Chapter 8
What will be Next?
Abstract
This chapter, the last of this book, discusses possible directions along which the electrical control of biological reactions and phenomena are likely to develop in the years to come. The potential contexts encompass both biomedical and energy applications, foreseeing the involvement of both animals (humans included) and plants. The technological development of micro- and nano-electrodes and diffused power supplies (nano-batteries), along with the advances in the field of redox biology, will for sure suggest many more phenomena where direct electrochemical control is possible and advantageous in view of an always more pervasive extension of human technological control of living matter.
Keywords
angiotensinogen; angiotensin II; redox-regulated blood pressure; embryonic stem cells; Oct-4; thioredoxin; p53; plant growth and metabolism; implantable bio-fuel cells8.0. A pervasive presence of redox-controlled biosystems
The largest majority of relevant biological reactions and systems that are prone to be directly or indirectly affected by the action of electric fields are, as we have seen, quite complex and often still far from being fully unraveled both in their mechanistic aspects and in the number of consequences and implications that each of them brings about.
Redox biology, that branch of biology that focuses on understanding phenomena involving changes in the oxidation state of molecular partners, is constantly expanding the number of relevant cases that appear to be redox-regulated; moreover, these cases belong to an increasing variety of biological situations and contexts, ranging from cellular energetics, to the control of gene expression level, plant growth and development, stem cell differentiation, blood pressure, activity of p53, etc., just to quote some of the most relevant cases. As the understanding of these numerous and diverse phenomena deepens, it is very likely that the possibility of implementing technological control over them will also develop in turn.
In the next sections we will present some of these cases that appear particularly promising to us. It is, however, our firm conviction that future developments in science and technology will provide many more opportunities and exploitable cases on this topic than we can now even imagine.
8.1. Redox-dependent control of blood pressure
Blood pressure is regulated by many signals arising from the kidneys, blood vessels, heart and brain and any imbalance among them can give rise to altered blood-pressure values. One of these signaling pathways is that realized by the renin-angiotensin system. The enzyme renin is a protease that catalyzes the cleavage of the 10-amino-acid terminus at the N-terminus of its substrate, angiotensinogen. The result of the cleavage step is further processed to give rise to angiotensin peptide hormones, among which the best characterized is angiotensin II, whose effect is to increase blood pressure in various ways. Even if this hormone can activate a variety of cellular processes while binding to its cellular receptors, a shared aspect is the formation of reactive oxygen species (ROS) (Rajagopalan et al., 1996). As an example, the activation of angiotensin receptors can induce the production of superoxide and cause oxidative stress in the main cardiovascular target organs such as the kidneys, blood vessels and brain. Superoxide represents indeed a highly reactive free radical that can be converted to lipid-soluble products such as hydrogen peroxide and peroxynitrite. It has been shown in rat and mouse models of angiotensin-induced hypertension that treatment with antioxidant can decrease blood pressure, thus providing further support for the hypertensive effects of ROS as generated by the renin-angiotensin system.
One of the effects of oxidative stress is its action on cysteine amino acid residues in proteins. For example, interaction between H2O2 and the thiol group of cysteines yields the formation of a sulfenic acid intermediate that can react with cysteines, giving rise to disulfide bonds that can alter protein structure and function. This kind of oxidative protein alteration appears to play a role also in the control of hypertension. Indeed, angiotensinogen’s amino terminal tail, along with the angiotensin cleavage site, also undergoes a conformational rearrangement that makes it accessible to renin. The essential aspect of this conformational change seems to be the formation of a disulfide bridge between a pair of cysteines (C18 and C138) of angiotensinogen. The oxidized state of this protein correlates with its increased affinity for renin, hence, with an enhanced production of angiotensin that results in increased hypertension (Zhou et al., 2010). Interestingly,the C18-C138 disulfide appears to be redox sensitive, being reducible by the action of glutathione, which regenerates two unbound cysteine residues. This fact can have important physiological implications since endogenous nitric oxide, which is a potent dilator of blood vessels, hence reducing blood pressure, can react with cysteine thiols by S-nitosylation (addition of NO group), altering their chemical nature. Indeed, it has been found that nitric oxide can react with and block the thiols of C18 and C138, possibly preventing further oxidation of the two cysteines.
These findings point to a tight regulation of the redox state of angiotensinogen in tissues by intracellular chemicals such as ROS and nitric oxide. As a conclusion, the rate of angiotensin formation can be determined by the redox state of tissues and blood and by the absolute amount of angiotensinogen and renin present.
The described results suggest this redox biosystem as a potential target for direct electrochemical control and open up the way for considering this kind of approach also for application as a preventive/therapeutic means in humans.
8.2. Redox regulation of embryonic stem cell transcription factors by thioredoxin
We have already met the key role of bacterial thioredoxins in ruling the redox-dependent mechanism of gene expression regulation in photosynthetic bacteria (see section 6.2). Thioredoxins play a prominent role also in the control of gene expression in eukaryotic organisms and hereby an example is reported of their key role in regulating embryonic stem cell transcription factors.
Also in eukaryotic organisms some transcription factors are regulated by variations in environmental redox state. This is the case, for instance, for PEBP2, AP-1, p53, NF-κB. Their redox regulation occurs through conserved cysteine residues in the DNA-binding domains of these proteins (Hirota et al., 1997, 1999; Ueno et al., 1999; Akamatsu et al. 1997). Two intracellular enzymes exist that can restore the DNA-binding ability of oxidized transcriptional regulators by reducing critical cysteines: thioredoxin and APE-1/Ref-1.
Also lineage commitment and stage progression during embryonic stem cell development is controlled by transcription factors. Particularly, Oct-4 is one of the regulators, required to maintain the totipotentiality of embryonic stem cells. Indeed, down-regulation of its activity is required for proper differentiation of the blastocyst during uterine implantation. Uterine implantation and subsequent vascularization increase the developing embryo’s exposure to oxygen, altering, as a consequence, the intracellular redox state.
Oct-4 activity can be regulated by these changes in redox state. Particularly, oxidation abolishes Oct-4 binding to DNA, at variance with what happens in the case of another embryonic cell transcription factor – FoxD3. The role of thioredoxin appears ideally suited to restore the DNA-binding activity of Oct-4, whereas it is much less effective in restoring that of FoxD3. A mechanistic understanding of this fact traces the reasons for this difference back to the ability of thioredoxin (but not Ape-1) to associate with cysteines in the POU domain of Oct-4 (Guo et al., 2004). This fact indicates that there may be some specificity to this restoring effect and that individual redox regulators have specific targets. Furthermore, thioredoxin also has preferential targets in its reducing activity (e.g., acting much more effectively on Oct-4 than on FoxD3). Therefore, the intracellular redox state may produce different effects on distinct transcription factors that require specific redox partners to restore their activities.
The suggestive picture that arises from these results predicts that Oct-4 is preferentially expressed in the inner cell mass of the blastocyst that will give rise to the embryo. This inner cell mass is characterized by a lower oxygen tension than the surrounding cells, thus enhancing Oct-4 DNA-binding activity.
Since the blastocyst is also the location of thioredoxin requirements during gastrulation (Matsui et al., 1996; Kobayashi-Miura et al., 2002), the redox state of each cell in the blastocyst could define which cell is going to become part of embryonic tissue versus extraembryonic trophoectoderm. Upon uterine implantation and subsequent vascularization, the exposure to increased oxygen tension can down-regulate Oct-4 in the inner cell mass, enabling other less sensitive transcription factors to specify gastrulation and subsequent lineage differentiation.
The charming scenario described suggests a stimulating role of direct electrochemistry in controlling and actuating lineage differentiation in stem cells, allowing one to foresee extremely interesting scenarios in tomorrow’s regenerative medicine.
8.3. Role of p53 redox states in DNA binding
p53 is a transcription factor involved in maintaining the integrity of the genome. It responds to DNA damage by promoting cell cycle arrest in G1 phase (Cox & Lane, 1995; Elledge & Lee, 1995; Soussi & May, 1996) and in some cases by inducing apoptosis. In the last two decades, p53 has become one of the most important molecules in the field of cancer research. Indeed, p53 mutants, especially involving mutations in the protein core domain (which is deputed to binding to the p53 consensus sequence; Fojta et al., 1999), have been found in about 50% of human malignancies.
Generally speaking, the diverse functions of p53 are connected to its ability to bind DNA. This ability, in turn, is modulated by the domains of the action protein other than the core one. For example, deletion of the C-terminus gives rise to a p53 that is still active for sequence-specific DNA binding (Hupp et al., 1992; Jayaraman et al., 1997a) and in general the critical role of the basic C-terminus residues in regulating p53-DNA binding has been thoroughly studied. Furthermore, the redox/repair protein Ref-1 (Jayaraman et al., 1997b) is a strong activator of p53 sequence-specific DNA-binding both in vitro and in vivo. From a structural standpoint, it has been shown by X-ray crystallography of the human p53 core domain bound to DNA that the protein contains a zinc ion coordinated by amino acids C176, H179, C238 and C242 (Cho et al., 1994; Arrowsmith & Morin, 1996; Prives 1994). Moreover, chelation of Zn2+ abolishes sequence-specific DNA binding by p53 (Srinivasan et al., 1993; Hainaut & Milner, 1993; Hainaut, Butcher, & Milner, 1995).
The fact that the binding of p53 to its DNA consensus sequence is modulated by the redox state of the protein is of particular interest for the ideas presented in this book. Indeed, whereas the reduced protein binds to DNA, the oxidized counterpart appears to hamper irreversibly its ability to bind. This fact is traced back to the loss of the aforementioned Zn ion that is released upon cysteine’s oxidation and causes an irreversible structural destabilization in the p53 core domain.
The sensitivity of the p53 DNA-binding activity to its redox state suggests, therefore, the possible efficacy of direct electrochemistry in conditioning its state in order to control the protein’s activity in vivo.
8.4. Redox regulation in plants
Redox reactions are essential in plant cell metabolism, being involved in the majority of anabolic and catabolic processes. They take place in many cell sites, such as thylakoid membranes, plastid envelope, plasma membrane and in aqueous cell compartments such as the stroma, the thylakoid lumen and the cytosol in general. Electron-transport systems in cell membranes, i.e., in the photosynthetic and respiratory electron-transport chains, make use of various redox cofactors such as iron-sulfur clusters, quinones and photoexcitable systems that can generate reactive oxygen species. In aqueous-phase cell compartments, the redox state is ruled by metabolites that include NADH, NADPH, glutathione and thiol/disulfide proteins (Foyer & Noctor, 2009). In all the possible electron-transfer reactions that involve the mentioned redox molecules, oxygen can act as electron acceptor, giving rise to potentially harmful reactive oxygen species.
In cells there exists a specialized mechanism that balances redox metabolism and minimizes the formation of reactive oxygen and nitrogen species; this is a redox signaling network that can sense redox imbalance in the environment and react by readjusting redox homeostasis or repairing oxidative damage. Such a network is formed by redox sensors, redox input elements, redox transmitters and redox targets (Dietz, 2008). The basic structure and many components of the thiol-disulfide regulatory network are conserved among cells and cell compartments. In some cases its role is well established, whereas in others its significance is still patchy, due to the ongoing discovery of novel redox targets.
8.4.1. Redox control of plant metabolism
The overall reducing power in plant cells come from the light-induced electron transfer from water to NADP+ that is performed by the photosynthetic apparatus in the thylakoid membranes of chloroplasts. By this process (see section 3.7.3) the electrons are able to cross a total redox potential difference of 1.13 V, which is high enough to power all the subsequent redox reactions in the cell.
The generated reducing power is used by the plant cell mainly in three different types of processes that are interconnected. These are anabolic reactions of metabolism, the antioxidant systems and the redox regulatory system. In metabolism, NADPH is often directly exploited as a cofactor in enzymatic reactions, mainly in anabolic reactions synthesizing molecules of higher complexity or energetic content, for example carbohydrates in the Benson-Calvin cycle or reduced intermediates in sulfur or nitrogen metabolism. Reduced substrates can be used to generate reduction equivalents in the dark or in non-photosynthetic tissues, thereby enabling the plant to uncouple redox-dependent reactions and light reactions of photosynthesis.
Among the aforementioned three types of processes where the reducing power generated by photosynthesis is used, the redox regulatory dithiol/disulfide system is by far the most complex one. It consists of a large number of molecular components that are organized in a hierarchical and highly interconnected network.
Thiol regulation of the activity of Benson-Calvin-cycle enzymes connects light-dependent electron pressure in photosynthetic reactions to ATP and NADPH consumption in reductive carbohydrate metabolism. The regulatory mechanism may be regarded as a prototypical feed-forward activation loop. Indeed, the thiol state-dependent regulation of carbon fluxes through the Benson-Calvin cycle and their link to thioredoxin (thioredoxin-f)-mediated activation of chloroplast FBPase marked the starting point of long-lasting (over 30 years) research into redox regulation in metabolism (Buchanan & Balmer, 2005). In addition to FBPase, sedoheptulose- 1,7-bisphosphatase, activities of ribulose-5-P kinase, glyceraldehyde-3-P dehydrogenase and rubisco activase are controlled by thioredoxin. Thioredoxin-f donates electrons to target proteins characterized by a broad range of redox midpoint potentials (Hutchison et al., 2000). Differential inactivation of target proteins, e.g., in the Calvin cycle, is not related to the value of midpoint potential but is highly relevant for photoinhibition under non-optimal environmental conditions such as low temperatures (Hutchison et al., 2000). This complexity is partially explained by the fact that thiol modulation is connected to additional metabolic control systems, e.g., the presence of Fru-1,6-BP is needed for FBPase thiol activation (Reichert et al., 2000). Two main carbon pathways drain carbon from the Benson-Calvin cycle, namely Suc synthesis following export of triose phosphate to the cytosol and starch synthesis in the plastids. The committed step of starch synthesis is catalyzed by AGPase. AGPase is activated by reduction of a disulfide bridge between the two slightly smaller subunits of the tetrameric holoenzyme in vitro (Ballicora et al., 2000) and in vivo (Tiessen et al., 2002). Reduction is achieved by thioredoxin-f and thioredoxin-m in vitro and allows for a 4-fold stimulation of ADP-Glc synthesis (Ballicora et al., 2000). A good correlation has been found between Suc concentration, reduction state of the chloroplast and starch synthesis (Tiessen et al., 2002; Geigenberger et al., 2005). NTRC also reductively activates AGPase. NTRC-deficient Arabidopsis show less redox-dependent stimulation of AGPase activity and lower starch synthesis rates in high light and upon external feeding of Suc. Inhibition in NTRC knockout plants ranges between 40% and 60% in leaf chloroplasts and reaches 90% in non-photosynthetic amyloplasts (Michalska et al., 2009).
In addition to redox regulation in carbohydrate metabolism, proteomic and biochemical data indicate that thiol modifications also control other major metabolic pathways such as nitrogen assimilation, tetrapyrrole synthesis and lipid synthesis (Lindahl & Kieselbach, 2009).
Lipid synthesis that occurs in the plastids is a strong sink for electrons. Synthesis of palmitic acid (C16) from acetyl-CoA requires 14 molecules of NADPH and seven molecules of ATP. The plastid redox state affects lipid metabolism. Acetyl-CoA carboxylase (ACCase) catalyzes the committed step of malonyl-CoA production in plastid lipid synthesis. Isolated ACCase in vitro is inactive without reductant and activated after addition of DTT or reduced thioredoxin-f or thioredoxin-m (Sasaki et al., 1997). Reductive activation is supported by a pH shift to alkalinization and by increasing Mg2+ concentrations.
The chloroplast ACCase consists of four polypeptides, the biotin carboxylase, biotin carboxyl carrier protein, transcarboxylase a-subunit and transcarboxylase b-subunit, with three, one, two and five cysteine residues, respectively (Sasaki et al., 1997). One of the a- or b-subunits is suggested to mediate the redox regulation (Kozaki & Sasaki, 1999). The biotin carboxyl carrier subunit of ACCase in Chlamydomonas reinhardtii is subjected to S-thiolation with glutathione (Michelet et al., 2008). Biotin carboxylase is the target of glutathionylation in Arabidopsis cell culture (Dixon et al., 2005). Thus, each of the subunits of ACCase is potentially controlled by redox regulation using diverse mechanisms.
This fact underlines the connection between redox state and lipid metabolism. Envelope-bound monogalactosyldiacylglycerol synthase (MGD) synthesizes monogalactosyldiacylglycerol from diacylglycerol and UDP-Gal. Monogalactosyldiacylglycerol is a major lipid component of chloroplasts. In vitro MGD activity that depends on the presence of reductants (e.g., DTT) is inhibited by thiol-alkylating agents, and is modulated by thioredoxin acting on intramolecular disulfide bonds (Yamaryo et al., 2006). Plant MGD possesses nine conserved cysteine residues. Its regulation by the thiol redox state is thought to enable galactolipid synthesis along with photosynthetic activity and to foster replacement of oxidized lipids under conditions that cause oxidative stress (Yamaryo et al., 2006).
8.4.2. Redox regulation of gene transcription in plastids
Plastids possess their own genome, the plastome, and a complete molecular machinery to express the genetic information in it. Although this plastome encodes approximately just 120 genes (in vascular plants) the expression mechanisms appear to be rather complex and highly regulated. This includes a number of redox control mechanisms that influence regulatory proteins at any important level of gene expression, i.e., transcription, posttranscriptional modification and translation initiation (Pfannschmidt & Liere, 2005).
A major target of photosynthetic redox signals is the plastid-encoded RNA polymerase (PEP). Unbalanced excitation of the two photosystems generates either a reduced or oxidized pool of PQ that act as signals that control the phosphorylation of the light-harvesting complexes of PSII via the thylakoid-associated kinase STN7 (Lemeille & Rochaix, 2010; Pesaresi et al., 2010).
The same signals also trigger a phosphorylation cascade towards the PEP enzyme that results in alterations of photosynthesis gene expression (Allen & Pfannschmidt, 2000). Both processes have the effect of counteracting the unbalanced excitation and maintaining the highest photosynthesis efficiency. The phosphorylation cascade probably includes the action of a number of further kinases (STN8, an ortholog of STN7; CSK, the chloroplast sensor kinase; PTK, the plastid transcription kinase), generating a phosphorylation network. In a simplified view, the reduction of the PQ pool activates STN7, which provides an input signal for the subsequent kinase network. This controls the phosphorylation state of the sigma factor Sig1 that in turn regulates the relative transcription of the photosynthesis reaction center genes psbA (encoding the D1 protein of PSII) and psaA/B (encoding the P700 apoprotein of PSI; Shimizu et al., 2010). This view is consistent with the observation that CSK, PTK and Sig1 are able to interact with each other in the yeast (Saccharomyces cerevisiae) two-hybrid system (Puthiyaveetil et al., 2010). In organello transcription experiments in the presence of kinase inhibitors and/or the reductant DTT, however, indicate that this phosphorylation-dependent signal interacts with a second, thiol-dependent signal (Steiner et al. 2009). PTK, a casein-kinase 2 type enzyme, has been reported to be under control of the redox state of glutathione (Ogrzewalla et al., 2002), but its activity could not be modulated with DTT. This suggested the involvement of a further regulator.
Recently, two independent studies identified a novel thioredoxin-like protein that probably represents the sought-for additional player (Arsova et al., 2010; Schröter et al., 2010). The novel thioredoxin was named thioredoxin-z because of its distinct evolutionary position in relation to thioredoxin-x and thioredoxin-y. In a yeast two-hybrid screen it has been identified as an interacting protein of two chloroplast-located phosphofructokinase-like proteins called FLN1 and FLN2. The Arabidopsis knockout mutant line of thioredoxin-z exhibits pale-white leaves and is viable only on Suc-supplemented medium, a unique phenotype since the thioredoxin system is highly redundant and can easily compensate for the loss of single components. Gene expression analyses indicated the same plastid gene expression profiles as in PEP-deficient mutants, pointing to an important role of thioredoxin-z in plastid development and gene expression (Arsova et al., 2010). These observations have been complemented by mass spectrometry results that demonstrate that both the thioredoxin-like protein and the FLN2 kinase are intrinsic subunits of the PEP enzyme of chloroplasts (Schröter et al., 2010). This provides a direct explanation for the phenotype and the expression profiles in the knockout mutant. A lack of thioredoxin-z prevents proper assembly of the PEP enzyme and, as a consequence, the developmental transition from the nuclear-encoded RNA polymerase-driven transcription to the PEP-dependent transcription does not take place. The precise functional role of thioredoxin-z within the PEP complex, its relation to FLN1 and FLN2, as well as its regulatory impact remain to be elucidated. Furthermore, it is still a matter of debate how it relates to the other known redox regulators mentioned above. In summary, our understanding of photosynthetic redox signal transduction towards the level of gene expression is still at the beginning. The increasing number of identified regulatory components unravels a complex mechanistic redox tool-box enabling chloroplasts to respond to a wide range of environmental conditions in a dynamic and flexible manner. The elucidation of the role of the regulatory components will help in selecting the pathways that will be more prone to be directly under external electrochemical control.
8.5. The electrified snail
We conclude the review of possible biological systems that appear to be potentially suitable to direct electrochemical control with a beautiful example (Halámková et al., 2012) that involves many of the concepts we have introduced in the chapters of this book. It demonstrates a bio-fuel cell directly implanted on a living snail that is fueled by the glucose the snail gets with its food. This is the first example of time-sustainable generation of electrical power in vivo and shows that metabolic activity can “recharge” the living battery for continuous production of electricity.
The critical input provided on the one hand by a deep knowledge of bioelectrochemistry and surface biofunctionalization, and the potential expressed by the involvement of nanotechnologies on the other hand, allowed the setting-up of a biocompatible, efficient and robust electrode pair made of compressed carbon nanotubes modified with PBSE linker (a heterobifunctional cross-linker, 1-pyrenebutanoic acid succinimidyl ester) and then functionalized with laccase or PQQ-GDH (pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase) to yield the biocatalytic cathode or anode, respectively. The implantable electrodes were inserted into the snail through two holes cut in the shell and placed into the hemolymph (i.e., the snail’s blood) between the body wall and internal organs (visceral mass). The implanted electrodes performing the bioelectrocatalytic reactions (glucose oxidation at the anode and O2 reduction at the cathode) were connected to a circuit composed of an external variable-load resistance; voltage and current were measured during biofuel cell operation in vivo.
The open-circuit voltage and short-circuit current achieved in the biofuel cell were 530 mV and 42.5 μA (current density 170 μA cm−2), respectively. The sustainability of the implanted biofuel cell was tested by measuring the voltage and current produced over time on the optimum load resistance of 20 kΩ (corresponding to the internal resistance of the cell and providing a maximum power of 7.45 μW (i.e., a power density of about 30 μW cm−2). It turned out that the electrical output decreased rapidly upon cell operation; however, it was effectively restored when the current extraction was interrupted for 30−60 min to allow the snail to rest. The biofuel cell operation was reproducible even after a period of 2 weeks and was not affected by enzyme inactivation and/or biofouling due to the biological environment.
This experiment demonstrates clearly the state of development of the enabling technology that is needed for connecting living beings to an external electrical circuit. Its success represents an encouraging starting point for future, more sophisticated implementations of direct electrochemical control of biological systems and reactions in vivo. We firmly believe that we are at the beginning of a new, charming adventure in human technology that will soon provide results that are, to date, still far beyond our imagination.
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