The propagation of hamster-adapted scrapie PrP Sc can be enhanced by reduced pyridine nucleotide in vitro Song Shi, Chen-Fang Dong, Chan Tian, Rui-Min Zhou, Kun Xu, Bao-Yun Zhang, Chen Gao, Jun Han and Xiao-Ping Dong State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Beijing, China Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are lethal neurodegener- ative diseases, including Creutzfeldt–Jakob disease, Gerstmann–Straussler–Scheinker syndrome and fatal familial insomnia in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep and goats, and chronic wasting disease in deer and elk [1,2]. TSEs are caused by a proteinaceous infectious agent, termed a prion, which is considered to consist of a misfolded and aggregated protease-resistant isomer of a host- encoded glycoprotein (PrP C ) [3,4]. The pathological isomer present in the tissues of infected individuals is called PrP Sc . Although the clinical and pathological characteristics of TSEs have been recognized for a long time, the mechanisms underlying prion conversion are only partially settled. Recently, some endogenous factors encoded by prion hosts have been proposed as essential during the propagation of prions in studies in animal models and cell-culture systems, i.e. protein X and some chaperons [5–10]. Polyanions and sulfated glycans are also thought to be involved in converting PrP C into the abnormal isomer in vitro [11–14]. Moreover, pyridine nucleotides, a group of coenzymes ubiquitous in bio- synthesis and metabolism, are associated with the aggregation of recombinant prion protein (rPrP). Following incubation with reduced pyridine nucleo- tides, e.g. NADPH, rPrP can accumulate in fibrils and acquire weak proteinase resistance [15,16]. Several studies have shown that the amount of NADPH- diaphorase increases during the early stage of prion disease [17], but there is no direct molecular evidence Keywords PMCA; prion; propagation; pyridine nucleotide; transmissible spongiform encephalopathies Correspondence X P. Dong, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Ying-Xin Rd 100, Beijing 100052, China Fax: +86 10 63532053 Tel: +86 10 83534616 E-mail: dongxp238@sina.com (Received 16 September 2008, revised 15 November 2008, accepted 22 December 2008) doi:10.1111/j.1742-4658.2009.06871.x Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders caused by an infectious agent termed a prion, which can convert normal cellular prion protein (PrP C ) into a patho- logically misfolded isoform (PrP Sc ). Taking advantage of protein misfolding cyclic amplification (PMCA), a series of experiments was conducted to investigate the possible influences of pyridine nucleotides on the propaga- tion activities of hamster-adapted scrapie agents 263K and 139A in vitro using normal hamster brain homogenates and recombinant hamster PrP as the substrates. The results showed that PrP Sc from both scrapie agent 263K- and 139A-infected brains propagated more efficiently in PMCA with the addition of reduced NADPH, showing an obvious dose-dependent enhancement. Reduced NADH also prompted PrP Sc propagation, whereas NADP, NAD and vitamin C failed. Moreover, following incubation with NADPH, recombinant hamster PrP could be efficiently converted into the proteinase K-resistant form when exposed to the trace of PrP Sc from infected hamsters. Our data provide evidence that the reduced pyridine nucleotide plays an important role in the propagation of prion and this process seems to target PrP C molecules. Abbreviations NBH, 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 FEBS to clarify the mechanism by which reduced pyridine nucleotides affect PrP molecules. A novel technology, named protein misfolding cyclic amplification (PMCA) has been described in recent years [18], providing an efficacious, unique and conve- nient experimental approach to evaluating the replica- tion and infectivity of newly formed PrP Sc in vitro [19]. Following incubation of PrP Sc from TSE-infected ani- mals with homologous PrP C from normal brains, large quantities of PrP Sc can be achieved after a short cycle of alternate sonication and incubation [20,21]. This tech- nology is considered the most appropriate approach to understanding the fundamental mechanisms involved in PrP Sc propagation, aggregation and neuroinvasion in vivo [22]. In this study, possible influences of pyridine nucleotides on the propagation of PrP Sc from scrapie- infected hamsters in PMCA were investigated. We found that reduced pyridine nucleotide NADPH pro- moted PrP Sc -propagating activity in PMCA using PrP C from normal hamster brains as the substrate, and that this was a dose-dependent response. Other reduced ana- logues of pyridine nucleotides also showed enhanced capacity, whereas an oxidized analogue failed. Further- more, we proposed that after incubation with NADPH, recombinant hamster PrP could be converted to the proteinase K (PK)-resistant isoform in the presence of scrapie PrP Sc in PMCA. Results The propagating activities of PrP Sc in PMCA are remarkably enhanced in the presence of NADPH To see the influence of NADPH on the ability of PrP Sc to propagate in PMCA, a serial PMCA protocol con- sisting of seven rounds was conduced (Fig. 1A). Prior to mixing with a 10 )2 dilution of ScBH prepared from scrapie 263K-infected hamsters (SHa-263K), hamster NBH was mixed with 10 lm NADPH (referred as NBH + NADPH, lanes 3–9) and subjected to 12 PMCA cycles. Thereafter, 10 lL of PMCA product was added to 90 lL of fresh NBH with 10 lm NADPH and the next round was performed. This was repeated several times, leading to the original ScBH being diluted to 10 )8 . Meanwhile, mixture containing only NADPH and ScBH in the conversion buffer, but without NBH (referred as NADPH control, lanes 3–9), or one containing NBH and ScBH but without NADPH (referred as NBH control, lanes 3–9), was prepared as above. Each PMCA product was digested with PK and subjected to western blotting. PK-resis- tant PrP signal was detected only in the first NADPH control preparation (upper gel), representing the initial input PrP Sc (Fig. 1A, lane 2 in all gels). In the NBH control, the PrP Sc signal was strongest during round 1, weakened gradually and vanished from round 4 (mid- dle gel), indicating lower PMCA-propagating activity under these conditions. By contrast, in the presence of NADPH, obvious PrP Sc signals were seen in all prepa- rations from round 1 to round 7 (Fig. 1A, lower gel), highlighting a more actively propagating capacity for PrP Sc . This suggests that NADPH can prompt the propagation of scrapie agent 263K. In addition, no sig- nificant propagation of PrP Sc was observed in seeded preparations without sonication (Fig. S1A) or in PMCA samples without seeding ScBH (Fig. S1B), Fig. 1. NADPH induced more efficient propagation of PrP Sc from scrapie agents in PMCA. Ten microliters of a 100-fold dilution of ScBH of SHa-263K or SHa-139A were used as the seed in each prep- aration. PMCA was conducted over 12 cycles per round, with seven rounds in total. (A) Propagation of PrP Sc from hamster-adapted scra- pie agent 263K in PMCA. The NADPH control (upper gel) contained 10 l M NADPH in conversion buffer and ScBH, but without NBH. The NBH control (middle gel) contained NBH and ScBH, but without NADPH. NBH plus NADPH (lower gel) contained NBH, ScBH and NADPH. (B) Propagations seeded with hamster-adapted scrapie agent 139A underwent the same PMCA procedure containing NBH alone (upper gel) or NBH plus NADPH (lower gel). In all gels, NBH was loaded directly in the first lane without proteolysis as a refer- ence for comparison of electrophoretic mobility (PrP C PK)). All other samples were treated with 50 lgÆmL )1 PK (PK+). 0 indicates reac- tions containing ScBH before PMCA process. Round numbers are given on top. The molecular markers are indicated on the right. S. Shi et al. NADPH can enhances PrP Sc propagation FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1537 implying that NADPH can neither enhance PrP Sc propagation without PMCA nor induce spontaneous conversion of PrP C into PrP Sc with PMCA in vitro. To test whether NADPH-related enhancement was also appropriate to another scrapie agent, hamster- adapted scrapie strain 139A (SHa-139A) was used in PMCA following the same procedure. In order to avoid possible environmental contamination, all mate- rials and reagents were freshly prepared, including the homogenizer, chemicals and conversion buffer. NBH and ScBH (SHa-139A) were prepared in another labo- ratory that had never been exposed to prions. Like SHa-263K, PK-resistant signals were detected in reac- tions from round 1 to round 7 in the presence of NADPH (Fig. 1B, lower), but only in the first three rounds without NADPH (Fig. 1B, upper). These results indicate that propagation of SHa-139A in vitro can also be prompted by NADPH, and therefore this enhancement of PrP Sc propagation in PMCA is not limited to only one prion strain. NADPH increased the sensitivity for detection of PrP Sc in brain homogenates by PMCA To test the potential of using NADPH as an assistant chemical in increasing the detection sensitivity of PMCA, comparative analysis of serial PMCA, with or without NADPH, was conducted using serially diluted ScBH from agent 263K as the seed. Dilutions of the original ScBH ranged from 10 )4 to 10 )12 by 100-fold serial dilution. One round of PMCA consisted of 24 cycles (24 h), which was considered to be more effi- cient at increasing PrP Sc aggregation [19] and allowed low levels of PrP Sc to be detected [23]. After one round of PMCA, 10 lL of product was mixed with 90 lLof fresh NBH for the next round, up to seven rounds. Figure 2 shows that PrP Sc signals were detected at dilutions of 10 )6 and 10 )8 in the second round in the presence of 10 lm NADPH (second gel, lanes 2 and 3), whereas PrP Sc signals could be observed at a dilu- tion of 10 )6 in the third round (third gel, lane 7) and 10 )8 in the fifth round (fifth gel, lane 8) in the absence of NADPH. After seven rounds of PMCA, clear PrP Sc signals were observed at ScBH dilutions of 10 )4 to 10 )10 , and even at a 10 )12 -fold dilution a weak band was observed in the presence of NADPH (lower gel, lane 5). However, no PrP Sc signal was detected at dilutions of 10 )10 and 10 )12 without NADPH (lower gel, lanes 9 and 10). This indicates that NADPH can increase, by at least 10 2 -fold, the sensitivity for the detection of PrP Sc in brain homogenates under these experimental conditions. At the concentration of PrP Sc in ScBH used in this study (see Materials and methods), a 1 · 10 )10 dilution of ScBH contained  7.265 · 10 )19 gÆlL )1 PrP Sc , or 12.5 molecules of ini- tial PrP Sc per lL. Thereby, one can speculate that  12.5 monomeric PrP Sc molecules can successfully induce detectable amplification in a seven-round PMCA with NADPH, whereas in the absence of NADPH at least 1250 molecules of PrP Sc are required for the same amplification. PrP Sc generated from PMCA in the presence of NADPH had further propagation ability To determine whether newly generated PMCA PrP Sc by addition of NADPH could further propagate in the PMCA system in the absence of NADPH, 10 lLof PMCA product (PMCA-product NADPH ) from a 10 )10 dilution in the seventh round (Fig. 2, lane 4) was diluted in 90 lL of NBH and subjected to one round of PMCA (24 cycles) without NADPH (i.e. eight rounds in total). Obvious PrP Sc signals were detected Fig. 2. Comparative evaluation for serial PMCA detecting the sensi- tivity of PrP Sc in 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 )10 and 10 )12 . Each dilution was divided equally into two PCR tubes, one mixed with 10 l M NADPH and the other added to an equal volume of NaCl ⁄ P i . After 24 cycles PMCA as the first round, 10 lL of product was mixed with 90 lL of fresh NBH for the next round of PMCA. The five left-hand lanes are fresh NBH plus NADPH (NADPH+) and the five right-hand lanes are without NADPH (NADPH-). After seven rounds, the digested PMCA products of each round were analysed by western blotting. ScBH dilutions are shown on top. PMCA round numbers (1–7) are shown on the left. All samples were digested with 50 lgÆmL )1 PK. Molecular markers are indicated on the right. NADPH can enhances PrP Sc propagation S. Shi et al. 1538 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS in all samples from rounds 1–8, without significant alterations in signal intensity (Fig. 3). This indicates that PMCA-product NADPH is able to utilize native PrP C as a substrate to replicate in PMCA in the absence of NADPH. Enhancement of NADPH for PrP Sc propagation in PMCA was dose–response and oxidation/deoxidation related To estimate the influence of the NADPH concentra- tion on promoting propagation of PrP Sc in vitro, two- fold serially diluted NADPH from 160 to 1.25 lm was mixed with NBH, and seeded with 1000-fold diluted ScBH of 263K. Preparations were subjected to PMCA for 24 cycles (24 h) followed by PK treat- ment. Western blot analysis showed that the effi- ciency of PrP Sc propagation was enhanced by the addition of NADPH, which was closely related to increasing NADPH concentrations (Fig. 4A, compare lane 1 and lanes 2–9). After densitometric quantifica- tion of the PrP Sc signals from three independent assays, the relationship between the concentration of NADPH and PrP Sc propagation was analysed. We found that PrP Sc propagation was gradually enhanced with increasing NADPH concentration (from 1.25 to 10 lm), and reached a plateau at 10 lm NADPH (Fig. 4A, lane 5; Fig. 4B). Calculat- ing the signal intensity of the reactions of 0 and 10 lm NADPH revealed that the efficiency of PMCA was increased by  2.59-fold in the presence of NADPH. To evaluate whether the effect of NADPH on PrP Sc propagation was due to its reduced state, NADPH oxidized by overnight incubation at room temperature was serially diluted and amplification was performed as above. Interestingly, although PrP Sc signals could be detected in all preparations, there was no promotion effect of PrP Sc formation in PMCA compared with the preparation without NADPH (Fig. 4C, compare lane 1 and lanes 2–9). This suggests that oxidized NADPH cannot enhance the propagation of PrP Sc , and only its reduced state is enhances PrP Sc propagation in vitro. The reduced, but not oxidized, structural analogue of NADPH possessed similar promoting ability on PrP Sc propagation as NADPH As a pyridine nucleotide, NADPH is believed to be an electron donor in some biochemical reactions [24]. To Fig. 3. NADPH-induced PMCA product (PMCA-product NADPH ) prop- agated efficiently in further PMCA in the absence of NADPH. Ten microliters of PMCA product from the 10 )10 dilution in the seventh round with NADPH (Fig. 2, lane 4 in the lower gel) was mixed 90 lL of hamster NBH without NADPH. PMCA was conducted over 24 cycles per cycle, eight rounds in total, the dilution of the original ScBH reaching 10 )18 in the final round. The PMCA products of each round were digested with 50 lgÆmL )1 PK and analysed by western blotting. NBH without PK treatment was loaded directly onto the gel as a reference for comparison of electrophoretic mobil- ity (PrP C PK)). PMCA round numbers (0–8) are shown along top. PK+ represented the preparations digested with PK. Molecular markers are indicated on the right. Fig. 4. The enhancement of NADPH for PrP Sc propagation in PMCA was dose dependent and oxidation ⁄ deoxidation related. (A) Samples seeded with a 1000-fold dilution of ScBH (SHa-263K) were mixed with different concentrations of NADPH (0, 1.25, 2.5, 5, 10, 20, 40, 80 and 160 l M). PMCA was conducted over 24 cycles. (B) Quantitative analyses of each gray numerical value of PrP Sc . PrP Sc signals from each preparation were quantified densitometrically. Relative gray values of the PrP Sc signals in each experimental con- dition were normalized by division with that of the respective reac- tion without NADPH [0 l M, lane 1 in (A)]. The average values were calculated from three independent experiments and presented with as 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 )1 PK. NADPH concentrations are shown on top of the gels. Molecular markers are shown on the right. S. Shi et al. NADPH can enhances PrP Sc propagation FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1539 address whether other pyridine nucleotides or an elec- tron donor also help PrP Sc to propagate in PMCA, some NADPH structural analogues, including NADP, NADH and NAD, were selected and subjected to serial 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). NADH enhanced PrP Sc propagation in the reactions from rounds 1–5 (fourth gel, lanes 3–7), comparable with NADPH (second gel) which induced PrP Sc propaga- tion in all reactions. However, compared with the NBH control (upper gel), both NADP (third gel) and NAD (fifth gel) failed to promote PrP Sc propagation in PMCA. In addition, ascorbate, which is also believed to be an electron donor in some biological reduction–oxidation reactions, was applied as a func- tional analogue of NADPH in this experiment. Nota- bly, ascorbate did not significantly enhance PrP Sc propagation in PMCA (lower gel). These results indi- cate that only reduced pyridine nucleotides, i.e. NADPH and NADH, promote PrP Sc propagation in vitro. rSHaPrP could be converted into the PK-resistant isoform by addition of NADPH in PMCA To investigate whether NADPH helps native PrP Sc to induce the conformation change in recombinant PrP, purified recombinant hamster PrP23–231 and human PrP23–230 (rSHaPrP and rHuPrP) were incubated with 10 lm NADPH in PMCA conversion buffer. After mixing with 10 )3 to 10 )6 -fold diluted ScBH, the preparations were subjected to PMCA (24 cycles). Each product was exposed to PK digestion and wes- tern blotting (Fig. 6). Compared with the PK-resistant signal of preparations treated without NADPH, in which only a faint signal at M r 25 kDa appeared in the 10 )3 dilution (lane 2, upper gel), more PK-resistant bands 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 represent the PK-resistant fragment of rSHaPrP. Unseeded assays revealed that rSHaPrP could not be induced spontaneously by NADPH and alternative sonication ⁄ incubation to PK-resistant forms (lane 10, upper gels), even serial PMCA failed to induce the PK-resistance of rPrP spontaneously (Fig. S2). This indicates that rSHaPrP can be used as a substrate for PrP Sc propagation in the presence of NADPH using PMCA. Fig. 5. Influence of other analogues of NADPH and ascorbic acid on PrP Sc propagation in PMCA. ScBH of SHa-263K were mixed with NBH at 1 : 100 dilutions by the addition of 10 l M of NADPH, NADP, NADH, NAD or ascorbate, respectively. NBH control seeded with ScBH was also prepared (upper gel). PMCA was conducted with 12 cycles per round, for a total of six rounds. Identical aliquots of each PMCA product of each round were treated with 50 lgÆmL )1 PK and subjected to western blotting. NBH without PK treatment was directly loaded into gel as a reference for compari- son of electrophoretic mobility (PrP C PK)). PMCA round numbers (0–6) are shown at the top. PK+ represents the preparations digested 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 a PK-resistant isoform in the presence of NADPH. Aliquots of ScBH of SHa-263K were serially diluted into solutions of rSHaPrP or rHu- PrP, with final ScBH dilutions of 10 )3 ,10 )4 ,10 )5 and 10 )6 . Each preparation was divided equally into two PCR tubes, one incubated with 10 l M NADPH and the other with NaCl ⁄ P i . PMCA was con- ducted with 24 cycles per round. Identical aliquots of each PMCA product 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 with NADPH, but without ScBH (lane 10 in all gels). Arrow indicates the PK-resistant fragment of rPrP. ScBH dilutions are shown at the top. PK+ represents preparations digested with 20 lgÆmL )1 PK. Mole- cular markers are indicated on the right. NADPH can enhances PrP Sc propagation S. Shi et al. 1540 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS One of the hallmarks of prions is their species speci- ficity in propagation [24]. Our previous experiments confirmed that PrP Sc from scrapie agent 263K-infected hamsters could not utilize PrP C from mice or rabbits as a substrate to replicate in PMCA (data not shown). To test the possible species barrier when recombinant PrPs were used as the substrate in PMCA, especially in the presence of NADPH, rHuPrP was subjected to PMCA using the above protocol. Almost no PK-resis- tant signals were detected any preparation, regardless of the presence or absence of NADPH (Fig. 6, lower gel). These results correspond well with the pheno- menon that PrP Sc from 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 break this limitation. Discussion The crucial event in prion infection is the conforma- tional change of PrP C into PrP Sc . It has been reported that several compounds or chemicals can enhance the conversion of PrP C into aggregations in vitro [26,27]. In this study, we have proposed that in the presence of reduced pyridine nucleotide chemicals, NADPH and NADH, two scrapie agents propagate more efficiently in PMCA. This implies that endogenous reduced pyri- dine nucleotide chemicals may play an important role in the infection process of prions in vivo. Our observa- tions also indicate that reduced pyridine nucleotide chemicals as ubiquitous agents may convert normal PrP C into PrP Sc more easily when PrP C is exposed to exotic PrP Sc . Because both native PrP C in brain homo- genates and recombinant PrP can be converted more efficiently into PK-resistant isoforms by PrP Sc with the help of NADPH, enhancing the conversion from PrP C to PrP Sc in PMCA seems to target PrP C molecules. Our study has also shown that NADPH-enhanced PrP Sc propagation in PMCA increases the detection sensitivity of PrP Sc by ‡ 10 2 -fold and does not lead to any false positives. This suggests the potential use of this chemical as an accelerant in PMCA to detect trace levels of prions in biosamples. Reduced pyridine nucleotides have been shown to be involved in many reduction–oxidation reactions. NADPH and NADP + can bind the active site of glucose phosphate dehydrogenase competitively to regulate the speed of the pentose phosphate pathway [28] and NADPH also participates in the synthesis of cholesterol [29]. Recent studies have revealed that NADPH-diaphorase, which is known to catalyse NADPH to transfer electrons to their targets, is associ- ated with neuronal death [30]. Increasing hippocampal NADPH-diaphorase has been observed at early stages in the murine model of scrapie agent ME7, however, the level of NADPH-diaphorse decreases in the late stages of TSEs in animal models [17]. Interestingly, we also find that the presence of NADPH enhances de novo PrP Sc propagation in PMCA. PMCA-derived PrP Sc maintains a stable propagating capacity in nor- mal brain homogenates after NADPH is removed. Because the increased level of NADPH-diaphorase may correlate with the active synthesis of NADPH, one may think that in the early stage of TSE more NADPH in brain tissue helps the trace levels of exotic PrP Sc replicate more efficiently. Although NADPH synthesis may decrease along with the loss of neurons and spongiform degeneration during the pathogenesis of TSE, PrP Sc maintains its replication. Our data showed that oxidized NADPH will lose its enhancing activity for PrP Sc propagation in PMCA, indicating that the role as an electron donor might contribute to this enhancement. This enhancement is also observed in other reduced structural analogues, such as NADH. Neither oxidized structural analogue, such as NADP and NAD, nor ascorbate, which merely works as an electron donor, possess this activity. Therefore, it outlines that this enhancement depends on both electron transfer and structural similarity. Nevertheless, the presence of oxidized pyridine nucleo- tides does not influence the efficacy of PrP Sc propaga- tion compared with the reaction without pyridine nucleotides. It is clear that pyridine nucleotides are not absolutely necessary during prion propagation. Because a large amount of NADPH is oxidized during the long incubation time in PMCA, it also will be interesting to know whether there is a competitive rela- tionship between reduced pyridine nucleotide and its oxidized counterpart. Our study illustrates that in the presence of NADPH, hamster rPrP is successfully converted to the PK-resistant form when seeded with native PrP Sc from experimental scrapie hamsters. Similarly, many other chemicals or compounds may function as co-factors to facilitate the propagation of PrP Sc in vitro, including sulfated glycan, RNA molecules, DNA molecules and chaperons [6]. More strikingly, it has been proposed that polyanions, especially poly(A) RNA, can induce normal hamster PrP C to convert into infectious agents directly without any prion in PMCA, which may mimic the process of Creutzfeldt– Jakob disease [31]. NADPH has the ability to bind with recombinant PrP and induce PrP aggregation when the rPrP are refolded by some ions, such as Cu 2+ ,Zn 2+ and Mn 2+ [32,33]. We cannot exclude S. Shi et al. NADPH can enhances PrP Sc propagation FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1541 the possibility that the NADPH-induced formation of PK-resistant rPrP in PMCA is due to interaction with other unknown cellular components of the ScBH. This implies that reduced pyridine nucleotides mainly act on the PrP molecules directly, because farthing of ScBH (10 )6 dilution) used in PMCA is able to pro- duce large quantity of PK-resistant rPrP. Therefore, we speculate that certain binding domains of pyridine nucleotides may exist within the PrP molecule. Through transferring electrons, the energy class of PrP may change, leading to an unstable status. Another aspect may be the potential metal-catalysed oxidation of PrP. Reduced Cu + from Cu 2+ by NADPH can react with H 2 O 2 and produce . OH in vivo, which contributes to the structural alternation seen in a variety of diseases [34]. NADPH may cause copper-bound PrP to be a transient reduced state, which might result in uncertain structural changes in PrP C or rPrP, leading to the protein being more easily converted to PrP Sc . The level of extracellular NADPH or NADH has not been determined [35], so it is unclear whether PrP C on the cell surface can be influenced by this chemical. However, PrP C is not exclusively a cell-surface protein. Detectable cytoplasm PrP in neurons [36] leads us to presume that the abundant NADPH in cytoplasm [37] may affect PrP C molecules during the protein trafficking in endo- plasmic reticulum or Golgi. These processes may result the PrP C misfolds to PrP Sc spontaneously. Materials and methods Preparation of brain homogenates Frozen scrapie agents 263K- and 139A-infected brains [38] were homogenized in PMCA conversion buffer, containing 1· NaCl ⁄ P i , 1% Triton X-100, 5 mm EDTA, 150 mm NaCl and protease inhibitor cocktail tablets (Roche Applied Sci- ence, Basel, Switzerland) as described elsewhere [23]. Crude homogenates were centrifuged for 30 s at 5000 g, aliquots of 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 )12 dilution as the stock seeds. These homogenates were used immedi- ately in subsequent experiments. Whole normal brains were removed surgically from purchased 5-week-old male hamsters. Brains were washed thoroughly in cold NaCl ⁄ P i containing 50 m m EDTA to remove as much blood as possible. After homogenization in PMCA conversion buffer, 10% (w ⁄ v) NBH were centrifuged for 30 s at 5000 g, and aliquots of the super- natant were immediately frozen at )80 °C for subsequent experiments. All processes of experiments, including anaesthetic and surgical procedures, as well as animal man- agement, have been reviewed and approved, and were performed in accordance with the relevant China national legislations and regulations. Recombinant protein expression and purification The recombinant plasmid pQE-haPrP 23–231 expressing hamster PrP residues 23–231 and pQE-huPrP 23–230 expressing human PrP residues 23–230 were generated as described previously [39]. The expression and purification of the recombinant His-tagged PrP proteins (rPrP) were performed by using Ni-NTA affinity chromatography (Qia- gen, Hilden, Germany) as described previously [40]. Refolding of the recombinant PrP After filtering with a 0.22 lm filter membrane (Millipore, Bedford, MA, USA), the concentration of the purified pro- tein solution was estimated by measuring A 280 . Each recom- binant PrP was refolded by a 10-fold molar ratio of Cu 2+ oxidation of the disulfide bond at room temperature for 3 h. Thereafter, the protein solution was dialysed into a 100-fold volume of NaCl ⁄ P i , pH 6.0, containing 10 mm EDTA for 4 h. The protein solution was transferred into fresh dialysis buffer (NaCl ⁄ P i , pH 6.0) without EDTA and this 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 measuring A 280 . Estimates of rPrP purity were made by SDS ⁄ PAGE and Coomassie Brilliant Blue staining. All final protein preparations were diluted to 0.2 mgÆmL )1 in PMCA con- version buffer and frozen at )80 °C. Solutions were kept at 4 °C once thawed. In vitro amplification procedure PMCA was performed on a water-bath sonicator (Misonix sonicator 3000; Misonix, Farmingdale, NY, USA), which had a microplate horn for PCR tubes. Ninety microliters of NBH or 0.2 mgÆmL )1 rPrP were mixed with 10 lL of vari- ous concentrations of ScBH in a volume of 100 lLin 0.2 mL PCR tubes. The tubes were positioned on the soni- cator and amplification cycles were programmed as described elsewhere [20,21]. One PMCA cycle consisted of sonication 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 each round, 10 lL of amplified product was added to 90 lLof fresh normal homogenates (10-fold dilution), or 1 lLof product was added to 99 lL of fresh normal homogenates (100-fold dilution). New mixtures were subjected to another round. This was repeated several times to reach an ideal amplification. NADPH can enhances PrP Sc propagation S. Shi et al. 1542 FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS Treatment of chemicals Chemicals, including NADPH, NADP, NADH, NAD and ascorbate, were purchased from Sigma-Aldrich (St Louis, MO, USA). (The structures of the chemicals are shown in Table S1.) All chemicals were freshly prepared as 10· stock solutions in NaCl ⁄ P i before each experiment. Vari- ous concentrations of chemicals were added to NBH or rPrP solutions and gently shaken at room temperature for 10 min, followed by mixing with ScBH and subjecting to PMCA. PK digestion and western blotting assay PMCA products were digested with PK at 37 °C for 1 h. The concentration of PK was 50 lgÆ mL )1 for brain homo- genates or 20 lgÆmL )1 for 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 and electronically transferred to poly(vinylidene diflouride) membranes (Immobilon-P; Millipore) at 10 V for 1 h. For immunoblotting, the membrane was blocked with 5% non- fat milk in NaCl ⁄ P i -T and incubated with 1 : 5000 diluted PrP mAb 3F4 (Dako, Glostrup, Denmark) in 0.5% nonfat milk in NaCl ⁄ P i -T at room temperature for 2 h. After washing three times in NaCl ⁄ P i -T, the membrane was immersed in a 1 : 5000 diluted horseradish peroxidase con- junct anti-mouse IgG (Boehringer, Ingelheim, Germany) in NaCl ⁄ P i -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). PrP Sc quantitation To estimate the concentration and number of PrP Sc mole- cules, serial dilutions of scrapie-infected brain homogenate were analysed by western blots in the same gel as aliquots of known concentrations of recombinant PrP (Fig. S3A) according to the admitted protocol [18,23]. Signal intensities were evaluated by densitometry, and the concentration of PK-treated PrP Sc in these samples was calculated by extrap- olation of the calibration curve calculated with recombinant PrP (Fig. S3B). To minimize errors due to saturated or weak signals, several dilutions were analysed and the experi- ment was repeated three times for each dilution. 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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-fusion form in a baculovirus system and conversion of expressed PrP-sen to PrP-res in a cell-free system. Virus Res 87, 145–153. 41 Jackson GS, Hill AF, Joseph C, Hosszu L, Power A, Waltho JP, Clarke AR & Collinge J (1999) Multiple folding pathways for heterologously expressed human prion protein. Biochim Biophys Acta 1431, 1–13. Supporting information The following supplementary material is available: Fig. S1. (A) Evaluation the possible influence of NADPH on PrP Sc without sonication. (B) Evaluation of the possible influence of NADPH on NBH during PMCA. Fig. S2. Evaluation of the possible influence of NADPH on rSHaPrP during PMCA. Fig. S3. Quantification of PrP Sc in scrapie-infected brain homogenate. Table S1. Structure of chemicals. This supplementary material can be found in the online version of this article. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article. S. Shi et al. NADPH can enhances PrP Sc propagation FEBS Journal 276 (2009) 1536–1545 ª 2009 The Authors Journal compilation ª 2009 FEBS 1545 . The propagation of hamster-adapted scrapie PrP Sc can be enhanced by reduced pyridine nucleotide in vitro Song Shi, Chen-Fang Dong, Chan Tian, Rui-Min. be involved in converting PrP C into the abnormal isomer in vitro [11–14]. Moreover, pyridine nucleotides, a group of coenzymes ubiquitous in bio- synthesis