Metalloprotease-mediated OPA1 processing is modulated by the mitochondrial membrane potential.

BACKGROUND INFORMATION: Human OPA1 (optic atrophy type 1) is a dynamin-related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1. RESULTS: We find that various mammalian cell types display a similar pattern of OPA1 isoforms [two L-OPA1 (long isoforms of OPA1) and three S-OPA1 (short isoforms of OPA1)] and that loss of the inner membrane potential, but not inhibition of oxidative phosphorylation or glycolysis, induces rapid and complete processing of L-OPA1 to S-OPA1. In isolated mitochondria, OPA1 processing was inhibited by heavy-metal chelators, pointing to processing by a mitochondrial metalloprotease. The pattern of OPA1 isoforms and its processing kinetics were normal in mitochondria devoid of the serine protease PARL (presenilins-associated rhomboid-like protein) - the human orthologue of Pcp1/Rbd1 - and in cells from patients carrying homozygous mutations in SPG7 (spastic paraplegia type 7), a gene encoding the matrix-oriented metalloprotease paraplegin. In contrast, OPA1 processing kinetics were delayed upon knock-down of YME1L (human yme1-like protein), an IMS-oriented metalloprotease. OPA1 processing was also stimulated during apoptosis, but inhibition of this processing did not affect apoptotic release of OPA1 and cytochrome c. Finally, we show that all OPA1 isoforms interact with Mfn1 (mitofusin 1) and Mfn2 and that these interactions are not affected by dissipation of DeltaPsim (inner mitochondrial membrane potential) or OPA1 processing. CONCLUSIONS: Metalloprotease-mediated processing of OPA1 is modulated by the inner membrane potential and is likely to be mediated by the YME1L protease.     

Metalloprotease-mediated OPA1 processing is modulated by the mitochondrial membrane potential

Background information. Human OPA1 (optic atrophy type 1) is a dynamin‐related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1. Results. We find that various mammalian cell types display a similar pattern of OPA1 isoforms [two L‐OPA1 (long isoforms of OPA1) and three S‐OPA1 (short isoforms of OPA1)] and that loss of the inner membrane potential, but not inhibition of oxidative phosphorylation or glycolysis, induces rapid and complete processing of L‐OPA1 to S‐OPA1. In isolated mitochondria, OPA1 processing was inhibited by heavy‐metal chelators, pointing to processing by a mitochondrial metalloprotease. The pattern of OPA1 isoforms and its processing kinetics were normal in mitochondria devoid of the serine protease PARL (presenilins‐associated rhomboid‐like protein) – the human orthologue of Pcp1/Rbd1 – and in cells from patients carrying homozygous mutations in SPG7 (spastic paraplegia type 7), a gene encoding the matrix‐oriented metalloprotease paraplegin. In contrast, OPA1 processing kinetics were delayed upon knock‐down of YME1L (human yme1‐like protein), an IMS‐oriented metalloprotease. OPA1 processing was also stimulated during apoptosis, but inhibition of this processing did not affect apoptotic release of OPA1 and cytochrome c. Finally, we show that all OPA1 isoforms interact with Mfn1 (mitofusin 1) and Mfn2 and that these interactions are not affected by dissipation of ΔΨm (inner mitochondrial membrane potential) or OPA1 processing. Conclusions. Metalloprotease‐mediated processing of OPA1 is modulated by the inner membrane potential and is likely to be mediated by the YME1L protease.     

Relationship between OPA1 and cardiolipin in mitochondrial inner-membrane fusion

Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. The large GTPase optic atrophy 1 (OPA1) is identified as a core component of inner membrane (IM) fusion. OPA1 exists as the membrane-anchored L-OPA1 and the proteolytically cleavage soluble S-OPA1. Recently, we showed that OPA1 and mitochondria-localized lipid cardiolipin (CL) cooperate in heterotypic IM fusion [Ban et al., Nat. Cell Biol. 19 (2017) 856–863]. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 and S-OPA1 expressed in silkworm and found that L-OPA1 on one side of the membrane and CL on the other side were sufficient for mitochondrial fusion. L-OPA1 is the major fusion-prone factor in heterotypic fusion. However, the role of S-OPA1 remains unknown as S-OPA1 promoted L-OPA1-dependent heterotypic membrane fusion and homotypic CL-containing membrane fusion, but S-OPA1 alone was not sufficient for heterotypic membrane fusion. L-OPA1- and CL-mediated heterotypic mitochondrial fusion was confirmed in living cells, but tafazzin (Taz1), the causal gene product of Barth syndrome, was not essential for mitochondrial fusion. Taz1-dependent CL maturation might have other roles in the remodeling of mitochondrial DNA nucleoids.     

Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria

The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria     

Thermotolerance and molecular chaperone function of the small heat shock protein HSP20 from hyperthermophilic archaeon, <Emphasis Type="Italic">Sulfolobus solfataricus P2</Emphasis>

Small heat shock proteins are ubiquitous in all three domains (Archaea, Bacteria and Eukarya) and possess molecular chaperone activity by binding to unfolded polypeptides and preventing aggregation of proteins in vitro. The functions of a small heat shock protein (S.so-HSP20) from the hyperthermophilic archaeon, Sulfolobus solfataricus P2 have not been described. In the present study, we used real-time polymerase chain reaction analysis to measure mRNA expression of S.so-HSP20 in S. solfataricus P2 and found that it was induced by temperatures that were substantially lower (60°C) or higher (80°C) than the optimal temperature for S. solfataricus P2 (75°C). The expression of S.so-HSP20 mRNA was also up-regulated by cold shock (4°C). Escherichia coli cells expressing S.so-HSP20 showed greater thermotolerance in response to temperature shock (50°C, 4°C). By assaying enzyme activities, S.so-HSP20 was found to promote the proper folding of thermo-denatured citrate synthase and insulin B chain. These results suggest that S.so-HSP20 promotes thermotolerance and engages in chaperone-like activity during the stress response.     

Mdm35p imports Ups proteins into the mitochondrial intermembrane space by functional complex formation

Ups1p, Ups2p, and Ups3p are three homologous proteins that control phospholipid metabolism in the mitochondrial intermembrane space (IMS). The Ups proteins are atypical IMS proteins in that they lack the two major IMS‐targeting signals, bipartite presequences and cysteine motifs. Here, we show that Ups protein import is mediated by another IMS protein, Mdm35p. In vitro import assays show that import of Ups proteins requires Mdm35p. Loss of Mdm35p led to a decrease in steady state levels of Ups proteins in mitochondria. In addition, mdm35Δ cells displayed a similar phenotype to ups1Δups2Δups3Δ cells. Interestingly, unlike typical import machineries, Mdm35p associated stably with Ups proteins at a steady state after import. Demonstrating that Mdm35p is a functional component of Ups–Mdm35p complexes, restoration of Ups protein levels in mdm35Δ mitochondria failed to restore phospholipid metabolism. These findings provide a novel mechanism in which the formation of functional protein complexes drives mitochondrial protein import.     

Enhanced Thermotolerance of <Emphasis Type="Italic">E. coli</Emphasis> by Expressed OsHsp90 from Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A gene encoding the rice (Oryza sativa L.) 90-kDa heat shock protein (OsHsp90) was introduced into Escherichia coli using the pGEX-6p-3 expression vector with a glutathione-S-transferase (GST) tag to analyze the possible function of this protein under heat stress for the first time. We compared the survivability of E. coli (BL21) cells transformed with a recombinant plasmid containing GST-OsHsp90 fusion protein with control E. coli cells transformed with the plasmid containing GST and the wild type BL21 under heat shock after isopropyl β-d-thiogalactopyranoside induction. Cells expressing GST-OsHsp90 demonstrated thermotolerance at 42, 50, and 70°C, treatments that were more harmful to cells expressing GST and the wild type. Further studies were carried out to analyze the heat-induced characteristics of OsHsp90 at 42, 50, and 70°C in vitro. When cell lysates from E. coli transformants were heated at these heat stresses, expressed GST-OsHsp90 prevented the denaturation of bacterial proteins treated with 42°C heat shocks, and partially prevented that of proteins treated at 50 and 70°C; meanwhile, cells expressing GST-OsHsp90 withstood the duration at 50°C. These results indicate that OsHsp90 functioned as a chaperone, binding to a subset of substrates, and maintained E. coli growth well at high temperatures.     

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

We showed earlier that 15 deoxy Δ^12,14 prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion [1]. However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.     

Functional Differences Between Cyanobacterial DnaK1 Chaperones from the Halophyte Aphanothece halophytica and the Freshwater Species Synechococcus elongatus Expressed in Escherichia coli

DnaK chaperones participate in essential cellular processes including the assistance of the folding, structural maintenance, trafficking, and degradation of proteins, the control of stress responses, and so on. In contrast to the situation found in most other bacterial groups, the cyanobacteria contain multiple dnaK homolog genes whose cellular roles remain ambiguous. We compared in this work the in vivo chaperone capabilities of the DnaK1 members from the halophyte Aphanothece halophytica and the freshwater species Synechococcus elongatus. The corresponding dnaK1 genes were expressed in Escherichia coli, and the abilities of the encoded chaperones to provide for both general and specific functions conducted by E. coli DnaK were analyzed. Synechococcus DnaK1 was far more effective than A. halophytica DnaK1 in replacing E. coli DnaK in all activities tested in vivo, including changes in cell morphology and downregulation of the heat shock response, prevention of the aggregation of misfolded proteins, and restoration of thermotolerance to dnaK-deficient mutants. Thus, regardless of an extensive sequence similarity and comparable in vitro chaperone capabilities, the two cyanobacterial DnaK1 chaperones functionally differed under in vivo conditions. The overall results reinforce the notion that A. halophytica DnaK1 and Synechococcus DnaK1 evolved different substrate specificity since they separated from a common ancestor.     

Chaperone over-expression in Escherichia coli: Apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates

The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific.     

Expression and function of two chaperone proteins,AtGroEL and AtGroES,from Acidithiobacillus ferrooxidans ATCC 23270

Two molecular chaperone genes encoding the Acidithiobacillus ferrooxidans Hsp60 (AtGroEL) and Hsp10 (AtGroES), respectively were introduced into Escherichia coli using the pLM1 expression vector. Then the AtGroEL and AtGroES proteins were overexpressed successfully in Escherichia coli BL21 (DE3), and purified by one-step immobilized metal affinity chromatography. The ATPase assay showed that the proteins were in active form, and the ATPase activity of AtGroEL was temperature dependent with an optimal temperature of 50°C, but the co-chaperonin AtGroES inhibited the ATPase activity of AtGroEL. The chaperonin function of the recombinant proteins was examined using three different protein substrates in vitro, and the results showed that AtGroEL/AtGroES chaperone system could facilitate the refolding of the thermodenatured rusticyanin and recover the activity of thermodenatured ArsH protein. In addition, it could improve the thermal stability of xylanase. Molecular modelling for AtGroEL protein revealed that residues of Tyr199, Ser201, Tyr203, Phe204, Leu234, Leu237, Leu259, Val263 and Val264 were necessary for binding the denatured polypeptides.     

The thylakoid protease Deg1 is involved in photosystem‐II assembly in Arabidopsis thaliana

DegP proteases have been shown to possess both chaperone and protease activities. The proteolytic activities of chloroplast DegP‐like proteases have been well documented. However, whether chloroplast Deg proteases also have chaperone activities has remained unknown. Here we show that chloroplast Deg1 also has chaperone activities, like its Escherichia coli ortholog DegP. Transgenic plants with reduced levels of Deg1 accumulated normal levels of different subunits of the major photosynthetic protein complexes, but their levels of photosystem‐II (PSII) dimers and supercomplexes were reduced. In vivo pulse‐chase protein labeling experiments showed that the assembly of newly synthesized proteins into PSII dimers and supercomplexes was impaired, although the synthesis rate of chloroplast proteins was unaffected in the transgenic lines. Protein overlay assays provided direct evidence that Deg1 interacts with the PSII reaction center protein D2. These results suggest that Deg1 assists the assembly of the PSII complex, probably through interaction with the PSII reaction center D2 protein.     

Molecular chaperone activity of tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) endoplasmic reticulum-located small heat shock protein

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His₆-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

L‐OPA1 regulates mitoflash biogenesis independently from membrane fusion

Mitochondrial flashes mediated by optic atrophy 1 (OPA1) fusion protein are bioenergetic responses to stochastic drops in mitochondrial membrane potential (Δψ_m) whose origin is unclear. Using structurally distinct genetically encoded pH‐sensitive probes, we confirm that flashes are matrix alkalinization transients, thereby establishing the pH nature of these events, which we renamed “mitopHlashes”. Probes located in cristae or intermembrane space as verified by electron microscopy do not report pH changes during Δψ_m drops or respiratory chain inhibition. Opa1 ablation does not alter Δψ_m fluctuations but drastically decreases the efficiency of mitopHlash/Δψ_m coupling, which is restored by re‐expressing fusion‐deficient OPA1^K301A and preserved in cells lacking the outer‐membrane fusion proteins MFN1/2 or the OPA1 proteases OMA1 and YME1L, indicating that mitochondrial membrane fusion and OPA1 proteolytic processing are dispensable. pH/Δψ_m uncoupling occurs early during staurosporine‐induced apoptosis and is mitigated by OPA1 overexpression, suggesting that OPA1 maintains mitopHlash competence during stress conditions. We propose that OPA1 stabilizes respiratory chain supercomplexes in a conformation that enables respiring mitochondria to compensate a drop in Δψ_m by an explosive matrix pH flash.     

Improving the soluble expression of aequorin in Escherichia coli using the chaperone-based approach by co-expression with artemin

Abstract

Escherichia coli is a common host that is widely used for producing recombinant proteins. However, it is a simple approach for production of heterologous proteins; the major drawbacks in using this organism include incorrect protein folding and formation of disordered aggregated proteins as inclusion bodies. Co-expression of target proteins with certain molecular chaperones is a rational approach for this problem. Aequorin is a calcium-activated photoprotein that is often prone to form insoluble inclusion bodies when overexpressed in E. coli cells resulting in low active yields. Therefore, in the present research, our main aim is to increase the soluble yield of aequorin as a model protein and minimize its inclusion body content in the bacterial cells. We have applied the chaperone-assisted protein folding strategy for enhancing the yield of properly folded protein with the assistance of artemin as an efficient molecular chaperone. The results here indicated that the content of the soluble form of aequorin was increased when it was co-expressed with artemin. Moreover, in the co-expressing cells, the bioluminescence activity was higher than the control sample. We presume that this method might be a potential tool to promote the solubility of other aggregation-prone proteins in bacterial cells.     

Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.     

The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR^mt) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.     

Structural insights of proteins in sub-cellular compartments: In-mitochondria NMR

Many eukaryotic proteins exert their physiological function in specific cellular compartments. Proteins of the inter-membrane space (IMS) of mitochondria, for example, are synthesized in the cytoplasm and translocate to the IMS, where they are further processed to their mature form. In-cell Nuclear Magnetic Resonance (NMR) has proven to be an ideal approach to investigate eukaryotic proteins at the atomic level, inside the cytoplasm. Here we show that proteins inside intact mitochondria isolated from human cells can be structurally characterized by NMR (in-mitochondria NMR). By this approach, we characterized the folding and maturation state of two human proteins in the IMS, SOD1 and Mia40. Both observed proteins were in the folded state. Mia40 was in the oxidized, functional state, while SOD1 disulfide bond formation was promoted by increasing the level of the SOD1 chaperone, CCS, in the IMS.     

Functional mode of NtHSP17.6, a cytosolic small heat-shock protein fromNicotiana tabacum

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the α-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class I sHsp,NtHSP17.6, fromNicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation intoEscherichia coli and its over-expression, NtHSPI 7.6 was purified and examinedin vitro. This purified NtHSPI 7.6 exhibited typical chaperone activity in a light-scattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Non-denaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSPI 7.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.