2 3 Semiotic Selection of Mutated or Misfolded Receptor Proteins

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Biosemiotics ISSN 1875-1342Volume 6Number 2 Biosemiotics (2013) 6:177-190DOI 10.1007/s12304-012-9143-7

Semiotic Selection of Mutated or MisfoldedReceptor Proteins

Franco Giorgi, Luis Emilio Bruni &Roberto Maggio

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ORIGINAL PAPER

Semiotic Selection of Mutated or MisfoldedReceptor Proteins

Franco Giorgi & Luis Emilio Bruni & Roberto Maggio

Received: 28 September 2011 /Accepted: 25 November 2011 /Published online: 14 March 2012# Springer Science+Business Media B.V. 2012

Abstract Receptor oligomerization plays a key role in maintaining genome stabilityand restricting protein mutagenesis. When properly folded, protein monomers assem-ble as oligomeric receptors and interact with environmental ligands. In a gene-centered view, the ligand specificity expressed by these receptors is assumed to becausally predetermined by the cell genome. However, this mechanism does not fullyexplain how differentiated cells have come to express specific receptor repertoiresand which combinatorial codes have been explored to activate their associatedsignaling pathways. It is our contention that the plasma membrane acts as the locuswhere several contextual cues may be integrated. As such it allows the semioticselection of those receptor configurations that provide cells with the minimumessential requirements for agency. The occurrence of protein misfolding makes itimpossible for receptor monomers to assemble along the membrane and to sustainmeaningful relationships with environmental ligands. How could a cell lineage dealwith these loss-of-function mutations during evolution and restrain gene redundancyaccordingly? In this paper, we will be arguing that the easiest way for bacteria clonesto accomplish this goal is by getting rid of cells expressing mutated receptor proteins.The mechanism sustaining this cell selection is also occurring in many somatic tissuesand its function is currently believed to counteract in vivo protein mutagenesis. Ourdiscussion will be mainly focused on the significance and semiotic nature of theinterplay between membrane receptors and the epigenetic control of gene expression,as mediated by the control of mismatched repairing and protein folding mechanisms.

Keywords Receptors . Mismatch repairing . Protein misfolding . Cell death

Biosemiotics (2013) 6:177–190DOI 10.1007/s12304-012-9143-7

F. Giorgi (*)Neuroscience Department, University of Pisa, Pisa, Italye-mail: [email protected]

L. E. BruniDepartment of Architecture, Design and Media Technology, Aalborg University, Copenhagen, Denmark

R. MaggioDepartment of Experimental Medicine, University of L’Aquila, L’Aquila, Italy

Author's personal copy

Introduction

Receptor oligomerization can be defined as the molecular aggregation of two or morereceptors expressing a novel function as respect to that of the constituting oligomericsubunits. As such, it is known to play a key role in cell interactions. We have recentlyshown that, in this interactive process, the plasma membrane acts as a semioticthreshold. This is because cells are allowed to define an inside/outside border line(Kull 2009) and a first level of selectivity vis a vis the extracellular milieu (Giorgi etal. 2010). The ligand specificity expressed by membrane receptors is primarily due totheir ability to cluster as self-assembled supra-molecular structures held togetherby a number of feeble chemical bonds. The nature of these intermolecularinteractions along with their ligand specificity can be accounted for by a numberof chemical mechanisms that predominantly involve the transmembrane domainsof the receptor, rather than the hydrophilic loops leaning out of the membranelipid bilayer (Fanelli 2007).

Receptor-ligand interactions can be explained mechanistically in causal terms, andconsequently their occurrence and behavior can be easily predicted under a number ofphysiological conditions. However, regardless of the resoluteness of causal explana-tion, the question of how membrane receptors interact with only certain environmen-tal ligands and how their specificities have been gradually defined during evolution,cannot be answered mechanistically. We have previously argued that these types ofexplanations have to be complemented by a biosemiotic type of approach wherebyreceptor ligand interactions may also be interpreted as meaningful relationships(Giorgi et al. 2011) and be identified as sign(s) by cells behaving as semiotic systems(Gorlich et al. 2011). In a biosemiotic perspective, single cells as well as multicellularorganisms are conceived as autonomous agents capable of interpreting the signifi-cance of their surroundings. They are constitutively endowed with the ability tochoose, based on their encoded responding repertoire and anticipatory behavior(Giorgi and Bruni 2011).

For a protein domain to be functional, there has to exist a number of well definedclasses of correspondences such as to allow it to interact productively with othermolecular partners occupying the same spatial compartment or the same timeframe.Since these correspondences constitute a network of interacting codes, they ought tobe selected as wholes and not as separate units of energetically constrained pathways.This entails that whatever is being selected in nature – genes, cells or even organismsand species – they do so not because of their autonomous self-breeding capacity, butfor their being part of larger systems of correspondences (Bruni 2008).

In this paper, we will be reviewing some of the mechanisms that nature has devisedto allow cells to face the uncertainties of the external milieu. Any variation in theenvironmental setting – be it due to changes in temperature, pH or ligand composi-tion, or to mutational events - may be experienced as a stress, and consequentlysensed as a challenge for cell survival. Cells recovering from environmental distur-bances and overcoming any of these stressful effects are not simply restoring theiroriginal conditions. They are instead adjusting to potentially new situations whichmay lead them to evolve into more efficient living forms. Robustness and evolvabilityare thus two opposing poles within which life forms are allowed to change withoutcompromising their own survival. Robust systems are likely to persist, provided

178 F. Giorgi et al.


environmental conditions are stable enough not to challenge their tolerance range. Onthe other hand, evolvable systems are capable of adjusting to unforeseeable situa-tions, but they also run the risks of producing too many costly variations that mayultimately compromise their own survival. Balancing between these two extremeconditions is not a matter of controlling gene expression, i.e., which mechanism(s) toactivate in any particular circumstance and how to respond accordingly. Stressfulenvironmental conditions are first to be interpreted in the light of the respondingrepertoire available to the cell, and, secondarily cost-effective alternatives have to beselected to make stressful effects remediable. If no adequate responses are available,cells become irremediably damaged and eliminated.

In biosemiotic terms, we talk about interpretation because cells subject to stressfulconditions must prove capable of (1) monitoring the nature and the extent of thestress, (2) establishing whether mechanisms restoring functionality are to be activatedand (3) anticipating whether remediable interventions are preferable over activationof the cell death program. In this paper, the role played by receptor oligomerization incells facing stressful conditions will be evaluated in the light of the above arguments.In particular, we will ask how much receptor monomers are allowed to vary duringevolution – by either replication mismatch or genetic drift – and yet conserve theirinteracting propensity for the environmental ligands. We will also take into accountmechanisms that bring cells to an unavoidable death, and show how this process isaccomplished according to the tissue economy and proper cell turn-over require-ments. The assumption underlying these questions is that repairing mechanismsreinforce cell robustness, and at the same time make it impossible for heritablephenotypic variations to be produced in excess. By contrast, failure to restore theoriginal genetic situation may lead cells to explore new encoding functions or beselectively removed from the tissue context (Wagner 2008).

In synthesis, whenever stressed by adverse environmental conditions, cells may proverobust enough to restore their original genome condition(s), or be so evolvable to generatesufficient numbers of gene variations to ultimately establish new complex genomicsettings. In the event that both strategies fail, cells are irremediably destined to death. Inour view, whenever these alternatives are recognized as signs challenging their survival,cells prove capable of interpreting them as semiotically meaningful relationships.

Mismatch Repair

A major event causing loss-of-functions in membrane receptors is the occurrence ofmismatch base pairing during DNA replication. The extent of variation in the resultingtranslational product depends on both the position of the mismatched base pair and thechemical nature of the amino acid being eventually substituted. Given the importanceplayed by receptor oligomerization in the process of cell-to-cell interaction, the extent ofalteration induced in the receptor assembly is likely to be correlated with the proteindomain that is primarily affected by this substitution. A number of recent reports haveclearly shown that major phenotypic alterations of many receptor proteins are caused bymutations of the trans-membrane domains that make them incapable of interacting withtheir respective ligands (Biebermann et al. 2001; Gallagher et al. 2007). In the absenceof any repairing mechanisms, these events could be frequent enough to cause many of

Semiotic Selection of Receptors 179


the affected cells to die prematurely. On the other hand, the rarity of these recessivemutations suggests that mechanisms for repairing mismatched base pairings mighthave evolved in nature to minimize their phenotypic effects and reduce the incidenceof cell death. Any decrease in receptor activity or impediment of receptor ligandinteraction may ultimately affect the extent in which cells communicate with eachother, and thus alter such essential processes as cell adhesion and migration.

A proof-reading function is already known to reside in the exonuclease activity ofthe DNA polymerase that corrects mismatched pairing during replication (Hopfield1974). However, bacteria and eukaryotes are endowed with an additional repairingcapacity to detect mismatched base pairs in newly synthesized DNA daughter strandsand target them for proper base substitutions (Yang 2000). For this mechanism tocorrect efficiently any replication error, the newly synthesized DNA strand must beclearly distinguished from the parental one. This selectivity is attained by relying onmechanisms that discriminate the extent of adenine methylation of the two DNAstrands, a clear instance of categorical sensing and a key element in cell semioticcommunication due to the system’s ability to detect meaningful differences (Wagneret al. 1984; Dohet et al. 1985; Bruni 2007).

The complexity of the DNA mismatch repair and the underlying molecularinteractions required to sustain this mechanism have recently been clarified in greaterdetail. In general, methyl directed processes are part of what could be referred to as amethylation code which works by instantiating and recognizing methylation patternsduring DNA replication or cell differentiation. With the sole exception of develop-ment, where early embryonic cells can actually reset their epigenetic marks to targetdifferentiation along specific cell lineages, DNA methylation patterns are faithfullyinherited by daughter cells according to the cellular memories of their differentiatedepigenome (Bronner et al. 2009).

In conclusion, while DNA replication is a rather faithful process, DNA poly-merases make occasional mistakes by inserting the wrong nucleotide into a base pairsequence, disregarding the complementarity rules of DNA strand pairing. This mayhappen whenever nucleotides are chemically altered by tautomerization or ionization,such that their misinsertion upsets the pairing rules of the Watson–Crick geometry(Bebenek et al. 2011). If these replicating mistakes were not corrected, the mutatedDNA strand could serve as a template for subsequent replications to be maintained ascoding errors. Given the rate of polymerase base misinsertions and the dimension ofthe nucleotide sequences to be replicated in any organism, it may be easily compre-hended why DNA repairing mechanisms are so crucially important: they make DNAreplication compatible with life by restraining the number of genetically fixablemutations. Mismatched repairing proteins are in fact highly conserved in all organ-isms from bacteria to mammals, because any defective mismatch repair occurring inthis mechanism may inevitably lead to higher incidence of lethal or chronic diseases(Harfe and Jinks-Robertson 2000; Doutriaux et al. 1986). However, in spite of theexistence of these repairing mechanisms, a few mismatches may overcome theirselecting sieve and persist as bad mutations. But not all mutations are bad, as Pray(2008) says, since some of them may provide sufficient genetic variability forevolution to work on by natural selection. Therefore, while a faithful DNA replicationprocess would certainly produce no mutations, a slight degree of infidelity is never-theless required to make it compatible with the production of evolvable variations.



Adaptive Mutations

According to the above conclusion, evolution could only make use of those genevariations that are not remedied, and that are therefore capable of bypassing theprocess of mismatch repairing and maintain their functionality within the range ofvariability comprised of these fidelity limits. However, if this were the case, muta-tions could only arise randomly and be selected without any utility for the resultingphenotype. In spite of the long held belief that mutations originate spontaneouslyduring genomic replication, there is now a large body of evidence indicating thatbacteria exposed to stressful conditions are more likely to produce useful mutants,rather than blind variations. These type of mutations have been variously referred toas adaptive, selection-induced (Foster 1993), or even Cairnsian adaptive mutations(Roth et al. 2003). Utility in this context is defined by the ability to devise new copingstrategies from those phenotypic variations that are ultimately selected, even thoughmutations are still random and the extent of variations depends only on an increasedmutational rate. In this section, we will examine the evidence available today tosubstantiate the view that, under certain stressful circumstances, microbe populationsare more likely to enhance their rate of mutability and consequently to produce moreuseful variant forms.

New phenotypic traits may emerge from individuals bearing variable genotypes,while the overall population mutability results from the probability of the mutatedoffspring to be favorably selected. It follows that individual mutability may notnecessarily coincide with a gain in fitness for the whole population gene pool. Thetwo levels of gene expression should in fact be thought of as independent processes.A population’s mutability rate may turn out to be evolutionarily advantageous only ifdecoupled from that of the individual: what the population gains in fitness mayrequire an equivalent decrease in individual fitness (Lampert and Tlusty 2009). Thepopulation gene pool is in fact defined by a first level of information storageequivalent to the primary heritable knowledge expressed by a collection of specificgenotypes. Individuals require instead a second level of knowledge defined by alladaptable phenotypes that may result from a variable epigenesis (Vehkavaara 1998).

Any increase in the degree of population’s variability is a sine qua non conditionfor the selective pressure to install new adapting strategies. Newly appearing variantclones may in fact compete with one another and allows persistence of only those thatprove beneficial for population survival. Beneficial mutations may be fixed in largepopulations, provided the frequency of variation is high enough to overcome thegenetic drift and reduce the probability of extinction. Both these conditions arefavorably met when genomic mutability and clone selection allow competing bene-ficial mutations to be fixed in the same population even in the absence of generecombination (Sniegowski and Gerrish 2010). But how are adaptive mutationsproduced by an enhanced gene variability? The general consensus today is thatmicroorganisms subject to nonlethal selective conditions, respond by adjusting theactivity of an error-prone DNA polymerase (Layton and Foster 2005). As a rule,DNA polymerases are high-fidelity enzymes that accurately replicate the cell’sgenome, but low-fidelity DNA polymerases may also be activated under specificcircumstances and produce selectively advantageous gene variants. Thus, when risksand benefits of a low-fidelity DNA synthesis are properly filtered through the



selective pressure of a hostile environment, the survival strategy of the populationbecomes strictly correlated with the likelihood to be favorably selected (Rattray andStrathern 2003). In other words, new mutations may eventually be fixed in thegenome and be categorically sensed as adaptable responses to the contextual stateof affairs by simply fine-tuning the level of error tolerance of the DNA polymerase.

According to the so called quasispecies theory, originally put forward by Eigen(1971), there has to be a threshold in the mutation rate above which the replicatingpopulation – be it made of macromolecules, viral particles or bacteria – wouldinevitably be extinct. This critical point is reached when a population replicatingwith a high-fidelity genotype is gradually transformed into a multi-clone populationcharacterized by low-fidelity genotypes. Under these conditions, mutations can nolonger produce beneficial effects on the resulting phenotype, and no master genotypecan therefore be saved. Kamp et al. (2003) have experimentally determined that for avirus to remain viable, new gene mutations need to be generated every time the hostimmune system has just reacted to a new viral epitope. This optimal mutation rateallows the viral genome to stay always ahead of the immune system, and to renderany viral change adaptable to the co-evolving dynamics of the host. All theseconsiderations lead us to conclude that a major characteristic of natural systems istheir adaptability through the stochastic generation of genotypic variants.

In strictly causal terms, these mechanisms could be interpreted deterministically asif due to the expression of a pre-determined gene program, establishing or condition-ing in advance a sequence of gene activations. However, if this were the case, itwould be difficult to explain how stressful conditions could induce such a variety ofalternative choices, from mismatch repair to error-prone replication, or even to celldeath. It is not in the nature of the stimulus to determine the extent of the system’sresponding capability, but in the system ability to choose amongst various alternativesmade available by its responding repertoire. From a biosemiotic point of view, we aremore inclined to think of these cell behaviors as due to the exploration of anevolutionary sustainability, followed by the genomic fixation of those phenotypiceffects that have been positively explored. This indicates that no final objective canbe known in advance and only information and experience can make it sustainable bya process of gradual adaptation to ever changing circumstances. This strategy is notthe result of some kind of teleologically directed process or some randomly capturedcoincidence, but of natural selection acting on both the organism and its environmenttogether. This organism-environment co-evolutionary dynamics is what Bateson(1979) has referred to as unit of survival in order to emphasize the notion of flexibilityand reciprocal adaptability. In the long run, the occurrence of stochastic muta-tions may cause the appearance of different phenotypes, but it is only theenvironmental pressure that can determine which system trait may persist andwhat kind of functional optimization may be contributed to the whole popula-tion (Manrubia et al. 2010).

Protein Misfolding

So far only those genetic mistakes that arise during DNA replication have beenconsidered. However, cells subject to stressful environmental conditions are not only



challenged genetically, but also at the translational level. Under stressful conditions,some of the newly synthesized proteins in the endoplasmic reticulum may not foldproperly, undergo misfolding or even unfold. In any of these circumstances, proteinmisfolding is generally prevented by the intervention of members of the chaperonefamily to guarantee essential cell functions (Fink 1999). If misfolded proteins are notproperly refolded through chaperone assistance, they are removed from the endo-plasmic reticulum and brought to the proteasome for degradation. Even if theendoplasmic reticulum is not cleared of all misfolded proteins, the resulting conges-tion targets the cell for apoptosis, as due to excessive retention of molecular aggre-gates (Anelli and Sitia 2008). The question raised by these observations is howmisfolded and properly folded proteins can be distinguished, and, above all, howcells decide whether to refold or degrade them before viability is fully compromised.Recently, it has been demonstrated that specific aromatic sequences can be recog-nized in unfolded polypeptides of Escherichia coli and be directed to an ATP-dependent proteolysis, in spite of being resistant to proteolytic degradation whenfully folded in native proteins. This suggests that bacterial proteases may recognizeonly the string of unfolded amino acids and not those embedded in the hydrophobiccore (Gur and Sauer 2008).

The existence of protein folding mechanisms makes some phenotypic variationscausally independent of genetic polymorphisms. As long as biologists were explain-ing the acquisition of new functional traits as simply due to genetic differences, theywere implicitly assuming that only genes could determine the resulting phenotypicoutcomes. On the other hand, by understanding how genotypic variations are grad-ually transformed into phenotypes, and how they affect fitness, biologists have cometo appreciate the molecular mechanisms by which other forms of adaptation may beacquired. Protein misfolding is one of such mechanisms, whereby translationalmistakes are not genetically caused by replicative mismatches, but result insteadfrom the ribosome infidelity of the translational processing. As such, they areequivalent to simple epigenetic variations of error-free proteins, rather than togenetically-induced mutations.

To compensate for the phenotypic impact of protein misfolding affecting mem-brane receptor proteins, a strong selective pressure might have been exerted duringevolution on translational accuracy by positioning optimal codons in conservedamino acid sequence sites [see Hershberg and Petrov (2009) for a definition ofoptimal codons]. To account for these constraining factors, Drummond and Wilke(2008) have recently proposed that misfolded proteins may contain more translationalerrors than properly folded proteins, and that they are unlikely to occur in highlyexpressed genes. This implies that proteins playing essential roles in cell viability –such as membrane receptors specific for cell-to-cell and cell-to-environment inter-actions - may be less evolvable than other more abundant and redundant geneproducts. An interesting way to visualize pictorially these differences is through therepresentation of the so called bow-tie network (Csete and Doyle 2004), whereby avariety of metabolic stimuli are assumed to converge toward a sub-network ofcorrelated cell responses, so as to make their input–output relationship topologicallysimilar to the two lateral edges of a bow-tie figure. The nodes in the core region ofthis figure may vary, both in number and functionality, depending on the cellularcontext. For instance, the continuous flow of metabolic nutrients may be depicted as a



network of complex biochemical interactions, together forming a heterogeneous androbust nodal cluster in the central core. By comparison, the network of cell signalingpathways transducing environmental stimuli into specific cell responses appears toinclude fewer and more sparse interconnecting nodes. This remarkable differencemay be taken to mean that the smaller the number of molecules occupying the core ofthe bow-tie figure, the higher the possibility for external stimuli to be clustered intofewer separate classes (Polouliakh et al. 2009). And in turn, the fewer the nodesmediating these molecular interactions, the higher the learning capacity for catego-rizing a broad range of environmental stimuli into related cell responses. This impliesthat whenever stimulated by an extracellular signaling molecule, membrane receptorstransduce the signal into a variety of pleiotropic effects by simply up-regulating theCa2+ intracellular concentration or controlling the extent of cAMP synthesis ordegradation. This is why so much selective pressure has been placed on signaltransduction to restrict the number of alternative messenger pathways as respect tothe redundancy which characterize the metabolic pathways. While alternativemetabolic pathways may differ in energetic cost, but be functionally equivalent,each of the isolated signaling pathway or their combined cross talks provide thecell with functionally unique communication channels (Siso-Nadal et al. 2009).Metabolic redundancy may be an evolutionary favorable condition for alternativepathways to gain in efficiency. On the other hand, redundancy in cell signaling maynot be evolutionary advantageous (Soyer et al. 2006), the cell’s receptor systemhaving to provide functionally different responses to a potential overload ofsimultaneous stimuli.

For signal transduction to sustain the cell’s responsive capacity efficiently, thefolding capacity of membrane receptors has to be restricted only to those structuralconfigurations that proved capable of maintaining unaltered their interacting corre-spondence with secondary messengers. In this way, cells may develop the ability totransduce external stimuli according to a many-to-few relationship, as depicted by thenodal cluster of the bow-tie architecture. At the same time, this constrainedconfiguration on protein folding allows cells to develop a categorical sensingfor the external milieu: a necessary pre-condition for the constitution of anendosemiosis, i.e., the possibility to engage in intercellular communication onthe basis of the internal capacity to interpret external stimuli as meaningful signs(Bruni 2007). In a biosemiotically inspired view, cells and environment can thus beseen as interacting partners capable of coevolving through a process of reciprocaladaptation. As often underlined by Bateson (1972), we ought to talk about co-evolution because selection acts directly on partner relationship, and not on theproperties of each partner. In this way, the functional complementarities betweenpartners is maintained in order to conform with the essential requirements of thesystem they are part of (Smock et al. 2010).

Given the role played by receptor proteins in ensuring the necessary functionalcomplementarities for cell communication, the last question to be asked in thissection is whether natural selection acts against misfolded proteins because of theirfunctional detriment to cell viability or because of the toxicity caused by theirintracellular retention. Misfolding may cause proteins to lose their functionalitybecause: (1) they may be misrouted to other cell districts, (2) be removed from thecell interior by autophagy, or alternatively (3) be incompletely processed and retained



inside the endoplasmic reticulum (Conn et al. 2007). Each of these alternativesinvolves different remedial strategies, determining different impacts on either thefunctional recovering of the affected gene product or the eventual destiny of thebearing cell. Besides causing loss-of-function of many receptor proteins, misfoldingmay sometimes cause proteins to acquire new functions and perhaps become toxic tothe host cell. The fact that toxicity may not always be phenotypically manifest in thecell suggests that mutant proteins can be removed efficiently from the cytoplasm andavoid cell death. Menzies et al. (2011) have recently reviewed a large body ofevidence indicating that cells affected by this type of protein misfolding may recoverfrom the relative gain-of-toxic function by up-regulating the process of autophagy. Alikely hypothesis to justify the role played by autophagic degradation under thesecircumstances is that excessive amounts of misfolded proteins may not be cleared bythe ubiquitin–proteasome system as easy as is made possible by lysosomal interven-tion (Kimura et al. 2011). At the same time, confinement of misfolded proteins intothe membrane bound space of the autophagic vacuole allows cells to pursue a twofoldaim: (1) get rid of excess protein aggregates and (2) make their toxicity harmless.

But why should misfolded proteins become toxic for the endoplasmic reticulum?Is toxicity a new functional acquisition for some misfolded configurations or does itsimply result from their aggregational accumulation? As noted by Stefani (2008), theendoplasmic reticulum is a rather crowded environment, for newly synthesizedproteins tend naturally to aggregate. One of the consequences of this molecularovercrowding is the formation of large surface areas that may eventually affect thefluidity and compactness of the plasma membrane and the nearby exocytic vesicles,and serve as a recruiting nucleus for the aggregation of additional protein molecules.Thus, the toxicity of protein misfolding may ultimately result from the tendency ofprotein aggregates to alter membrane permeability within and between cell compart-ments, rather than by newly acquired chemical properties. Impairment of a number ofcell functions, eventually leading to cell death by apoptosis, is thus the ultimateexpression of a built-in program triggered by the disruption of intracellular compart-mentalization and of the associated protein partitioning process.

Cell Death

Excessive or prolonged stresses in the endoplasmic reticulum will eventually result incell death. Cell death or apoptosis is a process triggered by the activation of a numberof preexisting pro-caspases that are used to cleave a variety of cytoskeletal, nuclearand cytosolic proteins (Vernooy et al. 2000; Upton et al. 2008).

Regardless of the functional role played by programmed cell death in evolution, allliving organisms have adopted this suicidal program by availing of the same basicmechanism: a critical equilibrium between pro-death and pro-survival factors, sug-gesting that the cell-death program is also a semiotic process of contextual categoricalsensing, as already seen for the DNA-error replication modulation and the proteinmisfolding repair. One of the best examples of programmed cell death has been foundin Escherichia coli. It consists of two components: a stable toxin and an unstable, fastdegraded, anti-toxin, both encoded by a pair of adjacent genes, mazE and mazF.When challenged by stressful conditions, a few E.coli cells die by apoptosis for



failure of the unstable anti-toxin to counter balance the toxin effects (Engelberg-Kulka et al. 2006). Similar anti-death factors are also present in Drosophila mela-nogaster where the gene product DIAP1 triggers apoptosis by dis-inhibiting specificcaspases. Interestingly, the activation of these cell death factors is triggered by therelease of an extracellular peptide of five amino acids that is derived from theglucose-6-phosphate-dehydrogenase by partial proteolysis (Kolodkin-Gal et al.2007). Thus, this small pentapeptide plays the critical role of a cell death triggerwhenever nutrients are depleted under overcrowded conditions, while it is also acomponent of the pentose biosynthetic pathway. This should not come as a surprisesince this situation is appropriate for any molecule playing key metabolic roles. Anyvariation above or below critical thresholds may be perceived by the cell as a signindicating that either metabolites are about to be exhausted or are produced in excess.

In pluricellular organisms, activation of the suicidal program may occur by eitherextrinsic or intrinsic cell pathways. In the first instance, it is a trans-membrane receptorthat acts as a death-inducing signaling complex (DISC) to cleave pro-caspases by a two-step dimerization process (Hughes et al. 2009). In the intrinsic pathway, it is instead therelease of cytochrome c from mitochondria that causes the cytosolic protein Apaf-1 toassemble into a wheel-shaped molecular structure or apoptosome (Riedl and Salvesen2007). So even cytochrome c, which normally acts as an electron carrier of themitochondrial respiratory chain, may become an apoptotic trigger whenever releasedfrom mitochondria to the cytosol (Cheng and Hardwick 2007).

Mechanism(s) triggering apoptosis are likely to be even more complex than so farenvisioned. The present experimental approach to apoptosis is necessarily focused ona restricted number of casual factors, while the contextual background in whichapoptosis occurs remains essentially unknown. This may give the erroneous impres-sion that a single signal suffices to trigger the entire process, whereas many cues –both coadjuvating and triggering factors - may perhaps be required simultaneouslyfor the process to occur. Therefore, in a biosemiotic perspective, depletion of an anti-toxin component may simply be the contextual cue eventually installing an appro-priate cell response. When looked at this way, what is referred to as the apoptosomemay come to be seen as an analogical sign defining the whole synchronic assessmentof the cellular context. In this sense, the modularity of signaling molecules triggeringphysiologically different processes poses one of the central problems in biology forunderstanding how metabolic codes work. The question to be raised is thus, how canthe specificity of signaling molecules be established if the structure of the molecule(s)or the factor(s) that carry that information are not pre-determined and are ubiquitouslydispersed in the cytoplasm (Bruni 2007)? We believe that cell responses are nottriggered by the specific chemical properties of the effector molecules, but by thespatio-temporal instantiation of complex relationships that these molecules mayentertain with the whole responding apparatus of the cell. As an example of suchcomplexity, Reubold et al. (2009) have recently demonstrated that Apaf-1 does notact according to a simple key and lock model of protein interaction. Assembly of theapoptosome structure requires, in fact, the Apaf-1 protein to undergo complexconformational changes along with removal of the interlocking inhibition by cyto-chrome c and the simultaneous association with some phosphate moiety.

The selective elimination of genetically defective cells by cell death might havebeen one of the main reasons for such a suicidal program to be maintained invariant



throughout bacteria evolution (Engelberg-Kulka et al. 2004). Both the structuralintegrity of the bacterial chromosome and the genomic stability of the survivingpopulation may have been safeguarded by maintaining it within sustainable growthlimits and restricted genetic variations. Both these objectives are attained by selec-tively removing mutant cells and allowing only the fittest ones to survive and grow incritical environments. In pluricellular organisms, removal of apoptotic cells from thetissue does not only imply selective elimination of mutant or defective cells, but alsoselective removal of all cell debris derived from their apoptotic fragmentation.Interestingly, clearance of the extracellular environment in these situations isaccomplished by neighboring epithelial cells that undergo phagocytosis when-ever exposed to apoptotic bodies (Ren et al. 1995). Failure to clear dying cellsfrom the extracellular environment results in severe tissue damage and transforms theselective programmed cell death into a necrotic process. Under these conditions,dying cells may release toxic compounds and cause local tissue inflammation (Finkand Cookson 2005).

Conclusion

The fundamental reason why cells are allowed to choose between sustainable alter-natives is that they are facing an unpredictable environment. Stressful conditions arenot exceptional, but common events in an unreliable biological world. Perhaps theonly way for cells to deal with this unpredictability is to be fault-tolerant enough toaccept these messy conditions, and yet install a number of quality control mecha-nisms at all levels of their living hierarchy. Thanks to the existence of these qualitycontrol mechanisms, cells can thus resolve the dichotomy as to whether to correct anyfaulty behavior or allow the underlying structure to be degraded. The decision is notas straightforward as it may seem at first sight. It involves evaluation of the structurallevel at which corrections have to be made – whether at molecular, organellular orcellular levels – and to establish the temporal limits during which the controlmechanism(s) have to be accomplished to be effective. If mistakes are not caught atany level within the assigned timeframe, it becomes impossible for the quality controlmechanism to save the cell, in which case the cell is diverted to the degradationpathway to recycle its molecular and/or organellular constituents (Rorth 2008). In ourview, the choice of repair vs. destruction is a semiotic selection that cells are requiredto make to interpret the feasibility and cost effectiveness of either of these alterna-tives. The difficulty of pre-defining the beneficial effects of this choice lies in the factthat what could be initially perceived as a loss-of-function may turn out to be a gain-of-function, even though occasionally toxic for the hosting organism. The possibilityfor receptor proteins to acquire such new functionalities in the absence of anypreceding genetic variation demonstrates the powerfulness of the folding mechanismas a way of constraining the redundancy of phenotypic variants expressing differentaffinities (Van Craenenbroeck et al. 2011). The role of molecular chaperones incontrolling receptor expression by either (1) rescuing non-functional misfolding, (2)eliminating loss-of-function variants by cell death or (3) creating new gain-of-function variants may be seen as a semiotic response on behalf of cells coping withunforeseeable environmental challenges. To regard these cell responses as a kind of



semiotic engagement, requires us to consider them as processes mediated by signs,rather than as mechanisms mediated by signals or messages. While a signal is a vehicleconveying constrained information, the sign is everything that may cause meaningfulresponses by an interpreter capable of choosing (Gudwin 2001). A single signal does notcorrelate with the entire complexity of the cellular context in which it acts, as it mayoperate causally only in a linear mechanical fashion. On the other hand, signs can be saidto interpret the overall contextual complexity because contingently supported or consti-tuted by complex arrays of signals. Thus, with respect to mainstream thinking wherebyrelations are thought of as lawlike regularities instantiated by signal exchanges, semiosisoffers the alternative view of looking at relations as arbitrary associations of signs drivenby contingent and unconstrained necessities (Abel and Trevors 2005). In other words, acell system driven by signals would presuppose the existence of a pre-determined geneprogram led by a number of syntactic rules, while a cell system driven by semiotic signsshould be understood as (1) adaptable by experience to ever emerging circumstances and(2) capable of reducing the uncertainty of available opportunities into the certainty of averifiable outcome (Cariani 1998). As uncertainty is reduced in the course of semioticselection, a cell system becomes gradually capable of exploring its surrounding milieuthrough sign perception, and eventually to construct a meaningful developmentaltrajectory. Meaning is thus the ultimate utterance cells may accede when allowed tochoose between explorable alternatives, i.e. whenever their path dependency enablesthem to anticipate and to test the adequacy of their responding repertoire.

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2 3 Semiotic Selection of Mutated or Misfolded Receptor Proteins

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