Báo cáo khoa học: The propagation of hamster-adapted scrapie PrPSc can be enhanced by reduced pyridine nucleotide in vitro pdf

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The propagation of hamster-adapted scrapie PrPSccan beenhanced by reduced pyridine nucleotide in vitroSong Shi, Chen-Fang Dong, Chan Tian, Rui-Min Zhou, Kun Xu, Bao-Yun Zhang, Chen Gao,Jun Han and Xiao-Ping DongState Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Beijing, ChinaTransmissible spongiform encephalopathies (TSEs),also known as prion diseases, are lethal neurodegener-ative diseases, including Creutzfeldt–Jakob disease,Gerstmann–Straussler–Scheinker syndrome and fatalfamilial insomnia in humans, bovine spongiformencephalopathy in cattle, scrapie in sheep and goats,and chronic wasting disease in deer and elk [1,2]. TSEsare caused by a proteinaceous infectious agent, termeda prion, which is considered to consist of a misfoldedand aggregated protease-resistant isomer of a host-encoded glycoprotein (PrPC) [3,4]. The pathologicalisomer present in the tissues of infected individuals iscalled PrPSc. Although the clinical and pathologicalcharacteristics of TSEs have been recognized for a longtime, the mechanisms underlying prion conversion areonly partially settled.Recently, some endogenous factors encoded byprion hosts have been proposed as essential during thepropagation of prions in studies in animal models andcell-culture systems, i.e. protein X and some chaperons[5–10]. Polyanions and sulfated glycans are alsothought to be involved in converting PrPCinto theabnormal isomer in vitro [11–14]. Moreover, pyridinenucleotides, a group of coenzymes ubiquitous in bio-synthesis and metabolism, are associated with theaggregation of recombinant prion protein (rPrP).Following incubation with reduced pyridine nucleo-tides, e.g. NADPH, rPrP can accumulate in fibrils andacquire weak proteinase resistance [15,16]. Severalstudies have shown that the amount of NADPH-diaphorase increases during the early stage of priondisease [17], but there is no direct molecular evidenceKeywordsPMCA; prion; propagation; pyridinenucleotide; transmissible spongiformencephalopathiesCorrespondenceX P. Dong, State Key Laboratory forInfectious Disease Prevention and Control,National Institute for Viral Disease Controland Prevention, Chinese Center for DiseaseControl and Prevention, Ying-Xin Rd 100,Beijing 100052, ChinaFax: +86 10 63532053Tel: +86 10 83534616E-mail: dongxp238@sina.com(Received 16 September 2008, revised 15November 2008, accepted 22 December2008)doi:10.1111/j.1742-4658.2009.06871.xTransmissible spongiform encephalopathies (TSEs), or prion diseases, arefatal neurodegenerative disorders caused by an infectious agent termed aprion, which can convert normal cellular prion protein (PrPC) into a patho-logically misfolded isoform (PrPSc). Taking advantage of protein misfoldingcyclic amplification (PMCA), a series of experiments was conducted toinvestigate the possible influences of pyridine nucleotides on the propaga-tion activities of hamster-adapted scrapie agents 263K and 139A in vitrousing normal hamster brain homogenates and recombinant hamster PrP asthe substrates. The results showed that PrPScfrom both scrapie agent263K- and 139A-infected brains propagated more efficiently in PMCA withthe addition of reduced NADPH, showing an obvious dose-dependentenhancement. Reduced NADH also prompted PrPScpropagation, whereasNADP, NAD and vitamin C failed. Moreover, following incubation withNADPH, recombinant hamster PrP could be efficiently converted into theproteinase K-resistant form when exposed to the trace of PrPScfrominfected hamsters. Our data provide evidence that the reduced pyridinenucleotide plays an important role in the propagation of prion and thisprocess seems to target PrPCmolecules.AbbreviationsNBH, normal brain homogenate; PK, proteinase K; PMCA, protein misfolding cyclic amplification; rPrP, recombinant PrP; ScBH, scrapie-infected brain homogenate; SHa, Syrian hamster; TSEs, transmissible spongiform encephalopathies.1536 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBSto clarify the mechanism by which reduced pyridinenucleotides affect PrP molecules.A novel technology, named protein misfolding cyclicamplification (PMCA) has been described in recentyears [18], providing an efficacious, unique and conve-nient experimental approach to evaluating the replica-tion and infectivity of newly formed PrPScin vitro [19].Following incubation of PrPScfrom TSE-infected ani-mals with homologous PrPCfrom normal brains, largequantities of PrPSccan be achieved after a short cycle ofalternate sonication and incubation [20,21]. This tech-nology is considered the most appropriate approach tounderstanding the fundamental mechanisms involved inPrPScpropagation, aggregation and neuroinvasionin vivo [22]. In this study, possible influences of pyridinenucleotides on the propagation of PrPScfrom scrapie-infected hamsters in PMCA were investigated. Wefound that reduced pyridine nucleotide NADPH pro-moted PrPSc-propagating activity in PMCA using PrPCfrom normal hamster brains as the substrate, and thatthis was a dose-dependent response. Other reduced ana-logues of pyridine nucleotides also showed enhancedcapacity, whereas an oxidized analogue failed. Further-more, we proposed that after incubation with NADPH,recombinant hamster PrP could be converted to theproteinase K (PK)-resistant isoform in the presence ofscrapie PrPScin PMCA.ResultsThe propagating activities of PrPScin PMCA areremarkably enhanced in the presence of NADPHTo see the influence of NADPH on the ability of PrPScto propagate in PMCA, a serial PMCA protocol con-sisting of seven rounds was conduced (Fig. 1A). Priorto mixing with a 10)2dilution of ScBH prepared fromscrapie 263K-infected hamsters (SHa-263K), hamsterNBH was mixed with 10 lm NADPH (referred asNBH + NADPH, lanes 3–9) and subjected to 12PMCA cycles. Thereafter, 10 lL of PMCA productwas added to 90 lL of fresh NBH with 10 lmNADPH and the next round was performed. This wasrepeated several times, leading to the original ScBHbeing diluted to 10)8. Meanwhile, mixture containingonly NADPH and ScBH in the conversion buffer, butwithout NBH (referred as NADPH control, lanes 3–9),or one containing NBH and ScBH but withoutNADPH (referred as NBH control, lanes 3–9), wasprepared as above. Each PMCA product was digestedwith PK and subjected to western blotting. PK-resis-tant PrP signal was detected only in the first NADPHcontrol preparation (upper gel), representing the initialinput PrPSc(Fig. 1A, lane 2 in all gels). In the NBHcontrol, the PrPScsignal was strongest during round 1,weakened gradually and vanished from round 4 (mid-dle gel), indicating lower PMCA-propagating activityunder these conditions. By contrast, in the presence ofNADPH, obvious PrPScsignals were seen in all prepa-rations from round 1 to round 7 (Fig. 1A, lower gel),highlighting a more actively propagating capacity forPrPSc. This suggests that NADPH can prompt thepropagation of scrapie agent 263K. In addition, no sig-nificant propagation of PrPScwas observed in seededpreparations without sonication (Fig. S1A) or inPMCA samples without seeding ScBH (Fig. S1B),Fig. 1. NADPH induced more efficient propagation of PrPScfromscrapie agents in PMCA. Ten microliters of a 100-fold dilution ofScBH of SHa-263K or SHa-139A were used as the seed in each prep-aration. PMCA was conducted over 12 cycles per round, with sevenrounds in total. (A) Propagation of PrPScfrom hamster-adapted scra-pie agent 263K in PMCA. The NADPH control (upper gel) contained10 lM NADPH in conversion buffer and ScBH, but without NBH. TheNBH control (middle gel) contained NBH and ScBH, but withoutNADPH. NBH plus NADPH (lower gel) contained NBH, ScBH andNADPH. (B) Propagations seeded with hamster-adapted scrapieagent 139A underwent the same PMCA procedure containing NBHalone (upper gel) or NBH plus NADPH (lower gel). In all gels, NBHwas loaded directly in the first lane without proteolysis as a refer-ence for comparison of electrophoretic mobility (PrPCPK)). All othersamples were treated with 50 lgÆmL)1PK (PK+). 0 indicates reac-tions containing ScBH before PMCA process. Round numbers aregiven on top. The molecular markers are indicated on the right.S. Shi et al. NADPH can enhances PrPScpropagationFEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1537implying that NADPH can neither enhance PrPScpropagation without PMCA nor induce spontaneousconversion of PrPCinto PrPScwith PMCA in vitro.To test whether NADPH-related enhancement wasalso appropriate to another scrapie agent, hamster-adapted scrapie strain 139A (SHa-139A) was used inPMCA following the same procedure. In order toavoid possible environmental contamination, all mate-rials and reagents were freshly prepared, including thehomogenizer, chemicals and conversion buffer. NBHand ScBH (SHa-139A) were prepared in another labo-ratory that had never been exposed to prions. LikeSHa-263K, PK-resistant signals were detected in reac-tions from round 1 to round 7 in the presence ofNADPH (Fig. 1B, lower), but only in the first threerounds without NADPH (Fig. 1B, upper). Theseresults indicate that propagation of SHa-139A in vitrocan also be prompted by NADPH, and therefore thisenhancement of PrPScpropagation in PMCA is notlimited to only one prion strain.NADPH increased the sensitivity for detection ofPrPScin brain homogenates by PMCATo test the potential of using NADPH as an assistantchemical in increasing the detection sensitivity ofPMCA, comparative analysis of serial PMCA, with orwithout NADPH, was conducted using serially dilutedScBH from agent 263K as the seed. Dilutions of theoriginal ScBH ranged from 10)4to 10)12by 100-foldserial dilution. One round of PMCA consisted of 24cycles (24 h), which was considered to be more effi-cient at increasing PrPScaggregation [19] and allowedlow levels of PrPScto be detected [23]. After one roundof PMCA, 10 lL of product was mixed with 90 lLoffresh NBH for the next round, up to seven rounds.Figure 2 shows that PrPScsignals were detected atdilutions of 10)6and 10)8in the second round in thepresence of 10 lm NADPH (second gel, lanes 2 and3), whereas PrPScsignals could be observed at a dilu-tion of 10)6in the third round (third gel, lane 7) and10)8in the fifth round (fifth gel, lane 8) in the absenceof NADPH. After seven rounds of PMCA, clear PrPScsignals were observed at ScBH dilutions of 10)4to10)10, and even at a 10)12-fold dilution a weak bandwas observed in the presence of NADPH (lower gel,lane 5). However, no PrPScsignal was detected atdilutions of 10)10and 10)12without NADPH (lowergel, lanes 9 and 10). This indicates that NADPH canincrease, by at least 102-fold, the sensitivity for thedetection of PrPScin brain homogenates under theseexperimental conditions. At the concentration of PrPScin ScBH used in this study (see Materials andmethods), a 1 · 10)10dilution of ScBH contained 7.265 · 10)19gÆlL)1PrPSc, or 12.5 molecules of ini-tial PrPScper lL. Thereby, one can speculate that 12.5 monomeric PrPScmolecules can successfullyinduce detectable amplification in a seven-roundPMCA with NADPH, whereas in the absence ofNADPH at least 1250 molecules of PrPScare requiredfor the same amplification.PrPScgenerated from PMCA in the presence ofNADPH had further propagation abilityTo determine whether newly generated PMCA PrPScby addition of NADPH could further propagate in thePMCA system in the absence of NADPH, 10 lLofPMCA product (PMCA-productNADPH) from a 10)10dilution in the seventh round (Fig. 2, lane 4) wasdiluted in 90 lL of NBH and subjected to one roundof PMCA (24 cycles) without NADPH (i.e. eightrounds in total). Obvious PrPScsignals were detectedFig. 2. Comparative evaluation for serial PMCA detecting the sensi-tivity of PrPScin ScBH in the presence or absence of NADPH.Aliquots of SHa-263K ScBH were 100-fold serially diluted with ham-ster NBH, reaching dilutions of 10)4,10)6,10)8,10)10and 10)12.Each dilution was divided equally into two PCR tubes, one mixedwith 10 lM NADPH and the other added to an equal volume ofNaCl ⁄ Pi. After 24 cycles PMCA as the first round, 10 lL of productwas mixed with 90 lL of fresh NBH for the next round of PMCA.The five left-hand lanes are fresh NBH plus NADPH (NADPH+) andthe five right-hand lanes are without NADPH (NADPH-). After sevenrounds, the digested PMCA products of each round were analysedby western blotting. ScBH dilutions are shown on top. PMCA roundnumbers (1–7) are shown on the left. All samples were digestedwith 50 lgÆmL)1PK. Molecular markers are indicated on the right.NADPH can enhances PrPScpropagation S. Shi et al.1538 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBSin all samples from rounds 1–8, without significantalterations in signal intensity (Fig. 3). This indicatesthat PMCA-productNADPHis able to utilize nativePrPCas a substrate to replicate in PMCA in theabsence of NADPH.Enhancement of NADPH for PrPScpropagationin PMCA was dose–response andoxidation/deoxidation relatedTo estimate the influence of the NADPH concentra-tion on promoting propagation of PrPScin vitro, two-fold serially diluted NADPH from 160 to 1.25 lmwas mixed with NBH, and seeded with 1000-folddiluted ScBH of 263K. Preparations were subjectedto PMCA for 24 cycles (24 h) followed by PK treat-ment. Western blot analysis showed that the effi-ciency of PrPScpropagation was enhanced by theaddition of NADPH, which was closely related toincreasing NADPH concentrations (Fig. 4A, comparelane 1 and lanes 2–9). After densitometric quantifica-tion of the PrPScsignals from three independentassays, the relationship between the concentration ofNADPH and PrPScpropagation was analysed. Wefound that PrPScpropagation was graduallyenhanced with increasing NADPH concentration(from 1.25 to 10 lm), and reached a plateau at10 lm NADPH (Fig. 4A, lane 5; Fig. 4B). Calculat-ing the signal intensity of the reactions of 0 and10 lm NADPH revealed that the efficiency of PMCAwas increased by  2.59-fold in the presence ofNADPH. To evaluate whether the effect of NADPHon PrPScpropagation was due to its reduced state,NADPH oxidized by overnight incubation at roomtemperature was serially diluted and amplificationwas performed as above. Interestingly, althoughPrPScsignals could be detected in all preparations,there was no promotion effect of PrPScformation inPMCA compared with the preparation withoutNADPH (Fig. 4C, compare lane 1 and lanes 2–9).This suggests that oxidized NADPH cannot enhancethe propagation of PrPSc, and only its reduced stateis enhances PrPScpropagation in vitro.The reduced, but not oxidized, structuralanalogue of NADPH possessed similar promotingability on PrPScpropagation as NADPHAs a pyridine nucleotide, NADPH is believed to be anelectron donor in some biochemical reactions [24]. ToFig. 3. NADPH-induced PMCA product (PMCA-productNADPH) prop-agated efficiently in further PMCA in the absence of NADPH. Tenmicroliters of PMCA product from the 10)10dilution in the seventhround with NADPH (Fig. 2, lane 4 in the lower gel) was mixed90 lL of hamster NBH without NADPH. PMCA was conductedover 24 cycles per cycle, eight rounds in total, the dilution of theoriginal ScBH reaching 10)18in the final round. The PMCA productsof each round were digested with 50 lgÆmL)1PK and analysed bywestern blotting. NBH without PK treatment was loaded directlyonto the gel as a reference for comparison of electrophoretic mobil-ity (PrPCPK)). PMCA round numbers (0–8) are shown along top.PK+ represented the preparations digested with PK. Molecularmarkers are indicated on the right.Fig. 4. The enhancement of NADPH for PrPScpropagation inPMCA was dose dependent and oxidation ⁄ deoxidation related. (A)Samples seeded with a 1000-fold dilution of ScBH (SHa-263K) weremixed with different concentrations of NADPH (0, 1.25, 2.5, 5, 10,20, 40, 80 and 160 lM). PMCA was conducted over 24 cycles. (B)Quantitative analyses of each gray numerical value of PrPSc. PrPScsignals from each preparation were quantified densitometrically.Relative gray values of the PrPScsignals in each experimental con-dition were normalized by division with that of the respective reac-tion without NADPH [0 lM, lane 1 in (A)]. The average values werecalculated from three independent experiments and presented withas mean ± SD. (C) Oxidized NADPH, rather than fresh NADPH,was added into seeded preparations to undergo the PMCA proce-dure as in Fig. 4A. All samples were treated with 50 lgÆmL)1PK.NADPH concentrations are shown on top of the gels. Molecularmarkers are shown on the right.S. Shi et al. NADPH can enhances PrPScpropagationFEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1539address whether other pyridine nucleotides or an elec-tron donor also help PrPScto propagate in PMCA,some NADPH structural analogues, including NADP,NADH and NAD, were selected and subjected toserial PMCA using 100-fold diluted ScBH of SHa-263K as the seed. In total, six rounds were performed,each consisting of 12 cycles (Fig. 5, lanes 3–8). NADHenhanced PrPScpropagation in the reactions fromrounds 1–5 (fourth gel, lanes 3–7), comparable withNADPH (second gel) which induced PrPScpropaga-tion in all reactions. However, compared with theNBH control (upper gel), both NADP (third gel) andNAD (fifth gel) failed to promote PrPScpropagationin PMCA. In addition, ascorbate, which is alsobelieved to be an electron donor in some biologicalreduction–oxidation reactions, was applied as a func-tional analogue of NADPH in this experiment. Nota-bly, ascorbate did not significantly enhance PrPScpropagation in PMCA (lower gel). These results indi-cate that only reduced pyridine nucleotides, i.e.NADPH and NADH, promote PrPScpropagationin vitro.rSHaPrP could be converted into the PK-resistantisoform by addition of NADPH in PMCATo investigate whether NADPH helps native PrPSctoinduce the conformation change in recombinant PrP,purified recombinant hamster PrP23–231 and humanPrP23–230 (rSHaPrP and rHuPrP) were incubatedwith 10 lm NADPH in PMCA conversion buffer.After mixing with 10)3to 10)6-fold diluted ScBH, thepreparations were subjected to PMCA (24 cycles).Each product was exposed to PK digestion and wes-tern blotting (Fig. 6). Compared with the PK-resistantsignal of preparations treated without NADPH, inwhich only a faint signal at Mr25 kDa appeared inthe 10)3dilution (lane 2, upper gel), more PK-resistantbands were observed in all reactions with NADPH,among them a  17 kDa PK-resistant band was pre-dominant (lanes 6–9, upper gel), which may representthe PK-resistant fragment of rSHaPrP. Unseededassays revealed that rSHaPrP could not be inducedspontaneously by NADPH and alternativesonication ⁄ incubation to PK-resistant forms (lane 10,upper gels), even serial PMCA failed to induce thePK-resistance of rPrP spontaneously (Fig. S2). Thisindicates that rSHaPrP can be used as a substrate forPrPScpropagation in the presence of NADPH usingPMCA.Fig. 5. Influence of other analogues of NADPH and ascorbic acidon PrPScpropagation in PMCA. ScBH of SHa-263K were mixedwith NBH at 1 : 100 dilutions by the addition of 10 lM of NADPH,NADP, NADH, NAD or ascorbate, respectively. NBH control seededwith ScBH was also prepared (upper gel). PMCA was conductedwith 12 cycles per round, for a total of six rounds. Identical aliquotsof each PMCA product of each round were treated with50 lgÆmL)1PK and subjected to western blotting. NBH without PKtreatment was directly loaded into gel as a reference for compari-son of electrophoretic mobility (PrPCPK)). PMCA round numbers(0–6) are shown at the top. PK+ represents the preparationsdigested with PK. Individual chemicals are indicated on the left.Molecular markers are shown on the right.Fig. 6. rSHaPrP, but not rHuPrP, could be converted into aPK-resistant isoform in the presence of NADPH. Aliquots of ScBHof SHa-263K were serially diluted into solutions of rSHaPrP or rHu-PrP, with final ScBH dilutions of 10)3,10)4,10)5and 10)6. Eachpreparation was divided equally into two PCR tubes, one incubatedwith 10 lM NADPH and the other with NaCl ⁄ Pi. PMCA was con-ducted with 24 cycles per round. Identical aliquots of each PMCAproduct were treated with PK and subjected to western blotting.rPrP was the directly loaded recombinant protein without PK diges-tion as a reference for comparison of electrophoretic mobility(lane 1 in all gels). 0 represents the preparation of rPrP withNADPH, but without ScBH (lane 10 in all gels). Arrow indicates thePK-resistant fragment of rPrP. ScBH dilutions are shown at the top.PK+ represents preparations digested with 20 lgÆmL)1PK. Mole-cular markers are indicated on the right.NADPH can enhances PrPScpropagation S. Shi et al.1540 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBSOne of the hallmarks of prions is their species speci-ficity in propagation [24]. Our previous experimentsconfirmed that PrPScfrom scrapie agent 263K-infectedhamsters could not utilize PrPCfrom mice or rabbitsas a substrate to replicate in PMCA (data not shown).To test the possible species barrier when recombinantPrPs were used as the substrate in PMCA, especiallyin the presence of NADPH, rHuPrP was subjected toPMCA using the above protocol. Almost no PK-resis-tant signals were detected any preparation, regardlessof the presence or absence of NADPH (Fig. 6, lowergel). These results correspond well with the pheno-menon that PrPScfrom hamster cannot convert recom-binant mouse PrP into the PK-resistant form [25],showing clear species specificity at the level of recom-binant PrP in PMCA, while NADPH fails to breakthis limitation.DiscussionThe crucial event in prion infection is the conforma-tional change of PrPCinto PrPSc. It has been reportedthat several compounds or chemicals can enhance theconversion of PrPCinto aggregations in vitro [26,27].In this study, we have proposed that in the presence ofreduced pyridine nucleotide chemicals, NADPH andNADH, two scrapie agents propagate more efficientlyin PMCA. This implies that endogenous reduced pyri-dine nucleotide chemicals may play an important rolein the infection process of prions in vivo. Our observa-tions also indicate that reduced pyridine nucleotidechemicals as ubiquitous agents may convert normalPrPCinto PrPScmore easily when PrPCis exposed toexotic PrPSc. Because both native PrPCin brain homo-genates and recombinant PrP can be converted moreefficiently into PK-resistant isoforms by PrPScwith thehelp of NADPH, enhancing the conversion from PrPCto PrPScin PMCA seems to target PrPCmolecules.Our study has also shown that NADPH-enhancedPrPScpropagation in PMCA increases the detectionsensitivity of PrPScby ‡ 102-fold and does not lead toany false positives. This suggests the potential use ofthis chemical as an accelerant in PMCA to detect tracelevels of prions in biosamples.Reduced pyridine nucleotides have been shown to beinvolved in many reduction–oxidation reactions.NADPH and NADP+can bind the active site ofglucose phosphate dehydrogenase competitively toregulate the speed of the pentose phosphate pathway[28] and NADPH also participates in the synthesis ofcholesterol [29]. Recent studies have revealed thatNADPH-diaphorase, which is known to catalyseNADPH to transfer electrons to their targets, is associ-ated with neuronal death [30]. Increasing hippocampalNADPH-diaphorase has been observed at early stagesin the murine model of scrapie agent ME7, however,the level of NADPH-diaphorse decreases in the latestages of TSEs in animal models [17]. Interestingly, wealso find that the presence of NADPH enhancesde novo PrPScpropagation in PMCA. PMCA-derivedPrPScmaintains a stable propagating capacity in nor-mal brain homogenates after NADPH is removed.Because the increased level of NADPH-diaphorasemay correlate with the active synthesis of NADPH,one may think that in the early stage of TSE moreNADPH in brain tissue helps the trace levels of exoticPrPScreplicate more efficiently. Although NADPHsynthesis may decrease along with the loss of neuronsand spongiform degeneration during the pathogenesisof TSE, PrPScmaintains its replication.Our data showed that oxidized NADPH will lose itsenhancing activity for PrPScpropagation in PMCA,indicating that the role as an electron donor mightcontribute to this enhancement. This enhancement isalso observed in other reduced structural analogues,such as NADH. Neither oxidized structural analogue,such as NADP and NAD, nor ascorbate, which merelyworks as an electron donor, possess this activity.Therefore, it outlines that this enhancement dependson both electron transfer and structural similarity.Nevertheless, the presence of oxidized pyridine nucleo-tides does not influence the efficacy of PrPScpropaga-tion compared with the reaction without pyridinenucleotides. It is clear that pyridine nucleotides are notabsolutely necessary during prion propagation.Because a large amount of NADPH is oxidized duringthe long incubation time in PMCA, it also will beinteresting to know whether there is a competitive rela-tionship between reduced pyridine nucleotide and itsoxidized counterpart.Our study illustrates that in the presence ofNADPH, hamster rPrP is successfully converted tothe PK-resistant form when seeded with native PrPScfrom experimental scrapie hamsters. Similarly, manyother chemicals or compounds may function asco-factors to facilitate the propagation of PrPScin vitro, including sulfated glycan, RNA molecules,DNA molecules and chaperons [6]. More strikingly, ithas been proposed that polyanions, especially poly(A)RNA, can induce normal hamster PrPCto convertinto infectious agents directly without any prion inPMCA, which may mimic the process of Creutzfeldt–Jakob disease [31]. NADPH has the ability to bindwith recombinant PrP and induce PrP aggregationwhen the rPrP are refolded by some ions, such asCu2+,Zn2+and Mn2+[32,33]. We cannot excludeS. Shi et al. NADPH can enhances PrPScpropagationFEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1541the possibility that the NADPH-induced formation ofPK-resistant rPrP in PMCA is due to interaction withother unknown cellular components of the ScBH.This implies that reduced pyridine nucleotides mainlyact on the PrP molecules directly, because farthing ofScBH (10)6dilution) used in PMCA is able to pro-duce large quantity of PK-resistant rPrP. Therefore,we speculate that certain binding domains of pyridinenucleotides may exist within the PrP molecule.Through transferring electrons, the energy class ofPrP may change, leading to an unstable status.Another aspect may be the potential metal-catalysedoxidation of PrP. Reduced Cu+from Cu2+byNADPH can react with H2O2and produce.OHin vivo, which contributes to the structural alternationseen in a variety of diseases [34]. NADPH may causecopper-bound PrP to be a transient reduced state,which might result in uncertain structural changes inPrPCor rPrP, leading to the protein being more easilyconverted to PrPSc. The level of extracellular NADPHor NADH has not been determined [35], so it isunclear whether PrPCon the cell surface can beinfluenced by this chemical. However, PrPCis notexclusively a cell-surface protein. Detectable cytoplasmPrP in neurons [36] leads us to presume that theabundant NADPH in cytoplasm [37] may affect PrPCmolecules during the protein trafficking in endo-plasmic reticulum or Golgi. These processes mayresult the PrPCmisfolds to PrPScspontaneously.Materials and methodsPreparation of brain homogenatesFrozen scrapie agents 263K- and 139A-infected brains [38]were homogenized in PMCA conversion buffer, containing1· NaCl ⁄ Pi, 1% Triton X-100, 5 mm EDTA, 150 mm NaCland protease inhibitor cocktail tablets (Roche Applied Sci-ence, Basel, Switzerland) as described elsewhere [23]. Crudehomogenates were centrifuged for 30 s at 5000 g, aliquotsof the supernatant (10% scrapie-infected brain homogenate,indicated as ScBH) were serially diluted with PMCA con-version buffer by 10-fold dilution to reach a 10)12dilutionas the stock seeds. These homogenates were used immedi-ately in subsequent experiments.Whole normal brains were removed surgically frompurchased 5-week-old male hamsters. Brains were washedthoroughly in cold NaCl ⁄ Picontaining 50 m m EDTA toremove as much blood as possible. After homogenizationin PMCA conversion buffer, 10% (w ⁄ v) NBH werecentrifuged for 30 s at 5000 g, and aliquots of the super-natant were immediately frozen at )80 °C for subsequentexperiments. All processes of experiments, includinganaesthetic and surgical procedures, as well as animal man-agement, have been reviewed and approved, and wereperformed in accordance with the relevant China nationallegislations and regulations.Recombinant protein expression and purificationThe recombinant plasmid pQE-haPrP 23–231 expressinghamster PrP residues 23–231 and pQE-huPrP 23–230expressing human PrP residues 23–230 were generated asdescribed previously [39]. The expression and purificationof the recombinant His-tagged PrP proteins (rPrP) wereperformed by using Ni-NTA affinity chromatography (Qia-gen, Hilden, Germany) as described previously [40].Refolding of the recombinant PrPAfter filtering with a 0.22 lm filter membrane (Millipore,Bedford, MA, USA), the concentration of the purified pro-tein solution was estimated by measuring A280. Each recom-binant PrP was refolded by a 10-fold molar ratio of Cu2+oxidation of the disulfide bond at room temperature for3 h. Thereafter, the protein solution was dialysed into a100-fold volume of NaCl ⁄ Pi, pH 6.0, containing 10 mmEDTA for 4 h. The protein solution was transferred intofresh dialysis buffer (NaCl ⁄ Pi, pH 6.0) without EDTA andthis procedure was repeated five times as described else-where [25,41]. After filtering the pooled fractions, the con-centration of oxidized rPrP was determined by measuringA280. Estimates of rPrP purity were made by SDS ⁄ PAGEand Coomassie Brilliant Blue staining. All final proteinpreparations were diluted to 0.2 mgÆmL)1in PMCA con-version buffer and frozen at )80 °C. Solutions were kept at4 °C once thawed.In vitro amplification procedurePMCA was performed on a water-bath sonicator (Misonixsonicator 3000; Misonix, Farmingdale, NY, USA), whichhad a microplate horn for PCR tubes. Ninety microliters ofNBH or 0.2 mgÆmL)1rPrP were mixed with 10 lL of vari-ous concentrations of ScBH in a volume of 100 lLin0.2 mL PCR tubes. The tubes were positioned on the soni-cator and amplification cycles were programmed asdescribed elsewhere [20,21]. One PMCA cycle consisted ofsonication at 60% potency ( 140 W) for 40 s and fol-lowed by incubation at 37 °C for 59 min 20 s. In this study,one round of PMCA contained 12 or 24 cycles. After eachround, 10 lL of amplified product was added to 90 lLoffresh normal homogenates (10-fold dilution), or 1 lLofproduct was added to 99 lL of fresh normal homogenates(100-fold dilution). New mixtures were subjected to anotherround. This was repeated several times to reach an idealamplification.NADPH can enhances PrPScpropagation S. Shi et al.1542 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBSTreatment of chemicalsChemicals, including NADPH, NADP, NADH, NAD andascorbate, were purchased from Sigma-Aldrich (St Louis,MO, USA). (The structures of the chemicals are shown inTable S1.) All chemicals were freshly prepared as 10·stock solutions in NaCl ⁄ Pibefore each experiment. Vari-ous concentrations of chemicals were added to NBH orrPrP solutions and gently shaken at room temperature for10 min, followed by mixing with ScBH and subjecting toPMCA.PK digestion and western blotting assayPMCA products were digested with PK at 37 °C for 1 h.The concentration of PK was 50 lgÆ mL)1for brain homo-genates or 20 lgÆmL)1for recombinant PrP solutions.Reactions were stopped by the addition of an equal vol-ume of 2· SDS loading buffer and boiled for 10 min.Samples were separated in 0.75 mm, 15% SDS ⁄ PAGE andelectronically transferred to poly(vinylidene diflouride)membranes (Immobilon-P; Millipore) at 10 V for 1 h. Forimmunoblotting, the membrane was blocked with 5% non-fat milk in NaCl ⁄ Pi-T and incubated with 1 : 5000 dilutedPrP mAb 3F4 (Dako, Glostrup, Denmark) in 0.5% nonfatmilk in NaCl ⁄ Pi-T at room temperature for 2 h. Afterwashing three times in NaCl ⁄ Pi-T, the membrane wasimmersed in a 1 : 5000 diluted horseradish peroxidase con-junct anti-mouse IgG (Boehringer, Ingelheim, Germany) inNaCl ⁄ Pi-T and incubated at room temperature for 2 h.Signal detection was performed with an ECL detection kit(GE Healthcare Bio-Sciences, Piscataway, NJ, USA).Immunoblot images were scanned and quantified by densi-tometry using a Gel-Pro analyser (Binta 2020D; Binta,Beijing, China).PrPScquantitationTo estimate the concentration and number of PrPScmole-cules, serial dilutions of scrapie-infected brain homogenatewere analysed by western blots in the same gel as aliquotsof known concentrations of recombinant PrP (Fig. S3A)according to the admitted protocol [18,23]. Signal intensitieswere evaluated by densitometry, and the concentration ofPK-treated PrPScin these samples was calculated by extrap-olation of the calibration curve calculated with recombinantPrP (Fig. S3B). To minimize errors due to saturated orweak signals, several dilutions were analysed and the experi-ment was repeated three times for each dilution. In thisway, the average concentration of PrPScin scrapie-infectedbrain homogenate in this study was measured to be 7.265 ngÆlL)1. The number of molecules of PrPScwas cal-culated by a mathematical method, i.e. for 1 lL of ScBH,no. of molecules = 7.265 · 10)9⁄ monomeric PrP molecularmass 35 000 · Avogadro’s number = 1.25 · 1011.AcknowledgementsThis work was supported by National Science andTechnology Task Force Project (2006BAD06A13-2)National Basic Research Program of China (973 Pro-gram) (2007CB310505), Institution Technique R&DGrand (2008EG150300) and Chinese National NaturalScience Foundation Grants 30571672, 30500018,30771914 and 30800975.References1 Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95,13363–13383.2 Collinge J (2001) Prion diseases of humans and animals:their causes and molecular basis. 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J Neurosci 27, 852–859.37 Wise DD & Shear JB (2006) Quantitation of nicotin-amide and serotonin derivatives and detection of flavinsin neuronal extracts using capillary electrophoresis withmultiphoton-excited fluorescence. J Chromatogr A 1111,153–158.38 Gao JM, Gao C, Han J, Zhou XB, Xiao XL, Zhang J,Chen L, Zhang BY, Hong T & Dong XP (2004)Dynamic analyses of PrP and PrPScin brain tissues ofgolden hamsters infected with scrapie strain 263Krevealed various PrP forms. Biomed Environ Sci17, 8–20.39 Dong CF, Shi S, Wang XF, An R, Li P, Chen JM,Wang X, Wang GR, Shan B, Zhang BY et al. (2008)The N-terminus of PrP is responsible for interactingNADPH can enhances PrPScpropagation S. Shi et al.1544 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBSwith tubulin and fCJD related PrP mutants possessstronger inhibitive effect on microtubule assemblyin vitro. Arch Biochem Biophys 470, 83–92.40 Zhang FP, Zhang J, Zhou W, Zhang BY, Hung T &Dong XP (2002) Expression of PrP(C) as HIS-fusionform in a baculovirus system and conversion ofexpressed PrP-sen to PrP-res in a cell-free system. VirusRes 87, 145–153.41 Jackson GS, Hill AF, Joseph C, Hosszu L, Power A,Waltho JP, Clarke AR & Collinge J (1999) Multiplefolding pathways for heterologously expressed humanprion protein. Biochim Biophys Acta 1431, 1–13.Supporting informationThe following supplementary material is available:Fig. S1. (A) Evaluation the possible influence ofNADPH on PrPScwithout sonication. (B) Evaluationof the possible influence of NADPH on NBH duringPMCA.Fig. S2. Evaluation of the possible influence ofNADPH on rSHaPrP during PMCA.Fig. S3. Quantification of PrPScin scrapie-infectedbrain homogenate.Table S1. Structure of chemicals.This supplementary material can be found in theonline version of this article.Please note: Wiley-Blackwell is not responsible forthe content or functionality of any supplementarymaterials supplied by the authors. Any queries (otherthan missing material) should be directed to the corre-sponding author for the article.S. Shi et al. NADPH can enhances PrPScpropagationFEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1545 . The propagation of hamster-adapted scrapie PrPSc can be enhanced by reduced pyridine nucleotide in vitro Song Shi, Chen-Fang Dong, Chan Tian, Rui-Min. be involved in converting PrPCinto the abnormal isomer in vitro [11–14]. Moreover, pyridine nucleotides, a group of coenzymes ubiquitous in bio-synthesis
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