Redoxoma Highlights

Redoxoma Highlights by Maria F. Forni*

Known for over a century, mitochondria have become, during the last four decades, an important subject of research within several disciplines. This is mostly due to the fact that this organelle comprises the site of oxidative phosphorylation, the citric acid cycle, fatty acid oxidation, the urea cycle and the biosynthesis of iron-sulphur centres and haem. Moreover, mitochondria are an important redox-signaling node. Indeed, the bioenergetic status of a cell is dependent on the overall quality and relative abundance of the mitochondrial population it harbors. Recent evidence suggests that the control of mitochondrial mass and morphology occurs through the processes of fusion, fission, mitophagy and biogenesis, in a tightly regulated manner known as the “mitochondrial life cycle”. It is believed that these events also play an important role in the quality-control of this organelle, and that the failure to do so could lead to several pathologies and ageing. Nonetheless, very little is known about the impact of mitochondrial dynamics in the process of stem cell differentiation.

Adult stem cells are rare, ranging in proportion from 0.1 to 5% of the total population in many mammalian tissues. They are considered the gatekeepers of tissue homeostasis since they can proliferate in order to maintain their numbers in the resident tissue but, importantly, they can differentiate and originate specialized cells, maintaining organ integrity in face of cell death imposed by ageing or injury.

Using a murine model of stromal mesenchymal stem cells derived from the skin (specifically from the dermis) we have been trying to unveil some of the alterations associated with mitochondrial homeostasis during differentiation. These cells, when properly stimulated, can generate osteoblasts/osteocytes, adipocytes and chondrocytes. It seems predictable that during differentiation an extensive metabolic reconfiguration would occur. Strikingly, we found that very early on, during the commitment period, when these cells are irreversibly specified towards a specific cell fate, the mitochondrial network is actively being remodeled. Moreover, if the processes of fusion and fusion are intentionally manipulated, the differentiation outcome can be altered. These findings demonstrate that mitochondrial morphology and its regulating processes are modulated early on during commitment, leading to alterations in the bioenergetic profile during differentiation. Our results thus suggest that mitochondrial dynamics may play a central role in the maintenance/commitment of mesenchymal stem cells.

Contributed by: Maria F. Forni, PhD.

*MF Forni is a post-doctoral fellow at IQUSP (Alicia Kowaltowski’s lab)
Department of Biochemistry, Institute of Chemistry, University of São Paulo, Brazil

Contributed by: Maria Fernanda Forni, Ph.D. *MF Forni is a post-doctoral fellow at IQUSP (Alicia Kowaltowski’s lab) Department of Biochemistry, Institute of Chemistry, University of São Paulo, Brazil

Redoxoma Highlights by Denise de C. Fernandes

Correct protein folding is a vital and extremely regulated cellular function. Disulfide bonds are essential determinants of the correctly folded protein structure. During the folding of nascent proteins into the endoplasmic reticulum (ER) lumen, essential enzymes promote disulfide bond insertion (oxidation) and their eventual repositioning (isomerization) when they are initially formed between wrong cysteines. These reactions are catalyzed by PDIs (protein disulfide isomerases), a family of enzymes that contains more than 20 members, from yeast to humans [1]. Thus, PDIs do not have one specific substrate, but rather a large variety of un/misfolded protein substrates.

Using purified PDIA1 (the founder member of PDI family), it is possible to measure 3 PDI redox activities: disulfide reduction, oxidation or isomerization, depending on the substrate selected for the assay. In cells, although most of PDIA1 resides in the ER, a small quantity is able to bypass ER retention mechanisms by so far unknown mechanisms, and is found in cytosol, nuclei, mitochondria and plasma membrane. PDIA1 function(s) in these subcellular compartments is not so clear, but in plasma membrane there are good evidences that PDIA1 is preferentially a reductase, acting in glycoproteins such as integrins - with implications in virus infection, thrombus formation and coagulation [2]. Thus, the preferential in vivo PDIA1 redox activity depends on compartmentalized redox environment. While in the more oxidizing ER lumen PDIA1 is rather an oxidase/isomerase, in the more reducing surrounding of plasma membrane PDIA1 behaves mostly as a reductase. Besides the strictly redox activities, PDIA1 exhibits a chaperone activity (i.e., assisting other proteins to stabilize), not directly dependent on its redox thiols. In vitro,it is also possible to measure solely PDIA1 chaperone activity, if the substrate chosen is devoid of disulfide bonds.

Putting all together, while with purified protein is possible to measure up to 4 activities (chaperone, thiol reduction, oxidation and isomerization), in vivo all these activities should overlap into each other to achieve PDIA1 function in each specific cellular compartment. These issues were recently critically discussed in a minireview about methods for measuring PDI activities in vitro and in cells [3]. Not surprisingly, there are interferents when measuring PDI activity in cellular samples. These include endogenous interferents such as other reductase enzymes, e.g. thioredoxin itself. Intriguingly, we described that several surfactants used in common buffers in procedures of lysis or preparation for microscopy analysis may alter PDI assays by inhibiting its reductase activity.

These methodological hardships are not unusual in essentially every research field and the task of choosing a specific method requires a lot of scrutiny, great involvement with the project and design of several controls to avoid intrinsic interferents. Particularly, in redox research, in which protein/cellular sample manipulation generates artifactual oxidations (such as disulfide formation) due to molecular oxygen, this is a more challenging task, especially when the fascinating multifaceted PDI is involved.

1. F. R. Laurindo, L. A. Pescatore, D. C. Fernandes. Protein disulfide isomerase in redox cell signaling and homeostasis. Free Radical Biology & Medicine, 52 (9): 1954-69, 2012. | http://dx.doi.org/10.1016/j.freeradbiomed.2012.02.037
2. H. Ali Khan, B. Mutus. Protein disulfide isomerase a multifunctional protein with multiple physiological roles. Frontiers in Chemistry, 2: 70, 2014. | http://dx.doi.org/10.3389/fchem.2014.00070
3. M. M. Watanabe, F. R. Laurindo, D. C. Fernandes. Methods of measuring protein disulfide isomerase activity: a critical overview. Frontiers in Chemistry, 2: 73, 2014. | http://dx.doi.org/10.3389/fchem.2014.00073

Denise de Castro Fernandes, Ph.D. Associate Researcher of Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil

Redoxoma Highlights by Fernanda M. da Cunha

Mitochondria are believed to be former free living bacteria that established a successful symbiotic relationship with eukaryotic cells in such a way that today, besides being crucial for the biosynthesis of intermediary metabolites, calcium homeostasis, coordination of apoptosis and ATP synthesis, most mitochondrial proteins are encoded by nuclear rather than mitochondrial DNA. In that scenario, communication pathways that relay signals from the nucleus to mitochondria as well as from mitochondria to the nucleus (the retrograde way) are mandatory to secure energetic and metabolic homeostasis. In yeast, the best characterized retrograde signaling pathway, activated whenever there is some mitochondrial dysfunction, is the one dependent on RTG1, RTG2 and RTG3 proteins. Previous studies have shown that upon activation, RTG-dependent signaling leads to nuclear transcription of a number of genes with the purpose of adjusting cell metabolism to sustain survival even in the absence of a fully functioning Krebs cycle.

In a recently published article, Torelli et al. [1], describe interesting outcomes of retrograde signaling activation. The authors used yeast cells defective in RTG1 or 2 and showed that in the late stationary phase (when the cells are still alive but ceased multiplying due to glucose exhaustion in the culture medium) the mutants consumed twice of the oxygen/min when compared to wild type cells, suggesting an increased mitochondrial content. Additional assays demonstrated that mitochondria were indeed increased in mutants and the likely reason for this finding is that the mutants were less able to degrade mitochondria by mitophagy when compared to wild type cells. It is interesting to note that wild type cells’ mitochondria that escaped mitophagy produce significantly more hydrogen peroxide than those from RTG mutants. This is an intriguing finding, since mitophagy has been described as a strategy to eliminate dysfunctional and thus highly oxidant mitochondria. This increased hydrogen peroxide production by wild type cells’ mitochondria proved to be beneficial, as Torelli et al. [1] found out that wild type cells had increased catalase and glutathione peroxidase activities, making them more capable of degrading exogenous hydrogen peroxide and surviving after hydrogen peroxide challenge when compared to RTG mutant cells. Taken together, the results indicate that besides controlling mitochondrial activity, RTG-dependent retrograde signaling is determinant for the mounting of a redox resistance response through hormesis in stationary yeast cells.

1. N. Q. Torelli, J. R. Ferreira-Júnior, A. J. Kowaltowski, F. M. da Cunha. RTG1- and RTG2-dependent retrograde signaling controls mitochondrial activity and stress resistance in Saccharomyces cerevisiae. Free Radical Biology & Medicine, 81: 30-7, 2015. | http://dx.doi.org/10.1016/j.freeradbiomed.2014.12.025

Fernanda Marques da Cunha, Ph.D. Professor at Department of Biochemistry, Paulista School of Medicine, Federal University of São Paulo, Brazil.

Redoxoma Highlights by Luis. E. S. Netto

Peroxiredoxin (Prx) enzymes are becoming more and more popular among other reasons due to their high reactivity towards hydroperoxides and to their abundance. As a consequence, Prxs are proposed as biological sensors of hydrogen peroxide. It is interesting to observe that since their beginnings (in the end of the 60’s), one feature that called attention was their ability to form high molecular weight species, visible by electron microscopy [1]. It was almost twenty years later that the thiol-dependent peroxidase activity of Prx enzymes was described.

Among Prx family of proteins, 2-Cys Prx enzymes (those belonging to the AhpC/Prx1 group) can adopt a wide array of quartenary structures. The one most often found in crystallographic studies is a decamer, composed of five dimers arranged as a doughnut. Decamers (or even higher molecular weight species) and dimers are in equilibrium, switching back and forth. The physiological meaning of these distinct quartenary structures is yet poorly understood. Initially it was proposed that dimers display peroxidase activity, whereas the decamers (or the higher molecular weight species) would be endowed with chaperone (holdase) activity. However, recent evidences indicated that decamers and high molecular weight species are more active as peroxidases than the dimeric counterparts. Therefore, the picture is clearly very complex and the factors governing the switch between decamers and dimers are elusive. Anyway, the reduced Prx forms exist primarily as decamers and disulfide bond formation in 2-Cys Prx favors decamer dissociation into dimeric units. In contrast to such redox-dependent pathways, pH-associated mechanisms underlying switch between dimers and decamers were unknown.

A study led by Dr. Mario Murakami (Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas/SP) in collaboration with Dr. Marcos Oliveira (UNESP São Vicente) and Dr. Luis Netto (Instituto de Biociências – USP) from CEPID redoxoma described a pH-dependent modulation of decamer and dimer inter-conversion for a mitochondrial tryparedoxin peroxidase from Leishmania braziliensis (LbPrx1m) [2]. Previously, it was described that a 2-Cys Prx from Leishmania infantum confers thermotolerance to the parasite and displays in vitro chaperone activity, which appeared to be related to virulence [3]. Two crystallographic structures were reported in a dimeric (pH = 8.5) and decameric (pH = 4.4) states and support the proposed model. Besides crystallography, site-directed mutagenesis and biophysical studies supported a model in which the histidine residue 113 (His¹¹³) acts as a pH sensor that may trigger LbPrx1m decamerization at acidic pHs. It is well known that the pK_a of the histidine side chain lies in the physiological range (imidazole group has a pK_a of approximately 6.0) and, therefore, upon protonation gains a positive charge. Therefore, in acidic pHs, His¹¹³ is protonated and can interact with the negative charge of the Asp⁷⁶ side chain (with a negative charge) from other dimer. Remarkably, Asp⁷⁶ side chain is located at the same loop that contain the reactive cysteine (so called peroxidatic cysteine = Cys_P). The inter-conversion between dimer and decamer occurs in narrow pH interval (between 7.0 – 8.0) and might have physiological consequences. Noteworthy, both His¹¹³ and Asp⁷⁶ are highly conserved not only in protozoa like Leishmania braziliensis, but also in bacteria, archaea, mammals and plants. Accordingly, the pH-dependent dimer to decamer conversion in a narrow interval was also described for human Prx2 and the chloroplast 2-Cys Prx from Pisum sativum. Processes such as apoptosis in mammalian cells and CO₂ fixation in plants are under modulation by pH variations. Further studies are required to fully understand the biological meanings of these distinct quartenary structures as well as the mechanisms that regulate switches among them, while these subjects appear to be relevant chapters for redox biology.

1. J. R. Harris. Release of a macromolecular protein component from human erythrocyte ghosts. Biochimica et Biophysica Acta (BBA)  - Biomembranes, 150 (3): 534-7, 1968. | dx.doi.org/10.1016/0005-2736(68)90157-0
2. M. A. Morais, P. O. Giuseppe, T. A. Souza, T. G. Alegria, M. A. Oliveira, L. E. S. Netto, M. T. Murakami. How pH modulates the dimer-decamer interconversion of 2-Cys peroxiredoxins from the Prx1 subfamily. | dx.doi.org/10.1074/jbc.M114.619205 Journal of Biological Chemistry, 290 (13): 8582-90, 2015.
3. H. Castro, F. Teixeira, S. Romao, M. Santos, T. Cruz, M. Flórido, R. Appelberg, P. Oliveira, F. Ferreira-da-Silva, A. M. Tomás. Leishmania mitochondrial peroxiredoxin plays a crucial peroxidase-unrelated role during infection: insight into its novel chaperone activity. PLoS Pathogens,  7 (10): e1002325, 2011. | dx.doi.org/10.1371/journal.ppat.1002325

Luis Eduardo Soares Netto, PhD. Professor at Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, Brazil

Redoxoma Highlights by Sayuri Miyamoto

Cholesterol is an important component of cell membranes and plays essential structural and signaling roles. It is synthesized in the endoplasmic reticulum and distributed to other cell membranes/compartments through a tightly regulated trafficking system involving vesicular and non-vesicular processes [1]. Cholesterol distribution among intra-cellular membranes is not homogeneous. Mitochondria are cholesterol-poor organelles (less than 5 %). However, mitochondrial cholesterol is increased in cancer cell lines and treatment of these cells with statins (cholesterol lowering drugs) increases their susceptibility to chemotherapy [2].

How mitochondrial cholesterol could influence cell death mechanisms? Cholesterol is easily oxidized by reactive oxygen species, giving rise to oxidized products, especially hydroperoxides and aldehydes. The study by Genaro-Mattos et al., 2015 [3] showed that cholesterol hydroperoxides react with cytochrome c, inducing a fast decomposition of the heme group of this protein (heme bleaching) and also the production of protein radicals. During this process, cytochrome-c underwent oligomerization, producting dimeric and trimeric species. These findings demonstrate that cytochrome c is seriously damaged in the presence of cholesterol hydroperoxides and this could inhibit its ability to induce apoptosis. Similarly, in a previous work [4] our group showed that cholesterol derived aldehydes are able to attach covalently on Lys residues (Lys 22 and Lys 8) located at the surface of cytochrome c, creating a hydrophobic tail in the protein. This modification caused a reduction in cytochrome c detachment from a model membrane in vitro. Overall, these studies indicate that cytochrome c can be modified by cholesterol oxidation products and it is tempting to speculate that these effects could have potential implications on cell death mechanisms.

1. E. Ikonen. Cellular cholesterol trafficking and compartmentalization. Nature Reviews Molecular Cell Biology, 9 (2): 125-38, 2008. | dx.doi.org/10.1038/nrm2336
2. J. Montero, M. Mari, A. Colell, A. Morales, G. Basañez, C. Garcia-Ruiz, J. C. Fernández-Checa. Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties, permeabilization and cell death. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1797 (6-7): 1217-24, 2010. | dx.doi.org/10.1016/j.bbabio.2010.02.010
3. T. C. Genaro-Mattos, R. F. Queiroz, D. Cunha, P. P. Appolinario, P. Di Mascio, I. L. Nantes, O. Augusto, S. Miyamoto. Cytochrome c reacts with cholesterol hydroperoxides to produce lipid- and protein-derived radicals. Biochemistry, 54 (18): 2841-50, 2015. | dx.doi.org/10.1021/bi501409d
4. T. C. Genaro-Mattos, R. F. Queiroz, D. Cunha, P. P. Appolinario, P. Di Mascio, I. L. Nantes, O. Augusto, S. Miyamoto. Covalent binding and anchoring of cytochrome c to mitochondrial mimetic membranes promoted by cholesterol carboxyaldehyde. Chemical Research in Toxicology, 26: 1536-44, 2013. | dx.doi.org/10.1021/tx4002385

Sayuri Miyamoto, PhD. Assistant Professor at Department of Biochemistry, Institute of Chemistry, University of São Paulo, Brazil

Redoxoma Highlights by José Carlos Toledo

Possibly, most pathophysiological processes involving the radicals nitric oxide (NO^•) and superoxide (O₂^•–) depend on their simultaneous production and their favorable co-reaction to produce peroxynitrite. Peroxynitrite is an oxidant itself but in the presence of carbon dioxide it gives origin to radical species such as carbonate anion (CO₃^•–) and nitrogen dioxide (NO₂^•) radicals, both capable of stimulating oxidative and nitrosative events that damage biomolecules. The interplay of NO^• and O₂^•– in biological environments is complex, though. Using a fluorescent probe molecule to monitor nitrosation in cells upon extracellular NO^• and O₂^•– co-generation, Espey et al. [1] found an unexpected behavior shaped like a circumflex accent (^). That is, nitrosation increases, peaks and then decreases as NO^• levels grow in excess vs. a constant flux of O₂^•–. The decreasing non-nitrosating phase was attributed to extracellular reactions of NO^• with CO₃^•– and NO₂^• radicals. Thus, unexpectedly, in the extracellular space NO^• both stimulates and may avoid nitrosation. But, that is not the whole story. Recent work from the CEPID-Redoxoma group led by Toledo and collaborators [2] adopted a strategy similar to that of Espey et al. [1], except that they did not generate O₂^•– extracellularly and, as a result, observed a distinct behavior. Nitrosation requires intracellular O₂^•–, but it increases until reaching a plateau and never falls when the NO^• grows in excess to O₂^•– (generating a ramp-plateau behavior). The existence of the plateau phase led to the conclusion that NO^• cannot intercept CO₃^•– and NO₂^• intracellularly to prevent nitrosation due to competition with more abundant targets of these radicals, including the fluorescent probe. At the same time, this study shows that O₂^•–-dependent nitrosative processes taking place by the same chemical mechanisms may exhibit distinct patterns in cells as NO^• levels increase. Nitrosation may occur by additional different mechanisms, but observation of either the circumflex accent or the ramp-plateau shaped profiles appear to be good indicators for the involvement of O₂^•– in cellular nitrosation events.

1. M. G. Espey, D. D. Thomas, K. M. Miranda, D. A. Wink. Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide. Proceedings of the National Academy of Sciences of the United States of America, 99 (17): 11127-32, 2002. | http://dx.doi.org/10.1073/pnas.152157599
2. F. C. Damasceno, R. R. Facci, T. M. Silva, J. C. Toledo Jr. Mechanisms and kinetic profiles of superoxide-stimulated nitrosative processes in cells using a diaminofluorescein probe. Free Radical Biology and Medicine, 77: 270-80, 2014. | http://dx.doi.org/10.1016/j.freeradbiomed.2014.09.012

José Carlos Toledo Jr, PhD. Professor at Department of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, Brazil

Redoxoma Highlights by Mauricio da S. Baptista

On the recent years, we aimed to understand the effects of visible light on skin and hair. We showed that melanin is able to photosensitize the generation of singlet oxygen both in the UVA (355nm) and in the visible (532nm) with similar yields and that the photosensitization of melanin forming singlet oxygen is the main cause of damage in hairs under exposition to visible light [1]. These results also suggest the possible role of visible light in damaging human skin, similarly to the well accepted effect of UVA light. In order to understand the effects of the photoinduced melanin reactions on human skin, we compared the effect of visible light on two melanocyte cell lines: one of them producing lots of intracellular melanin and the other just a natural amount. These results were recently published in [2].

By quantifying the amount of singlet oxygen production by its characteristic emission at 1270nm, we showed that intracellular melanin leads to an increased level of intracellular singlet oxygen generation. We also wanted to prove that the singlet oxygen generation by melanin photosensitization is able to cause direct damage on nuclear DNA. Therefore, we performed a comet assay and we observed that in the presence of melanin and visible light there clearly is Fpg and Endo III-sensitive modifications. The presence of strand breaks after the treatment with Fpg and Endo III, demonstrated that melanin photosensitization by visible irradiation induces direct oxidative damage to nuclear DNA. he ratio of Fpg- to Endo III-sensitive modifications indicated that oxidative damage in DNA is most likely due to both type II and type I mechanisms.

Clearly, visible light affects skin health, but people are encouraged to stay under the sun if they use sufficient amounts of ‘‘good sunscreen’’ (i.e., sunscreens that provide effective protection against UVA and UVB). This recommendation is clearly a mistake because it ignores the effects of visible light, which penetrates more deeply into skin than does UVB and UVA. We hope our research will stimulate the development of new comprehensive strategies of skin protection that also considers the effect of visible light.

Comment on:

1. O. Chiarelli-Neto, C. Pavani, A. S. Ferreira, A. F. Uchoa, D. Severino, M. S. Baptista Generation and suppression of singlet oxygen in hair by photosensitization of melanin. Free Radical Biology and Medicine, 51 (6): 1195-202, 2011. | http://dx.doi.org/10.1016/j.freeradbiomed.2011.06.013
2. O. Chiarelli-Neto, A. S. Ferreira, W. K. Martins, C. Pavani, D. Severino, F. Faião-Flores, S. S. Maria-Engler, E. Aliprandini, G. R. Martinez, P. Di Mascio, M. H. G. Medeiros, M. S. Baptista Melanin photosensitization and the effect of visible light on epithelial cells. PLoS One, 9: e113266, 2014. | http://dx.doi.org/10.1371/journal.pone.0113266

Mauricio da S. Baptista
PhD. Professor

Department of Biochemistry, Institute of Chemistry
University of São Paulo, Brazil

Redoxoma Highlights by Paolo Di Mascio

Historically, it was thought that plants derived all nitrogen (N) nutrition from the inorganic forms of N, NO₃^- and NH₄⁺. However, it is now known that the principal form of N entering soils that do not receive inorganic fertilizer is organic N derived from microbial breakdown of organic matter, including amino acids, di and tri-peptides, DNA and proteins. Plants form symbiotic associations with endophytic bacteria within tissues of leaves, stems, and roots. It is unclear whether or how plants obtain nitrogen from these endophytic bacteria. One possible mechanism for N transfer to host is that plants may scavenge organic nitrogen by oxidation and degradation of endophytic bacteria or their proteins using reactive oxygen species to lyse cells and denature proteins. This mechanism has been termed ‘oxidative nitrogen scavenging’.

The experiments reported in this work were done to evaluate whether ¹⁵N incorporated into bacterial endophyte biomolecules such as proteins and nucleic acids could be traced into plant molecules and whether the transfer process involves evidence of oxidative degradation of microbes. Agave tequilana and its seed-transmitted endophyte [¹⁵N]-labeled bacteria Bacillus tequilensis were employed to elucidate incorporation of organic [¹⁵N]-labeled nitrogen to plants. Incorporation of ¹⁵N into tryptophan, deoxynucleosides and pheophytin derived from chlorophyll a were traced. Probes for hydrogen peroxide show its presence during degradation of bacteria in plant tissues, supporting involvement of reactive oxygen in the degradation process. Endophytic bacteria potentially transfer more nitrogen to plants and stimulate greater biomass in plants than heat-killed bacteria that do not colonize plants but instead degrade in the soil. Findings presented here support the hypothesis that some plants under nutrient limitation may degrade and obtain nitrogen from endophytic microbes.

Comment on:

• M. J. Beltran-Garcia, J. F. White Jr., F. M. Prado, K. R. Prieto, L. F. Yamaguchi, M. S. Torres, M. J. Kato, M. H. G. Medeiros, P. Di Mascio. Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria. Scientific Reports, 4: 6938, 2014. | http://dx.doi.org/10.1038/srep06938

Paolo Di Mascio
PhD. Professor

Department of Biochemistry, Institute of Chemistry
University of São Paulo, Brazil

Redoxoma Highlights by Ohara Augusto

ALS (amyotrophic lateral sclerosis) is a fatal disease characterized by the degeneration of motor neurons, resulting in progressive muscle weakness, atrophy, paralysis and death. Most cases of ALS are sporadic but about 10% of the cases have a genetic basis.ALS-causing mutations have been identified in several genes, but the mutation of Cu,Zn superoxide dismutase gene (SOD1) is the most studied and responsible for about 20% of the familial cases. SOD1-linked ALS patients exhibit pathology and symptoms similar to those of sporadic ALS patients, leading to the hypothesis that both forms of the disease have a common pathogenic mechanism. This hypothesis led to many advances in the understanding of the disease but its pathogenic mechanism remains unclear. Our group has been exploring a role for oxidative modifications of hSOD1 in ALS pathology, based on several lines of evidence. Among them, the remarkable stability of hSOD1WT and several of its ALS-associated suggests that hSOD1 oxidation precedes its conversion to the unfolded and aggregated forms consistently found in ALS patients. Also, hSOD1 possesses a bicarbonate-dependent peroxidase activity, which oxidizes its own solvent-exposed Trp32 residue. The resulting products are specific for simian SOD1s, which contain the Trp32 residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1WT and hSOD1G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp32 residue in the process. The results showed that the Trp32 residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp32 residue (bovine SOD1 and hSOD1W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp32 residue, particularly the covalent dimer cross-liked by the ditryptophan bond, in triggering the non-amyloid aggregation of hSOD1. Therefore, the ditryptophan cross-link may become a useful target for therapeutic intervention but, to this end, further studies will be required. Nonetheless, these studies provided a novel route for the non-amyloid aggregation of hSOD1, a pathway that relies on an oxidative modification caused by its own bicarbonate-dependent peroxidase activity.

Comment on:

• F. R. Coelho, A. Iqbal, E. Linares, D. F. Silva, F. S. Lima, I. M. Cuccovia, O. Augusto. Oxidation of the tryptophan 32 residue of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity triggers the non-amyloid aggregation of the enzyme. The Journal of Biological Chemistry, 289: 30690-701, 2014. | http://dx.doi.org/10.1074/jbc.M114.586370

Ohara Augusto
Director of CEPID Redoxoma, PhD Professor

Department of Biochemistry, Institute of Chemistry
University of São Paulo, Brazil

Redoxoma Highlights by Marilene Demasi

Protein polyubiquitination was first described as a post-translational modification to direct proteins for degradation. The ubiquitin molecule is covalently bound to the target protein and the polyubiquitin chain is created by successive attachments of ubiquitin through its carboxy-terminal Glycine mainly to Lysine48 (K48) residues of previously conjugated ubiquitin. Proteins tagged with a K48-linked polyubiquitin chain are directed for degradation. However, distinct ubiquitin chains are built up through other Lysine residues from ubiquitin, resulting in distinct structural patterns of ubiquitin complexes, which in most cases are unrelated to target protein degradation.

The role of the ubiquitin-proteasome system during the oxidative stress response has been investigated for decades. In particular, there are major controversies regarding the way that oxidized proteins are degraded, especially whether they are or are not polyubiquitinated. A work recently published by Silva et al. in Nat. Struct. Mol. Biol. [1] unravels a novel important mechanism of oxidative stress response regulated through the formation of Lysine 63 (K63)-based polyubiquitin chains. Using the yeast S. cerevisiae as model, the authors showed that K63-linked polyubiquitin chains specifically accumulate when cells are challenged with H₂O₂. Ribosomal proteins were identified as preferential targets of K63 polyubiquitination. Additionally, the deubiquitinating (DUB) enzyme Ubp2, which supports the hydrolysis of K63-linked ubiquitins, was identified as the redox sensor of such process.

Although an important class of DUBs is dependent on a reactive cysteine in the catalytic site, their reactivity towards pro-oxidants has been poorly explored. In this work, Ubp2 inactivation by H₂O₂ was shown to determine the maintenance of the K63-linked polyubiquitin chains, which in turn stabilize the ribosome. Ribosomal stabilization was demonstrated to ensure increased translation of proteins related to antioxidant defense (i.e., Thioredoxin1, Peroxiredoxin1, Glutaredoxin2, Glutaredoxin5) and of other stress-response proteins, such as chaperones.

These results provide an important novel element for consideration regarding models of redox-dependent cellular signaling.

Gustavo M. Silva, the first author of the publication, started his scientific career as undergraduate student at Luis E. S. Netto’s lab (Institute of Biosciences - USP), supervised by Marilene Demasi (Institute Butantan), both members of the Redoxoma network, followed by a Ph.D in the same group on the redox regulation of the 20S proteasome catalytic unit [2]. He is presently a postdoctoral fellow at New York University. We are very pleased that his former training with our groups has provided relevant fruitful developments.

References:

1. G. M. Silva, D. Finley, C. Vogel. K63 polyubiquitination is a new modulator of the oxidative stress response. Nature Structural & Molecular Biology, 22: 116-23, 2015. | http://dx.doi.org/10.1038/nsmb.2955
2. G. M. Silva, L. E. S. Netto, V. Simões, L. F. A. Santos, F. C. Gozzo, M. A. A. Demasi, C. L. P. Oliveira, R. N. Bicev, C. F. Klitzke, M. C. Sogayar, M. Demasi. Redox Control of 20S Proteasome Gating. Antioxidants & Redox Signaling, 16(11): 1183-94, 2012. | http://dx.doi.org/10.1089/ars.2011.4210

Marilene Demasi
PhD. Professor

Laboratory of Biochemistry and Biophysics
Butantan Institute, Brazil