Study on the dynamics stability of human prion protein

CHEN Xin, WEI Yaru, YU Xiaohan, ZHANG Jinglai

(Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical Engineering,Henan University, Kaifeng 475004, Henan, China)

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Study on the dynamics stability of human prion protein

CHEN Xin, WEI Yaru, YU Xiaohan, ZHANG Jinglai*

(InstituteofEnvironmentalandAnalyticalSciences,CollegeofChemistryandChemicalEngineering,HenanUniversity,Kaifeng475004,Henan,China)

Prion diseases are fatal and infectious neurodegenerative diseases affecting humans and animals.Especially human prion diseases have threaten human health and become more widespread in the past few years.The dynamics stability of human prion protein (hPrPc) was studied by the molecular dynamics (MD) and flow MD (FMD) simulations.Two symbol denatured states of hPrPc were produced by the FMD simulation for the following refolding process.The unfolding and refolding paths were carefully discussed with the residues, secondary structures, and residue-residue contact map.The threeα-helices of hPrPc were assembled to form a hydrophobic core, which played key roles on the unfolding and refolding of protein.The rigid helix-core worked as a scaffold to facilitate the refolding of hPrPc.The π-helix and 310-helix were appeared in the refolding process.And the extending ofβ-sheet was more readily to occur in the complete unfolding system of hPrPc.

dynamics stability; helix core; human prion protein (hPrPc); molecular dynamics (MD) simulation

Received date：2016-07-19.

Foundation item：Supported by the National Natural Science Foundation of China (21003037) and the National Science Foundation of the Education Department of Henan Province (13A150085).

Biography：CHEN Xin (1981-), female, majoring in the research of the stability of proteins and biomaterials.Corresponding author, E-mail: zhangjinglai@henu.edu.cn.

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism.The key event seems to be the conversion of the prion protein (PrP) from its normal cellular isoform (PrPc) to an abnormal scrapie isoform (PrPsc).The accumulation of PrPsc in the brain causes neurodegeneration[1-3].Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases[4-5].This kind of disease has been found to be associated with a conversion of theα-helix rich PrPc into aβ-structure rich insoluble conformer PrPsc.The pathogenic mechanisms of prion diseases are diverse, while the conformational conversion of PrPc into PrPsc is a central etiological event.The conversion of PrPc into PrPsc includes an obvious conformational transition of secondary structure.Then the investigation of the structure change mechanism of the PrPc is essential to the understanding of the molecular pathogenesis of prion diseases[6-8].

Recently, many accelerated factors of protein unfolding have been studied, such as the pH, pressure, temperature, and hydrodynamics.For example, it has been found that the misfolding of human prion protein involves a partial unfolding and misfolding step as PrPc converts to a PrPsc-like structure at low pH[9-11].We have performed MD simulations on the elevated temperature induced unfolding of animal prion protein[12].It was found that the unfolding of prion protein could be regulated by the temperature.OKUMURA et al has performed a multibaric-multithermal MD simulation of a 10-residue protein to dissect the difference of the mechanism between temperature and pressure denaturation[13].Pure high-temperature simulations were also used to bring about the unfolding of the globular domain.Particularly, it was found that the unfolding of protein could be accelerated by temperature without changing the unfolding pathway[14-15].CHEN et al found that theβ-sheet of human prion protein (hPrPc) was active with the elongation, twisting, and unfolding behaviors[16].And theβ-sheet of hPrPc was easily to be destabilized by the external force.It was also found that many prion diseases were stimulated by the site-mutation.For example, fatal familial insomnia (FFI) was found to be related with the site-mutation of D178N, and the M129V mutant was involved in the CJD[17-20].The mutation was shown to be readily to destabilize the PrPc and be helpful to the formation ofβ-sheet, which was rich in the PrPsc.Aβ-hairpin, 14-residues syrian hamster prion protein H1 peptide was performed for long time scale MD simulation[21].The diffusion in the essential space of partially folded intermediates toward the completely folded conformation was found.However, such a model might not be always applicable, as it is based on linear regime kinetics for the conformational transitions and a pre-equilibrium of secondary structure interconversions.

Flow MD (FMD) method was another recommendable technique to probe the protein unfolding under the shear flow.It has become one of the most effective supplementary means of in vitro single-molecular experiments[22-24].CHEN et al[25]has investigated the flow induced structural transition in theβ-switch region of glycoprotein Ib by generating stable water flow under constant temperature.We have successfully studied the forced-unfolding dynamics of the hPrPc under the forces in different directions[14].The unfolding pathway of PrPc was widely studied and the inducing factors of prion unfolding were also carefully discussed.However, the conversion mechanism is still unclear.The unfolding and refolding details of PrPc to PrPsc are inevitable in the conversion.So it is necessary to study the two dynamics processes to reveal the misfolding mechanisms of PrPc.To get more insight into the molecular basis of the stability of human prion protein, the classical MD simulation was introduced to study the unfolding and refolding pathway of hPrPc.FMD was used to produce the force-driving unfolding conformation of hPrPc.The dynamics stability and the internal interaction network of hPrPc were discussed in this paper.

1 Simulation details

1.1 Materials and methods

The initial structure of MD for hPrPc was generated from the crystal structure obtained from the protein data bank (PDB code: 1QM0)[26].In the hPrPc system, Helix 1 (Asp144-Glu152), Helix 2 (Asn173-Lys194), Helix 3 (Glu200-Gln227), Strand 1 (Met129-Leu130), and Strand 2 (Tyr162-Tyr163) ofβ-sheet were named as H1, H2, H3, S1, and S2, respectively.The energy minimization and MD simulation were implemented with NAMD version 2.7[27]and Charmm27 force field[28].As the study was focused on the unfolding and refolding details of hPrPc, the initial MD simulation was carried out for 2 ns to get a sub-stable state for the following FMD simulation.The hPrPc was immersed in a periodic water box with TIP3P[29]model water molecules.The size of the water box was 6.223 nm×6.025 nm×4.993 nm.The Particle Mesh Ewald (PME) method was used to calculate the long-range electrostatics interactions for the separation of direct and reciprocal space summation.A time step of 2 fs was selected and the coordinates were saved every 1 ps.During the MD runs, the Langevin method was turned on to keep the constant pressure at 101.3 kPa.

1.2 Flow molecular dynamics simulation

Considering the structure-function relationship, the nature, and function of the protein would be changed with the disorganization of the initial protein structure.Due to the complexity of the system and the time scale of unfolding (from 10 ms to 1 ms for the fastest folding proteins), it is difficult to track the complete unfolding process with experiments and realistic MD simulations.The FMD simulation could make the unfolding of prion protein to occur in the time scale of ps.Following the MD simulation, a series of FMD simulations were performed to study the protein unfolding with shear flow of water for 100 ps.In this simulation, a slice of water molecules with a thickness of 0.3 nm was tagged as forced atoms and constant external force was applied to them.The rest water molecules and prion protein were free.Since the module of protein may be roughly described as a rectangular box, six different starting orientations were considered in this paper.The first one, indicated as +x, is the initial face of prion protein alongx-axis, and the other five faces along the Cartesian axes were denoted as -x, +y, -y, +z, and -z, respectively.The snapshot of the hPrPc system in direction +xis shown in Fig.1 as an example.

In FMD simulation, all the parameters were carefully adjusted by the rigidity of the protein itself.The applying force to create the water flow was set to be 10 pN in the references 24 and 25.In that paper, the forced water molecules were driven to simulate the blood flow.And the SMD waters were pulled to help the transition of loop toβ-hairpin structure of a 17-residues fragment of Glycoprotein Ib.However, the hPrPc (125-228) studied in this work was found to be compact and rigid enough to protect the structure of protein from unfolding by the water turbulence.Then the same force parameter in reference 24 was not suitable for the large and rigid systems.In order to detect the observation window of prion protein, the total forces applied on the whole protein was carefully selected and it was set to be 1.6×104pN.The same force had been successfully used to investigate the mechanics property of hPrPc and its R220K mutation[14].Andγwas set to be 1 ps-1.One thousand frames of trajectories were deposited during the FMD simulation.

1.3 Additional molecular dynamics

The secondary structure of protein could be gradually unfolded by the shear flow.In order to detect whether the unfolding conformation under water turbulence could revert back to their native ones or not, additional free MD simulations following the FMD simulation were performed.Six different unfolding states of hPrPc were obtained from the FMD simulation, the most unfolding (+x) and the least unfolding (-y) conformations were selected to be conducted as the initial state of additional MD simulations.The refolding simulations on the least and most unfolding conformations were both carried out for 150 ns.The parameters and settings of these simulations were the same as the initial MD simulations.

2 Results and discussion

2.1 Equilibrium state

MD simulation is one of the most popular computational tools, can access simulation time up to ～1 μs for a solvated protein, thus providing direct information on protein folding/unfolding processes[30].A lot of equilibrium states could be obtained from the MD simulation.Generally, there are three basic criteria to judge whether the system achieved equilibrium or not, i.e.the root mean square deviation (RMSD), the potential energy (PE), and the radius of gyration (Rg) of protein during the simulation[31-33].It was found in Fig.2c that the RMSD line fluctuated around 0.23 nm in the last 1.0 ns of simulation, which showed that the hPrPc had reached one equilibrium state.In the MD process, the conformation of protein became compact with the decrease of the Rg value of hPrPc (Fig.2a).The PE of hPrPc was gradually decreased from -1 197.65 to -1 534.26 kcal·mol-1and was kept around -1 534.26 kcal·mol-1during the last 0.5 ns of the simulation.The phenomena from the three judgments showed that the structure of hPrPc had been stabilized in the MD process.

Fig.2 Evolution of the (a) Rg, (b) PE, and (c) RMSD of hPrPc during MD process

2.2 Flow-induced structural transition

After 2 ns MD simulation without any disturbances applied to the system, the equilibrium state of hPrPc was relegated to the FMD simulations as the initial structure.The SMD atoms were pulled in a directed direction and the other water molecules moved freely under periodic boundary conditions.The deformation subjected to the same force was various in different directions.However, the extending direction of protein was constrained by the applying force, which could be used for specific unfolding of certain secondary structure.Comparing the final helix content of hPrPc under the forces in different directions, the largest and smallest decrease of helix content were produced in the direction of +xand -y, respectively.Then the FMD processes in the two notable directions were selected as the examples to discuss the dynamics stability of hPrPc and they were named as F+xand F-y, respectively.The content ofα-helix was changed from 58.65% to 0% ( Fig.3b) and 17.30% in F+xand F-y, respectively.Complete unfolding and part unfolding were occurred in F+xand F-yunder the same external force.

2.2.1 F+x system

The unfolding path and details were shown in Fig.3a by the evolution of the secondary structures of hPrPc.Simultaneously, the contents ofα-helix andβ-sheet were also displayed in Fig.3b and 3c.The content ofα-helix was gradually decreased, however, the content ofβ-sheet was fluctuated between 0% and 8.65% during the whole FMD time.The motion mode of the two major secondary structures was rather different.The unfolding, refolding and extending ofβ-sheet under the external force was occurred alternatively.The unfolding details of theα-helix varied in the three helices.With the view of the FMD trajectory, the order of the SMD waters touching the helices was H2, H3, and H1.Beginning time of the unfolding of the three helices was 0.0, 38.2, and 1.8 ps for H1, H2, and H3, respectively.Yet the complete unfolding time of H1, H2, and H3 was 99.6, 98.9, and 93.2 ps.The unfolding of H1 begun from the Glu152-terminus, and the firstα-helix easily changed to 310-helix or the turn structures.The unfolding of H2 was appeared from the Lys194-terminus, and it mainly transformed to the turn structure.Both the two terminus of H3 were readily to unfold.It was also found that 310-helix and turn were the major conversion forms of H3.The outer-to-inner unfolding path was appeared in all the three helices.It was shown in the structural evolution that the fast unfolding of secondary structure begun at 12.5 ps.Similarly, both the contents of helix and sheet were found to be kept stable for several picoseconds at the initial of simulation.Then it was proposed that the fast unfolding of hPrPc was resulted by the accumulation of the water turbulence.With the analysis of the structure of hPrPc, it was found that the three helix columns crossed to form a hydrophobic core, and the crossed state of helices was compressed to be a strong barrier by the water flow in +xdirection, which resulted in the force-resistance of hPrPc.With the lasting turbulence of SMD waters, the helix-core was destroyed.And then the fast unfolding of helices began.

The unfolding path was also illustrated by the snapshot of the unfolding state of hPrPc in Fig.4a-4e.The force induced unfolding of hPrPc in the +xdirection begun from the C-terminal.One helix unit departed from the H3, which was tagged with the circle at 20 ps.The compact helix core was separated by the water turbulence at 50 ps and then the rigidα-helix was gradually untied during the last of the simulation.

2.2.2 F-y system

For the case of F-ysystem, hPrPc was not completely unfolded by the water turbulence during the 100 ps FMD simulation.The order of the helices meeting the SMD waters in the F-ysystem was H2, H3, and H1.Different from the unfolding results of F+x, H1 was the only one with complete unfolding at the end of the simulation (Fig.5a).H2 and H3 were partly untied by the external force.There was still 17.30% helix structure preserved at 100 ps (Fig.5b).The content of theβ-sheet was stable at 3.84% before 39.0 ps, and then it changed frequently between 5.76% and 0.0% from 39.0 to 70.0 ps.Finally, it was kept complete unfolding in the last 30.0 ps (Fig.5c).The same result could be obtained from the graph of structural evolution in Fig.5a, the rectangle highlighted the complete untying state of the twoβ-strands.Both the inner and the terminal unfolding of theα-helix were occurred in the unfolding path of F-y system.The inner disappearing of H2 and H3 was tagged with circles.

The burial of hydrophobic amino acids in the protein core is one of the most important driving forces in protein folding.During the unfolding process, the hydrophobic core was opened by the external force, and H2 was separated from the H1 and H3 scaffold.The snapshot of the FMD states during the simulation was displayed in Fig.6.To elucidate the hydrophobic and hydrophilic changes in the FMD process, the time evolution of the two kinds of solvent accessible surface areas (SASA) were illustrated in Fig.6f and 6g.The hydrophobic and hydrophilic SASA was named asSphoandSphi, respectively.SASA was calculated with the probe radius of 0.14 nm by VMD package.

TheSphogradually increased from 46.06 nm2to 53.28 nm2, which was resulted by the release of hydrophobic residues with the unfolding of protein.Experiments on hPrPc (90-231) had shown that the denaturation of the protein involved a massive increase in the exposure of hydrophobic surfaces[9,34], which was also found in our study.However, theSphidramatically fluctuated between 86.81 and 91.18 nm2during the simulation.As the unfolding proceeded, the three helices of hPrPc were separated, and the tertiary contacts were lost in the process.The resulting denatured ensemble has a large amount of secondary structure, but few high order contacts.Then the probe of SASA could detect more hydrophilic residues, which resulted in the increase ofSphi.With more hydrophobic residues were exposed on the surface of protein, the conformation of hPrPc was rearrangement and some hydrophilic residues was buried again.It was discerned in Fig.6g that the curve dropped to a lower SASA value in the last 18.0 ps compared to the 37-82 ns before.The external force produced a quite unstable state of hPrPc with most hydrophobic residues exposed to the water.

2.3 Refolding dynamics

MD simulations in principle provide detailed insight into all steps of the protein-folding pathway.In the hope of observing a number of folding and unfolding events under equilibrium conditions and investigating whether the denatured structure could revert back to their native ones when the external force is removed, additional MD simulations were performed on the two preferred FMD conformations of hPrPc.The additional MD systems were named as R+xand R-y, which was corresponding to the final state of F+xand F-y, respectively.MD simulation of the partly unfolded R-ysystem and the completely unfolded R+xsystem were all carried out for 150 ns to refold the denatured protein.

2.3.1 Refolding residues

The RMSD of R+x(Fig.7a) increased from 0.27 to 0.69 nm before 39.8 ns, and then it was stable at about 0.70 nm.From the inserted graph, we could find that only few parts of helix were reverted in 150 ns.Moreover, the platform on the RMSD curve was preserved for 110 ns, which showed that the conformation of hPrPc was trapped by an energy minimum.The folding details ofα-helix andβ-sheet in the MD process were listed in Table 1, and the values of the native hPrPc were added as a reference.In the R+xsystem, H1 was extended during the refolding procedure and three residues were folded into the helix structure at 150 ns.Residues 170-174 formed helix and kept stable during the simulation, however, only residues 173 and 174 belonged to the native H2.Seven residues 208-214 assembled to form the helix, and they were located in the middle of the native H3.Forβ-sheet, it was readily to elongate in the refolding process.As shown in Table 1, two residues participated in the extension of eachβ-strand.The content column also showed the high frequency of the elongation ofβ-sheet.However, the content ofα-helix was shown that it was not easily to revert back to the native state.The largest helix content of 24.04% was occurred at 80 ns, and it was rather less than that of the native hPrPc.It might provide some insights into the misfolding mechanism of hPrPc.

Table 1 Refolding residues and contents of α-helix and β-sheet in R+x system during the MD simulation

The symbol “-”respect that there is no specific secondary structure formed.

For the partly unfolded system R-y, the RMSD was gradually increased from 0.25 to 0.87 nm (Fig.7b).The RMSD was kept stable at about 0.77 nm in the last 84.0 ns, which showed that one equilibrium state of hPrPc was reached.And two notable increases of RMSD were appeared at 0.6 and 63.5 ns, which was associated with the key refolding of hPrPc.Most secondary structures of hPrPc refolded in the R-ysystem at the end of simulation (Table 2).For example, H1 was reformed by the residues 144-150, which is part of the native residues 144-152.Similarly, residues 200-227 of native H2 were partly refolded by the residues 200-221.The content ofα-helix in-creased to 45.19% at 60 ns, which was close to the native one.The elongation ofα-helix was not shown in the R-ysystem.And the content ofβ-sheet showed that theβ-stands were also not readily to extend in this case.The complete unfolding state was favorable for the extending ofβ-sheet.By comparing the two refolding details of R+xand R-y, it was found that the complete unfolding state was hard to revert back.And the helix-core was found to work as a scaffold to facilitate the unfolding of protein.For example, the existence of the helix-core in R-ysystem produced a rigid framework of the denatured protein and it induced more helix structures revert back.

Table 2 Refolding residues and contents of α-helix and β-sheet in R-y system during the MD simulation

The symbol “-”respect that there is no specific secondary structure formed.

2.3.2 Structural flexibility

The RMSF value of the residues in the denatured state was much larger than that in the native state.The shape of the RMSF line in R-ysystem (Fig.7d) was similar to that of the native state.In addition to the high values of the two terminus of hPrPc, three notable peaks were associated with the large flexibility of the loop structures, which connected the threeα-helices.Yet the shape of the RMSF line in R+xsystem was deviated from the native one.The second and the third peak were mixed, and the valley for the residues 180-188 was not appeared.It was resulted by the flexibility of the residues in the refolding process.These residues were the part of H2 in the native state.It could be found from Table 1 that residues 180-188 was not refolded in the simulation and it existed in the form of flexible turn and coil structures.However, the newly formed H2 in R-ysystem was composed of residues 172-188, and then the rigidity of helix reduced the RMSF values of residues 180-188.

2.3.3 Contact map

To get more insight into the topology of hPrPc in various states, the residue-residue contact map was displayed in Fig.8.The contact map of protein at the native state was illustrated in Fig.8a as a reference.During the initial MD simulation, the conformation of hPrPc was relaxed and the residue-residue contact in regions A and B was dismissed.With the analysis of MD trajectory, it was found that the loop (136-138) was away from the H1 (151-153), and then the contact in region A was disappeared.The contact disappearance of region B was resulted by the separation of H2 (190-193) and H3 (197-201).In the complete unfolding case of F+x(Fig.8c), more contacts were found to be removed.The separation of H3 (206-221) and S1 (130-140) produced the vacancy of region C.And the region D was resulted by the separation of H3 (203-225) and H1 (147-165).The contact points in Fig.8e were less than that in Fig.8f, which also showed the refolding structures in R+xsystem was less than that in R-ysystem.In the R+xsystem, the refolding region H was resulted by the approaching of H1 (133-141) to the loop (150-156) which connected H1 and S1.Similarly, the meeting of H3 (211-219) and S1 (129-135) reappeared the contact points in region I.For the R-ysystem, regions K and M were tagged to discern the unfolding parts of hPrPc during the refolding MD process.The regions K and M represented the contacts of H3-S1 and H3-S2, respectively.

2.3.4 Refolding path

The refolding path of the two denatured states of hPrPc was illustrated by the secondary structure evolution in Fig.9.It was found that H1 in R+xsystem (Fig.9a) was fast refolded at the beginning of simulation.The extending ofβ-sheet was occurred frequently in the refolding process.In the region of H2, theα-helix and π-helix were appeared alternatively, which showed that π-helix was one of the most important intermediates in the refolding process ofα-helix.And it was also appeared in the H3 refolding path.Simultaneously, 310-helix was found in the transition from turn toα-helix in the region of H3 belt.In the R-ysystem,β-sheet was readily to appear from 30 to 68 ns.All the threeα-helices were partly refolded, and the newly folded H3 was the most stable one during the simulation.The unstable π-helix was prone to appear in the H2 refolding process.As the flexibility of the terminus ofα-helix, it was much harder to be refolded than the inner part of helix.

3 Conclusions

In this work, free MD and flow MD simulations were combined to study the dynamics stability of hPrPc.The equilibrium conformation of hPrPc in water was obtained by the energy minimization and the initial MD simulation.Hydrodynamic disturbance was introduced to produce the denatured hPrPc and to probe the mechanics properties of the secondary structures in hPrPc.One complete unfolding state and one part unfolding state of hPrPc were selected as the samples for the additional refolding MD process.As the anisotropy of the topology of protein, the unfolding result was directly resulted by the direction of the applying force.In the F+xsystem, the protein was completely unfolded and the unfolding order was H3, H2, and H1.Yet in the F-ysystem, H1 was the only helix that was completely unfolded.The threeα-helices in hPrPc assembled to form a hydrophobic core, which play key roles on the unfolding and refolding of hPrPc.In the part unfolding system, the refolding of hPrPc was readily to be performed with the existence of helix-core.Yet the complete unfolding state of hPrPc was hard to reassemble the helix-core and it was trapped in an energy minimum.The π-helix is one of the most favorable transition intermediates ofα-helix.Yetβ-sheet was rather active and the extending ofβ-sheet was more readily to occur in the complete unfolding system.Moreover, this study sheds new light on the molecular basis of inherited prion stability, introducing novel structural features, which are of great importance for our understanding of these disorders and of the earliest molecular events leading to the conformational transition of PrPc into aβ-sheet-rich fold.

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[責(zé)任編輯:吳文鵬]

人類朊蛋白的動力學(xué)穩(wěn)定性研究

陳 欣，魏亞茹，于笑寒，張敬來*

(河南大學(xué) 化學(xué)化工學(xué)院，環(huán)境與分析科學(xué)研究所，河南 開封 475004)

朊蛋白病是一種能在人類或者動物之間傳播的致命的神經(jīng)退行性疾病.尤其是人類朊蛋白疾病在近幾年蔓延迅速，已經(jīng)威脅到人類的健康.在本文中，我們使用分子動力學(xué)(MD)和流體分子動力學(xué)(FMD)模擬相結(jié)合的方法研究了人類朊蛋白(hPrPc)的動力學(xué)穩(wěn)定性.我們通過FMD模擬產(chǎn)生了兩個典型的hPrPc的變性結(jié)構(gòu)，并進(jìn)一步研究了在自然狀態(tài)下這兩個變性結(jié)構(gòu)重折疊的過程，從關(guān)鍵殘基、二級結(jié)構(gòu)、殘基-殘基相互作用圖等方面詳細(xì)討論了hPrPc的解折疊和重折疊路徑.研究發(fā)現(xiàn)hPrPc的三個α-螺旋結(jié)構(gòu)組成了一個疏水核心，在蛋白質(zhì)的解折疊和重折疊過程中發(fā)揮了重要的作用.剛性的疏水核心就像是腳手架一般為hPrPc的重折疊提供便利.在重折疊過程中，π-螺旋和310螺旋出現(xiàn)幾率較高，并且β-折疊的延長也更多地出現(xiàn)在完全解折疊的hPrPc體系中.

動力學(xué)穩(wěn)定性；螺旋核心；人類朊蛋白；分子動力學(xué)模擬

O641.3

A

1008-1011(2016)06-0681-12