the inter-feron-inducible 2-5A pathway of mammalian cells Uponvirus infection or interferon (IFN) treatment a group ofenzymes collectively termed as 2101584051015840 oligoadenylate syntheta-ses (2-5OAS) activated by double-stranded RNA (dsRNA)often produced as viral replication intermediates synthesizeunique and labile cofactor called 2101584051015840-oligoadenylates (2-5A)from ATP This 2-5A cofactor binds to latent monomericRNase L and converts it into an active dimeric enzymewhichdegrades both viral and cellular single-stranded RNAs thuseliminating both virus and virus-infected cells RNase L is a741 aa protein (sim83 kDa) and has an interesting arrangementof the structural and functional domains The N-terminalregion consists of 9 ankyrin repeats (l-9 9th one incomplete)often involved in protein-proteinprotein-nucleic acid inter-actions while the C-terminal region consists of a protein-kinase- (PK-) homology domain a cysteine-rich region anda ribonuclease domain [1] Functions of the PK-homologyand cysteine-rich domains are uncertain RNase L undergoesconformational switching as it gets activated by binding of 2-5A to the ankyrin repeats 2ndash4 [2]

Interestingly RNase L gene (RNASEL) has been identifiedas a human prostate cancer susceptible (HPC1) locus andhas been suggested to function as a tumor suppressorby causing apoptosis of mammalian cells through RNAdegradation Point mutations in RNase L (eg R462Q) canpotentially decrease its RNase activity and possibly increasesusceptibility to prostate cancer [3] Recent reports suggestthat not only RNase L is a marker for HPC but also germlinemutations in RNASEL may predict an increased risk of headneck uterine cervix and breast carcinoma [4] In additionderegulation in 2-5A pathway has been documented inimmune cells of chronic fatigue syndrome (CFS) patientscharacterized by abnormally upregulated OAS and RNaseL activities and by the presence of a low molecular weight(LMW) 2-5A-binding protein of 37 kDa related to RNaseL produced by proteolytic degradation of the wild typeprotein This protein escapes the normal regulation of RNaseL leading to a cascade of unwanted cellular events probablydue to preferential production of 2-5A dimers instead ofhigher oligomers The origin of the 2-5OAS dysregulationand production of these 2-5A dimers still remain speculativeand the 37 kDa RNase L proteinmight be a result of improperactivation and cleavage due to these dimers [5] Moreoverinduction of RNase L protein by stress-inducing agents such

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 817024 9 pageshttpdxdoiorg1011552014817024

2 BioMed Research International

as double stranded RNA (poly rI rC) chemotherapeuticanticancer drugs H

2O2 calcium chloride and TNF-120572 in

mammalian cells also indicates a possible role for RNase Lin stress-responsive cellular functions [6] Similarly elevatedexpression of RNase L protein and mRNA in colorectaladenocarcinomas suggests its involvement in early events ofcolorectal carcinogenesis [7] Identification of upregulatedgenes in the scrapie-infected brain tissue by subtractivehybridization revealed the upregulation of 2101584051015840 oligoadeny-late synthetase as one of the many interferon-inducible genesand it suggested apoptotic loss of neuronal cells probably byhyperactivation of RNase L [8] Thus considering the broadrange of protective cellular functions of RNase L startingfrom the antiviral antiproliferative apoptotic antineoplasticand immunomodulatory effects to its role in cellular RNAmetabolism translational regulation [9] stress responseand antibacterial immunity [10] we decided to study theeffect of the naturally occurring nontoxic and polyphenolicantioxidant curcumin on RNase L activity

Curcumin an orange-yellow pigment obtained from thedried rhizomes of the perennial herb Curcuma longa Linn(family Zingiberaceae) is commonly used as a spice in Indianfood Curcumin chemically identified as 17-bis-[4 hydroxy-3-methoxy]-16 heptadiene-35-dione or diferuloylmethanehas been traditionally used primarily as an curative agentagainst infections by pathogens and inflammation due toinjury recently many of its therapeutic effects have been con-firmed as antioxidant anti-inflammatory anticarcinogenicantimicrobial hepatoprotective thrombosuppressive car-diovascular protective agent (protection against myocardialinfarction) hypoglycemic antiarthritic (protection againstrheumatoid arthritis) and neuroprotective [11] In this paperwe report that purified curcumin acts as a noncompetitiveinhibitor of the enzymatic activity of full-length (741 aa)recombinant human RNase L in the presence of its cofactor2-5A in vitro This inhibitory effect of curcumin on full-length RNase L activity may be extrapolated as a therapeuticapproach for the treatment of chronic fatigue syndromepatients in the early development of colorectal tumors andloss of neuronal cells in scrapie-infected brain tissues as wellas many immune dysfunctions due to excessive RNase Lactivity

2 Materials and Methods21 Reagents Plasmids Oligonucleotides and E coli StrainsE coli strains DH5120572 and XL-1 blue were used as host cellsfor cloning and expression of recombinant human RNaseL respectively The LB-medium and LB-agar plates weresupplemented with 100 120583gmL of ampicillin The pBluescriptII SK- (+) vector (Stratagene USA) was used for DNAcloning and pGEX 2TK-vector (Amersham USA) wasused for protein expression Oligonucleotides were commer-cially synthesized (Microsynth Switzerland) and Pfu-DNApolymerase (Finnzymes) was used for PCR amplificationof the RNase L cDNA Glutathione-agarose beads IPTGand reagents used for RNase-activity assay were from SigmaAldrich USA The human RNase L cDNA (pZC5) andthe 2-5A cofactor [pppA(21015840p51015840A)

3(tetramer)] [12] used in

the ribonuclease assay were kind gifts from Professor R HSilverman Cleveland Clinic Foundation OH

22 pGEX-hRNase L Construct Preparation Expression andPurification of GST-hRNase L The cloning expression andpurification of the GST-RNase L fusion protein was per-formed as described in detail in our earlier work [13]Briefly the blunt-ended PCR fragment amplified from pZC5[12] plasmid encoding the full-length human RNase L waskinased and ligated into pBluescript II SK- (+) vector at SmaI site generating pBS-hRNL plasmid The Bam HI fragmentcontaining RNase L frompBS-hRNLwas subcloned into BamHI site of pGEX-2TK-vector generating pGEX-hRNL clonesto express the RNase L as a GST-RNase L fusion protein Therecombinant plasmids were transformed into E coli DH5120572cells to prepare plasmid DNA and E coli XL-1 blue cells toexpress the GST-RNase L fusion protein

Briefly for GST-RNase L expression a freshly trans-formed colony of pGEX-hRNLXL-1 blue cells grownovernight at 37∘C was inoculated in primary culture of 10mLLBAmp and grown overnight at 37∘Cwith shaking at 220 rpmA secondary culture of 100mL LBAmp 2 glucose was inocu-lated with 3 inoculum from primary culture and incubatedat 37∘C220 rpm until the OD

600nm of the culture reached

sim06ndash08The cells were then induced at 18∘C220 rpm for 18ndash21 hours with 03mM IPTG after prior reduction of the cul-ture temperature to 18∘CThe cells were pelleted at 6000 rpmfor 10min at 4∘C and the cell pellet from 200mL culturewas washed once in 10mL buffer A [phosphate bufferedsaline (PBS) 10 glycerol 1mM EDTA 01mM ATP 5mMMgcl2 14mM 2-mercaptoethanol 2 120583gmL leupeptin and

2mM PMSF] The washed cell pellet was resuspended in10mL buffer A supplemented with lysozyme (100 120583gmL)and lysed by sonication on ice at 18 micron for 15 secfive times The protein was solubilized by incubation ona rocking platform at 4∘C for 30min after addition of 1(vv) Triton X-100 and the supernatant was collected aftercentrifugation at 13000 rpm for 15min at 4∘C Purificationof the fusion protein was performed in batch affinity as perthemanufacturerrsquos instructions of glutathione agarose (SigmaAldrich USA) with minor modifications The glutathioneagarose [250 120583L of a 50 (vv) slurry preequilibrated withbuffer A] was incubated with clarified cell lysate on ice ona rocking platform at 4∘C for 1 hour After washing theprotein-bead mixture three times with 10mL of buffer A andcentrifugation at 3000 rpm to recover the bound fraction thefusion proteins were eluted in 05mL of buffer B [20mMreduced glutathione 50mM Tris Cl (pH 88) 100mM KCl01 Triton X-100 2mM EDTA 14mM 2-mercaptoethanol02mM ATP 10mM Mgcl

2 and 1 120583gmL leupeptin] three

timeswith incubation at 4∘C for 15min each time 20 sterileglycerol was added to the eluted fraction and small aliquotswere stored at minus80∘C For total intrinsic fluorescence studiesthe buffer exchange and concentration of the affinity-purifiedprotein was performed on Centricon-10 column (Millipore)in buffer C (50mM Tris Cl pH 75 200mMNaCl and 5mM2-mercaptoethanol) The quality of the purified protein wasmonitored by SDS-PAGE followed by Coomassie blue R-250

BioMed Research International 3

staining [13] The protein concentration was determined byBradford assay using BSA as standard

23 RNase-Activity Assay of GST-hRNase L The RNase Lactivity assay was performed as per our earlier work [13]which is broadly based upon the RNase L assay performed byYoshimura et al [14] Mouse kidney total RNA was preparedby using the LiCl precipitation method [15] The RNA-degradation reaction mixture was set up in a volume exclud-ing the RNA substrate in the reaction buffer D [222mMTris Cl (pH 75) 111mM magnesium acetate 89mM 2-mercaptoethanol and 011M KCl] ATP (011mM) GST-hRNase L (50 ng20120583L) 2-5A cofactor (10 nM) curcumin(variable concentration3120583L) and double distilled deion-ized sterile water A 20mMmain stock of curcumin was pre-pared in absolute ethanol and then substocks from 0034mMto 1mM were prepared in sterile deionized water from themain stock To ensure negligible ethanol concentrations 3 120583Lcurcumin20120583L reactionwas added fromdifferent substockswhich accounted for a final concentration of ethanol from825 times 10

minus6 to 75 times 10minus3 (vv) This reaction mixturewas preincubated on ice for 30ndash45min for dimerizationand activation of GST-hRNase L Finally variable amount ofmouse kidney total RNA (1 120583g120583L) was added to the reactionmixture to make the final volume to 20120583L and incubatedat 30∘C for 10min The reaction was terminated by adding40 120583L of 6 X RNA loading dye [10mM Triscl (pH75)01 bromophenol blue 60mM EDTA and 60 glycerol]and 12 120583L (50 reaction) of each sample was resolved by12 agarose-TBE gel electrophoresis stained with 05 120583gmLethidium bromide Quantitation of RNA degradation (RNaseactivity of purified GST-hRNase L) was carried out bymeasuring the intensity of the residual intact bands of 28S and18S rRNAs in each reaction and subtracting it from that of thecontrol reaction which did not contain 2-5A cofactor ( 28Sand 18S rRNA degraded) The quantity and quality of RNAwas measured by taking the absorbance at 260 nm and thecorresponding ratios A

260280and A

260230 respectively The

percentage RNA degradation was converted into pmolminof the RNA substrate degraded in 20 120583L reaction volumeas per our earlier work [13] Densitometric measurementswere carried out by Alpha imager 3400 and AlphaEase FCsoftware Ki of curcumin binding to RNase L was determinedby performing a nonlinear regression analysis by fittingthe acquired data to noncompetitive equation for inhibitorbinding using GraphPad Prism (version 504 for WindowsGraphPad Software San Diego CA)

24 Total Intrinsic Fluorescence Measurement for CurcuminBinding with RNase L The homogeneously purified humanRNase L protein obtained after affinity purification andconcentration by Centricon-10 was used for studies onintrinsic fluorescence with the help of Varian fluorescencespectrophotometer (Cary Eclipse) at room temperatureFluorescence excitation was set at 280 nm and emissionspectra were recorded from 300 to 500 nm at a bandwidthof 1 nm The excitation and emission slit widths were setat 5 nm respectively Each recorded spectrum representsan average of three scans The spectra were recorded at

protein concentration of 1 120583M in the presence of 50mM TrisCl (pH 75) 200mM NaCl and 5mM 120573-mercaptoethanolwith increasing concentration of curcumin (025ndash16120583M)[Curcumin][RNase L] molar ratio was varied between 0125and 32 From a 20mM main stock of curcumin preparedin absolute ethanol substocks of 2mM and 02mM wereprepared in sterile deionized water and added into the finalreactionmix of 300 120583L such that ethanol concentration neverexceeded 04 times 10minus3 (vv) Control experiments with RNaseL and similar ethanol concentrations as the main reactionproved the effect of the organic solvent to be undetectableon the intrinsic fluorescence of the protein The backgroundemission was eliminated by subtracting the signal of thebuffer alone from the test samples The change in intrinsicfluorescence intensity [(119865

0minus119865)119865

0] at 340 nmwith increasing

curcumin concentration and in absence of curcumin wasplotted and the Kd values were determined from a nonlinearleast square regression analysis of the titration data by fittingof saturation curve to the Hill slope using GraphPad Prism(version 504 for Windows GraphPad Software San DiegoCA)

25 Docking of Curcumin to the Human RNase L CrystalStructure Molecular docking experiments were conductedto map the possible curcumin binding sites to humanRNase L For docking calculations recently published crystalstructure of Human RNAse L (PDB Id 4OAU) [16] and3D structure model of curcumin (CID 969569) were usedPotential binding sites in human RNAse L were identi-fied using metaPocket 20 server (httpprojectsbiotectu-dresdendemetapocket) [17] Molecular docking experi-ment was performed using AutoDock Vina [18] whichautomatically calculates the grid maps Each time when thedocking was performed the grids were centred so as tocover the pockets Since curcumin can undergo keto-enoltautomerism in solution the enolic form can exist in differentcis and trans isomeric forms depending on temperaturepolarity or hydrogen bonding nature of solvents [19] hence15 different conformers for curcumin were generated usingDiscovery Studio 35 (Accelrys Inc San Diego CA USA)For each conformer 10 cycles of molecular docking was per-formed For every torsion (for flexible bonds) of curcumin allover 150 cycles of docking calculations were done by Vina atevery cluster of pocket (binding site 1ndash5) Chimera 18 Pymoland AutoDock visualizer were used for analysis of structuraland docking results

3 Results

RNase L is an IFN-inducible endoribonuclease of the innateimmune system of vertebrates Curcumin has been reportedto possess a number of therapeutic properties against var-ious diseases [20] In this report we examined the effectof curcumin (17-bis-[4 hydroxy-3-methoxy]-16 heptadiene-35-dione) (Sigma C-1386) (Figure 1) on the recombinanthuman RNase L activity against total cellular RNA frommouse kidney by scoring 28S and 18S rRNA degradationThe recombinant GST-hRNase L protein produced had thetheoretical pI and molecular weight of 627 and 11063 kDa


Curcumin

O O

OHHO

CH3O OCH3

Figure 1 Chemical structure of curcumin

respectivelyThe effect of various concentrations of curcuminwas checked on 225 nM (50 ng) of purified GST-hRNaseL in presence of 10 nM 2-5A cofactor which was requiredto dimerize RNase L Interestingly RNase L activity waspreincubated with 2-5A at different concentrations of cur-cumin for 45min followed by incubation with increasingconcentrations of mouse kidney total RNA for 10min at30∘C Figures 2(a) and 2(b) which depict the negative controlreactions without the cofactor and with cofactor respectivelyas well as Figures 2(c) and 2(d) which depict the activityfor 5 120583M and 10 120583M curcumin respectively are parts of thesame gels 1 and 2 (both 18 lanes) with control lanes of 2120583gRNA loaded onto each gel for intensity normalization andempty lanes for background subtraction were used for dataacquisition Figures 2(a) to 2(d) depict the inhibitory effectof lower concentrations of curcumin (5 120583M and 10 120583M) onGST-hRNase L activity and the inhibition occurred in a dose-dependent manner The inhibition kinetics was then ana-lyzed by performing nonlinear regression analysis by fittingthe acquired data to noncompetitive equation for inhibitorbinding (Figure 2(e)) which indicated that curcumin acts asa noncompetitive inhibitor of RNase L After the nonlinearregression analysis with lower concentrations of curcumina Ki value of 4136 120583M was obtained (Figure 2(e)) Then theeffect of a broad range of curcumin concentrations was exam-ined on fixed concentrations of GST-hRNase L (225 nM) 2-5A (10 nM) and RNA (sim92 nMor 2120583g20 120583L of reactionmix)(Figures 3(a) and 3(b)) The inhibitions at 5 10 20 30 and40 120583M curcumin were observed as 613 49 323 182and 64 respectively Interestingly it showed maximuminhibition with lower concentrations of curcumin that is5 120583M and 10 120583M but as the concentration of curcumin wasincreased the inhibition was relieved till 40 120583M Further asthe concentration of curcumin was raised to 50 120583M 100 120583Mand 150 120583M there was again an increase in the inhibitionto 77 85 and 905 respectively (Figure 3(c)) showingbiphasic inhibition kinetics by curcumin This indicatedthat there is probably a conformational change of RNaseL in the presence of 5 to 40 120583M curcumin which relievesthe inhibition of the enzyme and there is also again aconformational switching occurring between 40 and 50 120583Mand above curcumin concentrations that again shows gt90inhibition at the given concentration of the enzyme cofactor(2-5A) and the substrate RNA

31 Binding of Curcumin to Human RNase L Binding ofcurcumin to several proteins have been studied earlier usingbiophysical techniques like intrinsic fluorescence quenchingand circular dichroism studies [19 22 23] To investigatethe binding affinity of curcumin with the human RNase

L protein we used total intrinsic fluorescence as well asfluorescence quenching technique and measured the 119870

119863for

the binding (Figures 4(a) and 4(b)) Curcumin quenched thetotal intrinsic fluorescence of RNase L in a concentration-dependent manner The intrinsic fluorescence of the humanRNase L protein is due to the presence of seven tryptophanseighteen tyrosines and twenty-eight phenylalanine residuesFigure 4(a) depicts the plot of fluorescence quenching datafor human RNase L in the presence of increasing con-centrations of curcumin (025 120583M to 16 120583M) The emissionmaximum of human RNase L in absence of curcumin wasobserved at 340 nm (987 au) which was blue shifted from340 nm to 332 nm in presence of 2ndash4120583M curcumin (272 and202 au resp) The emission maximum further blue shiftedto 331 nm from 340 nm at 8 120583M curcumin (18 au) but at16 120583M curcumin the emission maximum came close to thatof the native protein at 337 nm (199 au) (Figure 4(a)) Thehighest blue shift of 9 nm was observed until the concentra-tion of curcumin was increased to 8 120583M and then it revertedclose to its native emission maximum with a 3 nm blue shiftat 16 120583M curcumin This data clearly indicates that upto8 120583M curcumin there is strong binding and internalizationof curcumin in the hydrophobic region of RNase L but above8 120583M there is a change in conformation probably due tobinding of curcumin to different residues at some other sitesThe 119870

119863value of 0397 plusmn 0018 120583M was determined by fitting

the change in fluorescence intensity data at 340 nm by usingHill equation (Figure 4(b))

32 Docking of Curcumin to the Human RNase L In orderto determine the possible curcumin binding sites in humanRNase L we conducted the molecular docking experimentwith the recently solved crystal structure of human RNaseL RNase L forms the intertwined crossed homodimericstructure stabilized by ankyrin repeat (ANK) and kinasehomology (KH) domains thereby positioning the kinaseextension nuclease (KEN) domains in each subunit forRNA substrate recognition [16] The pseudokinase domain isinvolved in 2-5A sensing dimerization nucleotide bindingand ribonuclease functions which explains the evolutionaryadaptability of this eukaryotic protein-kinase fold [21] Thir-teen potential binding sites in human RNAse L were iden-tified using metaPocket 20 server (httpprojectsbiotectu-dresdendemetapocket) [17] for the RNase L protein(3OAU) and these 13 sites formed 5 clusters of pocket (Site 1ndash5) (Figure 5) Docking calculations explored two energeti-cally equivalent sites (binding site 2 and 4) where dockingenergy at site 4 was 09 kcalmole higher (minus85 kcalmol) thanthe lowest energy result at site 2 (minus76 kcalmol) (Table 1)Thecomputational analysis revealed that according to binding


RNA

GST-hRNase L

20 05

GST

10 20 30 40 50 60

28S rRNA

18S rRNA

(b)

(c)

(d) (e)

0

5

10

15

20

25

00 01 02 03

RNA concentration (120583M)

(minus) Curcumin

(minus) Curcumin

(minus) Curcumin

5120583M curcumin10120583M curcumin

28

S an

d18

S rR

NA

deg

rade

d (p

mol

em

in)

(+) Curcumin (5120583M)

(+) Curcumin (10120583M)

120583g20 120583L

50ng20120583L

50ng20120583L

+

+ + + + + + +minus

minus minus minus minus minus minus minus

(a)

(minus) 2ndash5 A

(+) 2ndash5 A (10nM)



Figure 2 Effect of curcumin on RNase L activity 12 agarose-TBE gel showing inhibition of RNase L activity with purified GST-hRNaseL 2-5A (10 nM) and in absence and presence of 5120583M and 10 120583M curcumin respectively Panel (a) control lanes with no 2-5A cofactorpanel (b) activity in presence of 2-5A cofactor and no curcumin panels (c) and (d) inhibition in activity in presence of 5120583M and 10120583Mcurcumin respectively Panels (a) and (b) as well as panel (c) and (d) are parts of the same gels 1 and 2 (both 18 lanes) with control lanes of2 120583g RNA loaded onto each gel for intensity normalization and empty lanes for background subtraction RNase L activity was calculated fromthe decrease in the intensity of ribosomal RNA bands which were converted into pmolmin RNA degraded as mentioned in materials andmethods (e) Plot generated by fitting the data to nonlinear regression analysis which gave a 119870119894 of 4136 120583M Square bracket encompassing28S and 18S rRNA bands depict the area used for intensity calculations

energy themost favorable docking solutions suggested place-ment of curcumin into the hydrophobic-binding pocket (site4) of RNase L (Figure 5) Several docking solutions with acomparable score are also mentioned (Table 1) The site 4is situated at interlobe pocket of kinase homology domainand RNase domain near to the active site where Val-532Phe-585 Trp-589 Arg-592 and Lys-694 residues come closeenough for making interactions Arg-592 is involved in Hbonding The site 2 is situated in the pocket formed by C-terminus of ankyrin repeat domain and extended loop and

helix region of N-terminus lobe of kinase homology domainThe directionality is much less pronounced at site 2 withno hydrogen-bonding interactions Amino acids involved inmaking interaction at site 2 are Leu-278 Asp-303 Pro-331His-351 Arg-352 Ile-353 Arg-355 and Lys-362

4 Discussion

We report that curcumin is a specific potent and noncom-petitive inhibitor of recombinant humanRNase L at fairly low


Table 1 Results of the molecular docking experiment (number of poses having binding energy)

S number Between minus4 andminus5 kcalmol

Between minus5 andminus6 kcalmol



Greater thanminus8 kcalmol

Site 1 8 22 114 6 0Site 2 0 87 49 14 0Site 3 5 78 67 0 0Site 4 0 0 29 103 18Site 5 0 89 61 0 0Clustering of highest number of poses with binding energy greater than minus7 kcalmol occurs mostly at sites 4 and 2 respectively

RNA

GST-hRNase L

Curcumin minus

minus minus

minus+ + + + + + + + +

+ + ++ + + + + + + +

+ + ++ + + + + +

+ + ++ + + + + + + +

00 5050 5 10 20 30 40 100 150

(10nM)

(120583M)

2ndash5 A(2120583g20120583L)

(50ng20120583L)

(a)

005

1015202530354045

C-0 C-5 C-10 C-20 C-30 C-40 C-50 C-100 C-150Curcumin (C) concentration (120583M)

RNas

e act

ivity

(V) (

pmol

min

)

3912 plusmn 285

1515 plusmn 183

1998 plusmn 255

2644 plusmn 289

3204 plusmn 196

3671 plusmn 196

911 plusmn 189

58 plusmn 066371 plusmn 083

(minus) 2ndash5 A

(b)

0

61349

323

182

64

767852

905

0102030405060708090

100


Inhi

bitio

n (

)

(c)

Figure 3 RNA-degradation activity assay showing the effect of increasing concentration of curcumin on RNase L activity (a) 12 agarose-TBEgel showingRNAdegradation profile at 30∘C for 10min RNA92 nM(2 120583g20120583L) asmouse kidney total RNA 2-5A (10 nM)GST-hRNaseL (225 nM each) (b) Graph depicting quantitative degradation of 28S and 18S rRNAs for GST-hRNase L with increasing concentrations ofcurcumin (5120583M to 150 120583M) expressed as pmolmin RNA degraded (c) Graph depicting percentage inhibition in GST-hRNase L activity withincreasing concentrations of curcumin

concentrations that is 5120583M and 10 120583M An active RNase Lcan cause extensive degradation of cellular RNAs leading toapoptosis hence the enzyme has to be critically regulatedat the level of either its synthesis or activity The expressionof RNase L under normal conditions is very limited in mostof the cells and tissues however very little is known aboutthe regulation of the RNase L activity RNase L undergoessubtle conformational changes with respect to its N-terminal2-5A-binding dimerization and hence its activation [2] N-terminal deletion of 1ndash335 aa leads to a constitutively activeRNase L protein [24] while deletion of C-terminal ribonucle-ase domainmakes it a dominant negativemolecule inhibitingthe activity of wild type RNase L [25] Pointmutants of RNase

L like K392R and R462Q in the protein-kinase homologydomain and Y712A and F716A in the ribonuclease domainrender the molecule partially or completely inactive againemphasizing its inherent conformational switching nature[26]

In order to combat the mechanism by which virusesevade the IFN pathways small molecule activators of RNaseL with antiviral nature were discovered which had similarmechanistic action towards RNase L activation as 2-5A andthey activated RNase L in vitro at micromolar concentrations[27] Curcumin is a potent inhibitor of TNF-mediatedphorbol ester and hydrogen-peroxide-mediated activation ofNF-120581B and it inhibits phosphorylation and degradation of


0

2

4

6

8

10

12

300 350 400 450 500

Wavelength (nm)

Fluo

resc

ence

inte

nsity

(au

)

0120583M025 120583M05 120583M1120583M

2120583M4120583M8120583M16120583M

(a)

0 5 10 15 20

00

02

04

06

08

10

(F0minusF

)F0

Curcumin (120583M)

(b)

Figure 4 Curcumin binds to human RNase L directly with high affinity (a) Effect of curcumin on the total intrinsic fluorescence of thepurified human RNase L (GST-RNase L) protein (1 120583M) in buffer (50mM TrisCl (pH 75) 200mM NaCl and 5mM 120573-ME) Fluorescenceemission spectra indicate quenching and blue shift of the fluorescence intensity of humanRNase L during the titration by increasing curcuminconcentration (025120583M to 16 120583M) (b) Saturation curves were generated from the intrinsic fluorescence data by plotting the change influorescence intensity at 340 nm as a function of increasing curcumin concentration where 119865

0and 119865 are fluorescence intensity in the absence

and presence of curcumin respectively119870119863 determined by the fit of fluorescence intensity on the Hill plot was 0397 plusmn 0018 120583M Excitation

wavelength of 280 nm was used

Site 4Site 1

Site 2

Site 3

Site 5N-terminus

C-terminus

PRO331

ARG352

LYS362

HIS351

ILE353

ARG355

TYR354

LEU278ASP303

LYS694

ARG592

PHE585

VAL532

TRP589

(a) (b)

Figure 5 Structure of human RNAse L (4OAU)-curcumin complex obtained after docking analysis as described in materials and methodsSites filled with magenta dots are pockets predicted as potential binding sites for curcumin Site 1 is occupied by 2-5A and site 3 by ADPin native structure Residues highlighted in blue cyan and green are active site residues showing position of active site [21] Inset Figures5(a) and 5(b) show interaction of curcumin (backbone C in orange color) at site 4 and site 2 respectively Oxygen atoms are shown in redand nitrogen in blue Green dotted lines indicate possible H-bonds Wireframe spheres are interaction spheres between atoms of ligand andreceptor


I120581B120572 hence the translocation of p65 subunit to the nucleus[28] Curcumin directly interacts and inhibits various pro-teins for example cyclooxygenase-2 (COX-2) lipoxygenase(LOX) glycogen synthase kinase (GSK)-3120573 phosphorylase-3 kinase xanthine oxidase N-aminopeptidase amyloidprotein human a1-acid glycoprotein autophosphorylationactivated protein kinase DNA polymerase focal adhesionkinase (FAK) pp60 src tyrosine kinase thioredoxin reductase(TrxR) tubulin topoisomerase II ubiquitin isopeptidaseand toll-like receptor- (TLR-) 4 [11] Previous studies havedemonstrated that curcumin accelerates oxidative foldingof cysteine-rich proteins like RNase A through multiplemechanisms probably by stabilizing the disulphide bondswhich do not involve redox chemistry [29] Recently we havereported a convenient method for expression purificationand characterization of the recombinant human RNase L as aGST-RNase L fusion protein [13]This will help in identifyingsmall molecular weight reagents binding to RNase L andmodulating its activity We have also identified several RNaseL-interacting proteins from natural mammalian tissue likemouse spleen [30] These RNase L-interacting proteins mayreveal natural biological role of RNase L in mammalian cellsand tissues

Curcumin basically interacts with the proteins via offer-ing moieties for hydrogen bonding and also the two planarphenyl rings of curcumin stacks with the planar rings of thearomatic amino acid residues of proteins thereby alteringthe conformation and their activity Human RNase L (741aa) has total of 14 cysteine (7 cysteines forming a cysteine-rich region in the PK-homology domain (293 to 444 aa)residues probably making disulphide linkage in the nativemolecule and 53 aromatic (28 phenylalanine 7 tryptophanand 18 tyrosine) aa residuesmaking it a target for curcumin-mediated inhibitionThemolecular docking study performedby us clearly indicates that curcumin fits strongly into thehydrophobic pocket (site 4) of human RNase L formed byldquoFFWTWErdquo (aromatic amino acids 585 and 589 that bindcurcumin are shown in bold) having two tryptophans andtwo phenylalanines (Figure 5) The occurrence of closelyspaced glutamic acid (E) at position 590 (adjacent to Trp-589) and aspartic acid (D) at positions 575 and 579 mightbe responsible for such strong fluorescence quenching due topresence of their carboxylate groups The second predictedsite (site 2) for curcumin binding within amino acids Leu-278 to Lys-362 which lies within the region that stabilizesthe crossed homodimeric structure of human RNase L [16]Thus the higher affinity of curcumin to the hydrophobicsite 4 which lies very close to the ribonuclease domain(key active site residues His-672 Arg-677 Asn-678 andTyr-655) [16] might perturb or block the active site regionat lower curcumin concentrations (5 120583M and 10 120583M) Thefluorescence quenching experiment revealed that there wassaturation in curcumin binding and blue shift in fluorescencemaxima from 4 120583M to 8 120583M curcumin which supported ourRNA-degradation assay with maximum activity at 5120583M andas the concentration of curcumin was increased to saturatingconcentration namely 16120583M in intrinsic fluorescence exper-iment therewas shift in fluorescencemaxima to higher wave-length (337 nm from 331 nm at 8 120583M) (Figure 4(a)) close to

the native (340 nm) and decrease in inhibition by curcuminfrom 5 120583M to 40 120583M in RNA-degradation assay (Figure 3(c))This clearly indicated that the enzyme RNase L has under-gone a conformational change such that different residues(or the same residues but in different environment) havebeen affected by increasing curcumin binding Simultaneousbinding of curcumin to site 2 might relieve the inhibitionfirst but as the concentration is increased above 50 120583M theremight be major switching in the conformation leading tocomplete loss in the activity of human RNase L rendering itapproximately 77 inactive Further higher concentrationsof curcumin that is 100 and 150 120583M cause inhibition of85 and 90 respectively probably due to disruption ofthe crossed homodimeric structure at site 2 by curcuminCurcumin binds to human RNase L with submicromolaraffinity and in none of the docking solutions did it bind tothe cysteine rich region (7 cysteines present from amino acid293 to 444) of the kinase homology (KH) domain of humanRNase L The biphasic response might complicate the useof curcumin as a therapeutic agent but since curcumin canbe consumed at higher levels in the diet and only 150120583Mcurcumin can almost fully inhibit RNase L it is a very goodcandidate for development of efficacious curcuminoids intreating RNase L-mediated immune deregulations Howeverour data indicates that curcumin is a potent inhibitor of puri-fied human RNase L at submicromolar levels which mighthave important physiological consequences particularly forsome types of chronic inflammation it may possibly be usedas therapeutic approach to treat RNase L abnormalities forexample CFS colorectal carcinogenesis scrapie infection inbrain and other defects of the RNase L-pathway

5 Conclusion

Curcumin a natural plant-derived antioxidant inhibitsrecombinant GST-RNase L enzymatic activity in vitro

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor R H Silverman ClevelandClinic Foundation OH USA for generously providing thehuman RNase L cDNA (pZC-5 plasmid) and the 2-5Acofactor [12] They also thank Ms Chhaya Singh School ofBiotechnology Banaras Hindu University for critical help inperforming and compilation of RNase L-curcumin dockingexperiment Research grantsfacilities to PramodC Rath andSLS (School of Life Sciences) under the Capacity BuildupUGC-Research Network Resource Centre program fromthe University Grants Commission (UGC) and Departmentof Science and Technology (DST)-Purse Government ofIndia are gratefully acknowledged Ankush Gupta receiveda JuniorSenior Research Fellowship from the Council of


Scientific and Industrial Research (CSIR) Government ofIndia

References

[1] C Bisbal and R H Silverman ldquoDiverse functions of RNase Land implications in pathologyrdquo Biochimie vol 89 no 6-7 pp789ndash798 2007

[2] M Nakanishi Y Goto and Y Kitade ldquo2-5A induces a con-formational change in the ankyrin-repeat domain of RNase LrdquoProteins Structure Function and Genetics vol 60 no 1 pp 131ndash138 2005

[3] R H Silverman ldquoImplications for RNase L in prostate cancerbiologyrdquo Biochemistry vol 42 no 7 pp 1805ndash1812 2003

[4] B E Madsen E M Ramos M Boulard et al ldquoGermlinemutation in RNASEL predicts increased risk of head and neckuterine cervix and breast cancerrdquoPLoSONE vol 3 no 6 ArticleID e2492 2008

[5] M Fremont K El Bakkouri F Vaeyens C V Herst K deMeir-leir and P Englebienne ldquo2101584051015840-Oligoadenylate size is critical toprotect RNase L against proteolytic cleavage in chronic fatiguesyndromerdquo Experimental and Molecular Pathology vol 78 no3 pp 239ndash246 2005

[6] M Pandey G D Bajaj and P C Rath ldquoInduction of theinterferon-inducible RNA-degrading enzyme RNase L bystress-inducing agents in the human cervical carcinoma cellsrdquoRNA Biology vol 1 no 1 pp 21ndash27 2004

[7] L Wang A Zhou S Vasavada et al ldquoElevated Levels of2101584051015840-linked oligoadenylate-dependent ribonuclease L occur asan early event in colorectal tumorigenesisrdquo Clinical CancerResearch vol 1 no 11 pp 1421ndash1428 1995

[8] C Riemer I Queck D Simon R Kurth and M BaierldquoIdentification of upregulated genes in scrapie-infected braintissuerdquo Journal of Virology vol 74 no 21 pp 10245ndash10248 2000

[9] F Le Roy T Salehzada C Bisbal J P Dougherty and S WPeltz ldquoA newly discovered function for RNase L in regulatingtranslation terminationrdquoNature StructuralampMolecular Biologyvol 12 no 6 pp 505ndash512 2005

[10] X-L Li H J Ezelle T-J Kang et al ldquoAn essential rolefor the antiviral endoribonuclease RNase-L in antibacterialimmunityrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 105 no 52 pp 20816ndash208212008

[11] B B Aggarwal and B Sung ldquoPharmacological basis for the roleof curcumin in chronic diseases an age-old spice with moderntargetsrdquo Trends in Pharmacological Sciences vol 30 no 2 pp85ndash94 2009

[12] A Zhou B A Hassel and R H Silverman ldquoExpression cloningof 2-5A-dependent RNAase a uniquely regulated mediator ofinterferon actionrdquo Cell vol 72 no 5 pp 753ndash765 1993

[13] A Gupta and P C Rath ldquoExpression purification and char-acterization of the interferon-inducible antiviral and tumour-suppressor protein human RNase Lrdquo Journal of Biosciences vol37 no 1 pp 103ndash113 2012

[14] A Yoshimura M Nakanishi C Yatome and Y Kitade ldquoCom-parative study on the biological properties of 2101584051015840-oligoadenylate derivatives with purified human RNase Lexpressed in E colirdquo Journal of Biochemistry vol 132 no 4 pp643ndash648 2002

[15] C Auffray and F Rougeon ldquoPurification of mouse immuno-globulin heavy-chain messenger RNAs from total myeloma

tumor RNArdquo European Journal of Biochemistry vol 107 no 2pp 303ndash314 1980

[16] H Yuchen J Donovan S Rath G Whitney A Chitrakar andA Korennykh ldquoStructure of human RNase L reveals the basisfor regulated RNA decay in the IFN responserdquo Science vol 343pp 1244ndash1248 2014

[17] B Huang ldquoMetapocket a meta approach to improve proteinligand binding site predictionrdquo OMICS A Journal of IntegrativeBiology vol 13 no 4 pp 325ndash330 2009

[18] O Trott and A J Olson ldquoAutoDock Vina improving the speedand accuracy of docking with a new scoring function efficientoptimization and multithreadingrdquo Journal of ComputationalChemistry vol 31 no 2 pp 455ndash461 2010

[19] A Majhi G M Rahman S Panchal and J Das ldquoBinding ofcurcumin and its long chain derivatives to the activator bindingdomain of novel protein kinase Crdquo Bioorganic and MedicinalChemistry vol 18 no 4 pp 1591ndash1598 2010

[20] A Goel A B Kunnumakkara and B B Aggarwal ldquoCurcuminas ldquoCurecuminrdquo from kitchen to clinicrdquo Biochemical Pharma-cology vol 75 no 4 pp 787ndash809 2008

[21] H Haung E Zeqiraj B Dong et al ldquoDimeric structure ofpseudokinase RNase L bound to 2-5A reveals a basis forinterferon-induced antiviral activityrdquoMolecular Cell vol 53 no2 pp 221ndash234 2014

[22] H Gradisar M M Keber P Pristovsek and R Jerala ldquoMD-2as the target of curcumin in the inhibition of response to LPSrdquoJournal of Leukocyte Biology vol 82 no 4 pp 968ndash974 2007

[23] F Zsila Z Bikadi andM Simonyi ldquoInduced circular dichroismspectra reveal binding of the antiinflammatory curcumin tohuman 1205721-acid glycoproteinrdquo Bioorganic amp Medicinal Chem-istry vol 12 no 12 pp 3239ndash3245 2004

[24] B Dong and R H Silverman ldquoA bipartite model of 2-5A-dependent RNase Lrdquo Journal of Biological Chemistry vol 272no 35 pp 22236ndash22242 1997

[25] B AHassel A Zhou C Sotomayor AMaran and RH Silver-man ldquoA dominant negative mutant of 2-5A-dependent RNasesuppresses antiproliferative and antiviral effects of interferonrdquoEMBO Journal vol 12 no 8 pp 3297ndash3304 1993

[26] M Nakanishi A Yoshimura N Ishida Y Ueno and Y KitadeldquoContribution of Tyr712 and Phe716 to the activity of humanRNase Lrdquo European Journal of Biochemistry vol 271 no 13 pp2737ndash2744 2004

[27] C SThakur B K Jha BDong et al ldquoSmall-molecule activatorsof RNase L with broad-spectrum antiviral activityrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 23 pp 9585ndash9590 2007

[28] S Singh and B B Aggarwal ldquoActivation of transcription factorNF-120581B is suppressed by curcumin (diferuloylmethane)rdquo TheJournal of Biological Chemistry vol 270 no 42 pp 24995ndash25000 1995

[29] G Gomez G Mansouraty J Gardea and M Narayan ldquoAccel-eration of oxidative protein folding by curcumin through novelnon-redox chemistryrdquo Biochemical and Biophysical ResearchCommunications vol 364 no 3 pp 561ndash566 2007

[30] A Gupta and P C Rath ldquoExpression of mRNA and protein-protein interaction of the antiviral endoribonuclease RNase Lin mouse spleenrdquo International Journal of Biological Macro-molecules vol 69 pp 307ndash318 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


as double stranded RNA (poly rI rC) chemotherapeuticanticancer drugs H

2O2 calcium chloride and TNF-120572 in

mammalian cells also indicates a possible role for RNase Lin stress-responsive cellular functions [6] Similarly elevatedexpression of RNase L protein and mRNA in colorectaladenocarcinomas suggests its involvement in early events ofcolorectal carcinogenesis [7] Identification of upregulatedgenes in the scrapie-infected brain tissue by subtractivehybridization revealed the upregulation of 2101584051015840 oligoadeny-late synthetase as one of the many interferon-inducible genesand it suggested apoptotic loss of neuronal cells probably byhyperactivation of RNase L [8] Thus considering the broadrange of protective cellular functions of RNase L startingfrom the antiviral antiproliferative apoptotic antineoplasticand immunomodulatory effects to its role in cellular RNAmetabolism translational regulation [9] stress responseand antibacterial immunity [10] we decided to study theeffect of the naturally occurring nontoxic and polyphenolicantioxidant curcumin on RNase L activity

Curcumin an orange-yellow pigment obtained from thedried rhizomes of the perennial herb Curcuma longa Linn(family Zingiberaceae) is commonly used as a spice in Indianfood Curcumin chemically identified as 17-bis-[4 hydroxy-3-methoxy]-16 heptadiene-35-dione or diferuloylmethanehas been traditionally used primarily as an curative agentagainst infections by pathogens and inflammation due toinjury recently many of its therapeutic effects have been con-firmed as antioxidant anti-inflammatory anticarcinogenicantimicrobial hepatoprotective thrombosuppressive car-diovascular protective agent (protection against myocardialinfarction) hypoglycemic antiarthritic (protection againstrheumatoid arthritis) and neuroprotective [11] In this paperwe report that purified curcumin acts as a noncompetitiveinhibitor of the enzymatic activity of full-length (741 aa)recombinant human RNase L in the presence of its cofactor2-5A in vitro This inhibitory effect of curcumin on full-length RNase L activity may be extrapolated as a therapeuticapproach for the treatment of chronic fatigue syndromepatients in the early development of colorectal tumors andloss of neuronal cells in scrapie-infected brain tissues as wellas many immune dysfunctions due to excessive RNase Lactivity

2 Materials and Methods21 Reagents Plasmids Oligonucleotides and E coli StrainsE coli strains DH5120572 and XL-1 blue were used as host cellsfor cloning and expression of recombinant human RNaseL respectively The LB-medium and LB-agar plates weresupplemented with 100 120583gmL of ampicillin The pBluescriptII SK- (+) vector (Stratagene USA) was used for DNAcloning and pGEX 2TK-vector (Amersham USA) wasused for protein expression Oligonucleotides were commer-cially synthesized (Microsynth Switzerland) and Pfu-DNApolymerase (Finnzymes) was used for PCR amplificationof the RNase L cDNA Glutathione-agarose beads IPTGand reagents used for RNase-activity assay were from SigmaAldrich USA The human RNase L cDNA (pZC5) andthe 2-5A cofactor [pppA(21015840p51015840A)

3(tetramer)] [12] used in

the ribonuclease assay were kind gifts from Professor R HSilverman Cleveland Clinic Foundation OH

22 pGEX-hRNase L Construct Preparation Expression andPurification of GST-hRNase L The cloning expression andpurification of the GST-RNase L fusion protein was per-formed as described in detail in our earlier work [13]Briefly the blunt-ended PCR fragment amplified from pZC5[12] plasmid encoding the full-length human RNase L waskinased and ligated into pBluescript II SK- (+) vector at SmaI site generating pBS-hRNL plasmid The Bam HI fragmentcontaining RNase L frompBS-hRNLwas subcloned into BamHI site of pGEX-2TK-vector generating pGEX-hRNL clonesto express the RNase L as a GST-RNase L fusion protein Therecombinant plasmids were transformed into E coli DH5120572cells to prepare plasmid DNA and E coli XL-1 blue cells toexpress the GST-RNase L fusion protein

Briefly for GST-RNase L expression a freshly trans-formed colony of pGEX-hRNLXL-1 blue cells grownovernight at 37∘C was inoculated in primary culture of 10mLLBAmp and grown overnight at 37∘Cwith shaking at 220 rpmA secondary culture of 100mL LBAmp 2 glucose was inocu-lated with 3 inoculum from primary culture and incubatedat 37∘C220 rpm until the OD

600nm of the culture reached

sim06ndash08The cells were then induced at 18∘C220 rpm for 18ndash21 hours with 03mM IPTG after prior reduction of the cul-ture temperature to 18∘CThe cells were pelleted at 6000 rpmfor 10min at 4∘C and the cell pellet from 200mL culturewas washed once in 10mL buffer A [phosphate bufferedsaline (PBS) 10 glycerol 1mM EDTA 01mM ATP 5mMMgcl2 14mM 2-mercaptoethanol 2 120583gmL leupeptin and

2mM PMSF] The washed cell pellet was resuspended in10mL buffer A supplemented with lysozyme (100 120583gmL)and lysed by sonication on ice at 18 micron for 15 secfive times The protein was solubilized by incubation ona rocking platform at 4∘C for 30min after addition of 1(vv) Triton X-100 and the supernatant was collected aftercentrifugation at 13000 rpm for 15min at 4∘C Purificationof the fusion protein was performed in batch affinity as perthemanufacturerrsquos instructions of glutathione agarose (SigmaAldrich USA) with minor modifications The glutathioneagarose [250 120583L of a 50 (vv) slurry preequilibrated withbuffer A] was incubated with clarified cell lysate on ice ona rocking platform at 4∘C for 1 hour After washing theprotein-bead mixture three times with 10mL of buffer A andcentrifugation at 3000 rpm to recover the bound fraction thefusion proteins were eluted in 05mL of buffer B [20mMreduced glutathione 50mM Tris Cl (pH 88) 100mM KCl01 Triton X-100 2mM EDTA 14mM 2-mercaptoethanol02mM ATP 10mM Mgcl

2 and 1 120583gmL leupeptin] three

timeswith incubation at 4∘C for 15min each time 20 sterileglycerol was added to the eluted fraction and small aliquotswere stored at minus80∘C For total intrinsic fluorescence studiesthe buffer exchange and concentration of the affinity-purifiedprotein was performed on Centricon-10 column (Millipore)in buffer C (50mM Tris Cl pH 75 200mMNaCl and 5mM2-mercaptoethanol) The quality of the purified protein wasmonitored by SDS-PAGE followed by Coomassie blue R-250





260280and A





0minus119865)119865




3 Results



Curcumin

O O

OHHO

CH3O OCH3





119863for






RNA

GST-hRNase L

20 05

GST

10 20 30 40 50 60

28S rRNA

18S rRNA

(b)

(c)

(d) (e)

0

5

10

15

20

25

00 01 02 03


(minus) Curcumin

(minus) Curcumin

(minus) Curcumin


28

S an

d18

S rR

NA

deg

rade

d (p

mol

em

in)

(+) Curcumin (5120583M)

(+) Curcumin (10120583M)

120583g20 120583L

50ng20120583L

50ng20120583L

+

+ + + + + + +minus


(a)

(minus) 2ndash5 A







4 Discussion










RNA

GST-hRNase L

Curcumin minus

minus minus

minus+ + + + + + + + +

+ + ++ + + + + + + +

+ + ++ + + + + +

+ + ++ + + + + + + +

00 5050 5 10 20 30 40 100 150

(10nM)

(120583M)

2ndash5 A(2120583g20120583L)

(50ng20120583L)

(a)

005

1015202530354045


RNas

e act

ivity

(V) (

pmol

min

)

3912 plusmn 285

1515 plusmn 183

1998 plusmn 255

2644 plusmn 289

3204 plusmn 196

3671 plusmn 196

911 plusmn 189


(minus) 2ndash5 A

(b)

0

61349

323

182

64

767852

905

0102030405060708090

100


Inhi

bitio

n (

)

(c)






0

2

4

6

8

10

12

300 350 400 450 500

Wavelength (nm)

Fluo

resc

ence

inte

nsity

(au

)

0120583M025 120583M05 120583M1120583M

2120583M4120583M8120583M16120583M

(a)

0 5 10 15 20

00

02

04

06

08

10

(F0minusF

)F0

Curcumin (120583M)

(b)





Site 4Site 1

Site 2

Site 3

Site 5N-terminus

C-terminus

PRO331

ARG352

LYS362

HIS351

ILE353

ARG355

TYR354

LEU278ASP303

LYS694

ARG592

PHE585

VAL532

TRP589

(a) (b)






5 Conclusion




Acknowledgments




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





260280and A





0minus119865)119865




3 Results



Curcumin

O O

OHHO

CH3O OCH3





119863for






RNA

GST-hRNase L

20 05

GST

10 20 30 40 50 60

28S rRNA

18S rRNA

(b)

(c)

(d) (e)

0

5

10

15

20

25

00 01 02 03


(minus) Curcumin

(minus) Curcumin

(minus) Curcumin


28

S an

d18

S rR

NA

deg

rade

d (p

mol

em

in)

(+) Curcumin (5120583M)

(+) Curcumin (10120583M)

120583g20 120583L

50ng20120583L

50ng20120583L

+

+ + + + + + +minus


(a)

(minus) 2ndash5 A







4 Discussion










RNA

GST-hRNase L

Curcumin minus

minus minus

minus+ + + + + + + + +

+ + ++ + + + + + + +

+ + ++ + + + + +

+ + ++ + + + + + + +

00 5050 5 10 20 30 40 100 150

(10nM)

(120583M)

2ndash5 A(2120583g20120583L)

(50ng20120583L)

(a)

005

1015202530354045


RNas

e act

ivity

(V) (

pmol

min

)

3912 plusmn 285

1515 plusmn 183

1998 plusmn 255

2644 plusmn 289

3204 plusmn 196

3671 plusmn 196

911 plusmn 189


(minus) 2ndash5 A

(b)

0

61349

323

182

64

767852

905

0102030405060708090

100


Inhi

bitio

n (

)

(c)






0

2

4

6

8

10

12

300 350 400 450 500

Wavelength (nm)

Fluo

resc

ence

inte

nsity

(au

)

0120583M025 120583M05 120583M1120583M

2120583M4120583M8120583M16120583M

(a)

0 5 10 15 20

00

02

04

06

08

10

(F0minusF

)F0

Curcumin (120583M)

(b)





Site 4Site 1

Site 2

Site 3

Site 5N-terminus

C-terminus

PRO331

ARG352

LYS362

HIS351

ILE353

ARG355

TYR354

LEU278ASP303

LYS694

ARG592

PHE585

VAL532

TRP589

(a) (b)






5 Conclusion




Acknowledgments




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Curcumin

O O

OHHO

CH3O OCH3





119863for






RNA

GST-hRNase L

20 05

GST

10 20 30 40 50 60

28S rRNA

18S rRNA

(b)

(c)

(d) (e)

0

5

10

15

20

25

00 01 02 03


(minus) Curcumin

(minus) Curcumin

(minus) Curcumin


28

S an

d18

S rR

NA

deg

rade

d (p

mol

em

in)

(+) Curcumin (5120583M)

(+) Curcumin (10120583M)

120583g20 120583L

50ng20120583L

50ng20120583L

+

+ + + + + + +minus


(a)

(minus) 2ndash5 A







4 Discussion










RNA

GST-hRNase L

Curcumin minus

minus minus

minus+ + + + + + + + +

+ + ++ + + + + + + +

+ + ++ + + + + +

+ + ++ + + + + + + +

00 5050 5 10 20 30 40 100 150

(10nM)

(120583M)

2ndash5 A(2120583g20120583L)

(50ng20120583L)

(a)

005

1015202530354045


RNas

e act

ivity

(V) (

pmol

min

)

3912 plusmn 285

1515 plusmn 183

1998 plusmn 255

2644 plusmn 289

3204 plusmn 196

3671 plusmn 196

911 plusmn 189


(minus) 2ndash5 A

(b)

0

61349

323

182

64

767852

905

0102030405060708090

100


Inhi

bitio

n (

)

(c)






0

2

4

6

8

10

12

300 350 400 450 500

Wavelength (nm)

Fluo

resc

ence

inte

nsity

(au

)

0120583M025 120583M05 120583M1120583M

2120583M4120583M8120583M16120583M

(a)

0 5 10 15 20

00

02

04

06

08

10

(F0minusF

)F0

Curcumin (120583M)

(b)





Site 4Site 1

Site 2

Site 3

Site 5N-terminus

C-terminus

PRO331

ARG352

LYS362

HIS351

ILE353

ARG355

TYR354

LEU278ASP303

LYS694

ARG592

PHE585

VAL532

TRP589

(a) (b)






5 Conclusion




Acknowledgments




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


RNA

GST-hRNase L

20 05

GST

10 20 30 40 50 60

28S rRNA

18S rRNA

(b)

(c)

(d) (e)

0

5

10

15

20

25

00 01 02 03


(minus) Curcumin

(minus) Curcumin

(minus) Curcumin


28

S an

d18

S rR

NA

deg

rade

d (p

mol

em

in)

(+) Curcumin (5120583M)

(+) Curcumin (10120583M)

120583g20 120583L

50ng20120583L

50ng20120583L

+

+ + + + + + +minus


(a)

(minus) 2ndash5 A







4 Discussion










RNA

GST-hRNase L

Curcumin minus

minus minus

minus+ + + + + + + + +

+ + ++ + + + + + + +

+ + ++ + + + + +

+ + ++ + + + + + + +

00 5050 5 10 20 30 40 100 150

(10nM)

(120583M)

2ndash5 A(2120583g20120583L)

(50ng20120583L)

(a)

005

1015202530354045


RNas

e act

ivity

(V) (

pmol

min

)

3912 plusmn 285

1515 plusmn 183

1998 plusmn 255

2644 plusmn 289

3204 plusmn 196

3671 plusmn 196

911 plusmn 189


(minus) 2ndash5 A

(b)

0

61349

323

182

64

767852

905

0102030405060708090

100


Inhi

bitio

n (

)

(c)






0

2

4

6

8

10

12

300 350 400 450 500

Wavelength (nm)

Fluo

resc

ence

inte

nsity

(au

)

0120583M025 120583M05 120583M1120583M

2120583M4120583M8120583M16120583M

(a)

0 5 10 15 20

00

02

04

06

08

10

(F0minusF

)F0

Curcumin (120583M)

(b)





Site 4Site 1

Site 2

Site 3

Site 5N-terminus

C-terminus

PRO331

ARG352

LYS362

HIS351

ILE353

ARG355

TYR354

LEU278ASP303

LYS694

ARG592

PHE585

VAL532

TRP589

(a) (b)






5 Conclusion




Acknowledgments




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









RNA

GST-hRNase L

Curcumin minus

minus minus

minus+ + + + + + + + +

+ + ++ + + + + + + +

+ + ++ + + + + +

+ + ++ + + + + + + +

00 5050 5 10 20 30 40 100 150

(10nM)

(120583M)

2ndash5 A(2120583g20120583L)

(50ng20120583L)

(a)

005

1015202530354045


RNas

e act

ivity

(V) (

pmol

min

)

3912 plusmn 285

1515 plusmn 183

1998 plusmn 255

2644 plusmn 289

3204 plusmn 196

3671 plusmn 196

911 plusmn 189


(minus) 2ndash5 A

(b)

0

61349

323

182

64

767852

905

0102030405060708090

100


Inhi

bitio

n (

)

(c)






0

2

4

6

8

10

12

300 350 400 450 500

Wavelength (nm)

Fluo

resc

ence

inte

nsity

(au

)

0120583M025 120583M05 120583M1120583M

2120583M4120583M8120583M16120583M

(a)

0 5 10 15 20

00

02

04

06

08

10

(F0minusF

)F0

Curcumin (120583M)

(b)





Site 4Site 1

Site 2

Site 3

Site 5N-terminus

C-terminus

PRO331

ARG352

LYS362

HIS351

ILE353

ARG355

TYR354

LEU278ASP303

LYS694

ARG592

PHE585

VAL532

TRP589

(a) (b)






5 Conclusion




Acknowledgments




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

2

4

6

8

10

12

300 350 400 450 500

Wavelength (nm)

Fluo

resc

ence

inte

nsity

(au

)

0120583M025 120583M05 120583M1120583M

2120583M4120583M8120583M16120583M

(a)

0 5 10 15 20

00

02

04

06

08

10

(F0minusF

)F0

Curcumin (120583M)

(b)





Site 4Site 1

Site 2

Site 3

Site 5N-terminus

C-terminus

PRO331

ARG352

LYS362

HIS351

ILE353

ARG355

TYR354

LEU278ASP303

LYS694

ARG592

PHE585

VAL532

TRP589

(a) (b)






5 Conclusion




Acknowledgments




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





5 Conclusion




Acknowledgments




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Curcumin, a Natural Antioxidant, Acts as ...

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