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Protein Multifunctionality and the Interactome

August 28, 2016

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A theme from several previous biopolyverse postings has highlighted the impressive economies of many aspects of complex biological systems, termed biological parsimony. As with the previous post, here the general notion of biological parsimony is explored in terms of the network of interactions that allows a complex organism to operate, or its interactome. For the present purposes, the emphasis is placed upon the role of multi-functional proteins in greatly extending the functional range of proteomes beyond what is directly encoded in their corresponding genomes. Protein functions routinely define some form of molecular interaction, so protein multifunctionality is one facet of the interactome.

 Proteins in the Moonlight

In any complex system, it is clearly an economy if a specific component has more than one function. This could arise from an inherent multifunctional property of the component, or via differential interactions of the component with other factors within the same complex system. Although there may be overlap between these two possibilities, they are not identical, and this is reflected in certain definitions. With respect to the latter ‘differential interactions’ category, we could reflect back to the previous post, where (among other things) the multi-faceted protein LIF (Leukemia Inhibitory Factor) was discussed. LIF could be considered ‘exhibit A’ as an example of pleotropism, but for the present purposes we can look to a different kind of multi-functionalism.

It is well-documented that many proteins go beyond multi-partner interactions and multiple signaling effects, and exhibit completely distinct functions under specific conditions. The first description of such protein multi-tasking was with the eye structural protein crystallin, which can also act as a specific enzyme. This effect, thus distinguishable from pleotropy, has been referred to a number of ways, but the term ‘moonlighting’ seems to have won the day. (This topic was noted briefly in a previous post devoted to the theme of parsimony (August 2015). The coining of the term is often attributed to Constance Jeffery in 1999, who has done much to promote the field, but at least one use of ‘moonlighting’ (in a similar context) in the literature prior to this date has also been recorded.

A key feature of moonlighting in its current conceptual definition is the functional use of a different region of a protein over the region(s) associated with its ‘original’ function. (The ‘original’ function in this context refers simply to what was historically described first, and says nothing about its relative biological importance). Thus, an enzyme showing ‘promiscuity’ in terms of being able to use more than one specific substrate at its active site is not moonlighting by this definition. With this caveat in mind, many examples of the moonlighting phenomenon have been described since the original discoveries.

How widespread is the propensity of proteins to ‘moonlight’? Inextricably entangled with this seemingly simple question is the deeper issue of knowledge limitations. A ‘non-moonlighting’ (monofunctional) protein is pigeon-holed as such owing to an absence of any evidence for additional functions beyond its standard role. Yet, as the old adage goes, absence of evidence is not the same as evidence of absence. It might be thought that genetic diseases (or knockout genes in animal models) with a single clear-cut and biochemically substantiated loss-of-function phenotype define protein products without moonlighting activities. But this in itself does not rule out additional roles for such a gene product, since the often-observed effect of genetic redundancy could in principle explain why the absence of the protein of interest does not produce additional loss phenotypes. In other words, if the moonlighting role of the protein of interest (Protein 1) was Function B, other ‘back-up’ proteins might perform Function B as well, and mask the absence of Protein 1. At the same time, it might well be noted that if the genetically observed role of Protein 1 was Function A, then obviously no functional redundancy for the A function could exist, or a single-loss genetic lesion producing the A-deficit would not have been observed in the first place. In turn, this hypothetical arrangement prompts the supposition that some proteins might have a ‘main’ role, and one or more ‘secondary’ roles for moonlighting duties. In some cases, this distinction might be relatively straightforward, but is unlikely to always be so. Issues such as this render the moonlighting concept not as simple as it is sometimes made out to be.

The converse of the ostensibly tidy scenario when a single gene defect produces a well-defined loss-phenotype is where the interpretation of a single genetic lesion is considerably complicated by the gene product’s propensity for moonlighting. This effect has been well-documented in the case of gene defects for metabolic enzymes, where specific cases might be expected to elicit a phenotype that is largely if not totally predictable in principle. Thus, if enzyme X acting on substrate U to create product V is genetically inactivated, then the resulting phenotype may be predicted on the basis of what effects a short-fall in production of V might have, or perhaps the consequences of failure to process and remove excess U. But such a prediction might at best tell only a part of the story, if X has a completely unpredicted but important moonlighting role in a very distinct cellular function.

 Intrinsically unstructured proteins and multifunctionality

In the context of multifunctionality and moonlighting, it is worth singling out an interesting category of proteins as being of special significance. Not so long ago (before the mid-1990s), proteins were regarded as always possessing a well-defined and highly ordered folded structure which enabled their functions, and in many cases this indeed applies. But it is now recognized that an important subset of proteins do not possess such an ordered structure, at least before they interact with a binding partner. Proteins of this nature may be completely disordered, or possess an ordered domain linked to a domain lacking specific order. Such is the extent of this phenomenon that up to 40% of eukaryotic proteins are mostly disordered, and >50% have a significant region of low folding order.

Intrinsically unstructured proteins or protein domains in some cases possess the ability to interact with more than one binding partner, in such a manner that the originally structured region assumes different conformations in different binding circumstances. Where this occurs, the binding partner protein may act as a template for the originally unstructured protein, in directing folding towards a specific configuration. Even if this templating effect for a particular unstructured protein is restricted to a limited set of partner proteins, the implications for protein moonlighting are clear, and this has been noted for over a decade. A protein that can assume alternative forms and functions based on the presence of different potential binding partners has in-built modularity, and is obviously capable of fulfilling even a rigorous definition of moonlighting

But not all moonlighting is carried out by proteins with initially poorly-defined structures. Some of the possible alternatives are considered in the Figure below:

MultiFunct-StructAspects

Fig. 1. Different forms of protein multi-functionality, and the processes resulting in multi-functional species from the same polypeptide. F1 and F2 denote distinguishable functions.Here a protein contains two separate and structurally definable segments, which perform well-demarcated and distinct functions under specific circumstances (depicted here as operating via different binding partners).

A. Here a protein contains two separate and structurally definable segments, which perform well-demarcated and distinct functions under specific circumstances (depicted here as operating via different binding partners).

B. In this depiction, an intrinsically unstructured polypeptide assumes different folds under the templating influence of different binding molecules. ‘Tails’ (curved lines) are shown on the altered originally unstructured proteins in the bound states, since it has been found that under such conditions the acquisition of an ordered structure is not necessarily complete.

C. This schematic depicts a protein that can, via a disordered intermediate, assume two quite distinct folding states, corresponding to the concept of ‘metamorphic’ proteins. These have only relatively recently been described and studied.

D. In this case, under the influence of a binding interaction, a part of a protein undergoes a distinct folding alteration, with the acquisition of new functional properties. This corresponds to the concept of ‘transformer’ proteins.

Whether all of these schemas or only some are classified as ‘moonlighting’ clearly comes down to a matter of definitions.

The description embodied by (A) is usually considered as a classic paradigm for protein moonlighting, as the two functions in this schematic are represented by different protein sites and mediate distinct functions. In practice, many different functions can be fulfilled by different protein regions. Perhaps the champion of polyfunctionality is the Large T protein of the mammalian SV40 virus, which contains at least seven different sites performing distinct functions. Intrinsically instructured proteins (as in [B] above) have indeed been discussed in the context of moonlighting, as noted above. The other categories (C) and (D) (metamorphic and transformer proteins, respectively) are not generally considered under the umbrella of the moonlighting concept, but certainly they are cases of novel mechanisms for protein multifunctionality.

__________________________________________________________________

 

Origins of moonlighting / multifunctionality

It is a natural question to ask why evolution should favor the development of multifunctional or moonlighting proteins, and this has been considered in some depth by those interested in the field. For example, one salient point has been raised to the effect that unused parts of a large protein surface may over evolutionary time tend to acquire new functions.

Consider a scenario where protein A performs task 1, and protein B does task 2. If another multidomain protein C can perform both tasks, but the size of C is < A + B, then the combined transcription / translation costs for C then also must be < A + B. Economies of bioenergetics arise where only one initiation event is required for both transcription and translation, rather than for two separate genes. Also, there may be a need for only single processing signals where applicable (for example, nuclear or organelle transport, membrane display, and so on). This kind of argument from the stance of energetics has a basic and logical appeal, and has been made repeatedly. Thus, by means of diverse forms of protein multifunctionality, a proteome of limited size can acquire an expanded functional range within the boundaries of the same energy budget as previously used.

But it is not quite as simple as it may seem at first glance. Again using the above example, Protein C must be integrated into a complex interactome, so in some cases having tasks 1 and 2 relegated to separate molecules may be actually advantageous, disfavoring multifunctional packing. Also, it has been noted that a conflict tends to exist between a protein with multiple functions and optimization of each function. Indeed, mutations which themselves minimize such ‘adaptive conflict’ may be key innovators in the development of successful moonlighting or other multifunctional mechanisms. All this is mediated through a complex balance sheet determining optimal fitness, such that the simplest and most economical alternatives that are evolutionarily accessible (parsimony) will always win.

There are clearly many unanswered questions when the evolution of protein multifunctionality are considered. For example, what is the timing of the evolutionary origins of moonlighting? In other words, at what point in the evolution of complex biosystems did it first appear? Systems with multi-tasked effectors would be presumably evolutionarily favored, but were they an early or late event during the course of molecular evolution?

These questions could lead to a final speculation: is the parsimony of moonlighting / multifunctionality not merely a edge-giving energy saver, but an essential requirement for the development of highly complex biosystems? If this proposal was true, then the only pathway for an organism to acquire increasingly complex structures and systems is via the introduction of the kind of parsimony that protein multifunctionality can confer. In this scenario, if all protein effectors were monofunctional, then biosystems would eventually hit an ‘energy wall’ beyond which they could not traverse, and all biology would be trapped at a relatively basic level of complexity and organization.

An interesting extension of this proposal can also be made with respect to the emergence of the extant world of protein-DNA-RNA from its precursor RNA World (a topic touched upon in several previous posts, for example, 21 June 2011 ). Since the functional range of folded RNA molecules is generally accepted as inferior to that which the larger protein alphabet can offer, an RNA world might likewise suffer from a deficiency in the ability of moonlighting ribocatalysts to form. An RNA world trapped with mostly monofunctional players might thus, by this logic, remain at a far-reduced level of complexity below that which the extant biosphere has attained. Obviously, many factors are likely to be involved in the ascendancy of the protein-DNA-RNA world, but restrictions on RNA multifunctionality might be one of them. As noted in other contexts in previous posts, these and related questions will actually become amenable to experimental testing, via advancements in synthetic biology and increasingly sophisticated model biosystems.

And a final word in a (biopoly)verse form:

 

A protein can wear more than one hat

It can do not just this, but now that

If its functional roles

Fulfill many goals

Then moonlighting is smoothly down pat.

 References & Details

(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).

The first description of such protein multi-tasking was with the eye structural protein crystallin…..’     See Piatigorsky & Wistow 1989.

The coining of the term [moonlighting] is often attributed to Constance Jeffrey…..’     See Jeffrey 1999.

‘………at least one use of ‘moonlighting’ in a similar context in the literature prior to this date has been recorded. ’     See Campbell & Scanes 1995, whose paper was entitled, “Endocrine peptides ‘moonlighting’ as immune modulators: roles for somatostatin and GH-releasing factor.”.

‘……an enzyme showing ‘promiscuity’ in terms of being able to use more than one specific substrate at its active site is not moonlighting…..’     This point was raised by Jeffrey 1999. See also an interesting review on enzyme promiscuity by Khersonsky & Tawfik 2010, where the distinction between promiscuity and moonlighting was also noted.

‘…….many examples of the moonlighting phenomenon have been described.’     See some informative reviews, including Gancedo et al. 2016; Henderson & Martin 2014; Huberts & Van der Klei 2010.

Issues such as this render the moonlighting concept not as simple as it is sometimes made out to be.’     For example, the respected molecular biologist and bioinformaticist Eugene Koonin has expressed the opinion, “I am actually inclined to think that all proteins perform multiple roles in organisms and are at some level moonlighting.”. (This statement is part of an open review by Koonin of a paper by Khan et al. 2014, entitled, “Genome-scale identification and characterization of moonlighting proteins” [Biol. Direct, 2014]).

This effect [complication of phenotypes produced from single-gene coding sequence mutations as a consequence of protein moonlighting] has been well-documented in the case of gene defects for metabolic enzymes…..’      See Sriram et al. 2005.

‘……..if enzyme X acting on substrate U to create product V is genetically inactivated, then the resulting phenotype may be predicted…….’      In practice, it has to be noted, even if a completely monospecific enzymatic function exists, it is not necessarily simple to predict the entirety of the phenotype that results from its functional knock-out. A good case in point in this regard is human Lesch-Nyhan syndrome, which results from mutational inactivation of the enyzme hypoxanthine-guanine phosphoribosyltransferase, involved in purine metabolism. This syndrome is characterized by a number of clinical manifestations, including effects on purine recycling, leading to excessive levels of uric acid. The mechanism of this is well-understood and quite predictable from the genetic lesion. However, in addition to this abnormality, severe Lesch-Nyhan syndrome produces motor disability, and a strange compulsion of afflicted individuals (almost all males, since the gene is on the X chromosome) to engage in serious self-mutilating behavior. Predicting the generation of such a higher-level neurological abnormality as a result of a deficit in a purine-salvage enzyme is another matter entirely, and despite much study, the mechanism for this behavioral pathology remains obscure. (See Jinnah et al. 2013; Dammer et al. 2015).

‘…..it is now recognized that an important subset of proteins do not possess such an ordered structure…..’      See Tompa et al. 2005; Fuxreiter et al. 2014 for useful reviews. An example of a protein with a disordered domain is the important transcription factor and oncoprotein c-Myc (Yu et al. 2016).

‘……up to 40% of eukaryotic proteins are mostly disordered, and >50% have a significant region of low folding order.’     These statistics were cited by Yu et al. 2016 and Fuxreiter et al. 2014, respectively.

‘…….the implications [of intrinsically unstructured proteins] for protein moonlighting are clear, and this has been noted for over a decade.’ See Tompa et al. 2005.

‘……..the concept of ‘metamorphic’ proteins. These have only relatively recently been described and studied.’      See Camilloni & Sutto 2009; Tyler et al. 2011.

This corresponds to the concept of ‘transformer’ proteins.’      See Knauer et al. 2012. It may be noted that the concept of ‘transformer’ proteins (Fig. 1D) can be considered as an extreme form of allostery, where a protein (or functional folded nucleic acid, for that matter) undergoes a conformational change in response to a covalent or non-covalent interaction. The general phenomenon of allostery is very widespread in nature and of fundamental importance, but the ‘transformer’ effect is at a different level of conformational change in comparison to the vast majority of allosteric circumstances.

Tails’ (curved lines) are shown on the altered originally unstructured protein in the bound states, since it has been found that under such conditions the acquisition of an ordered structure is not necessarily complete.’     When this is the case, the resulting protein complex may have a ‘fuzzy’ aspect. See Gruet et al. 2016.

‘……the champion of polyfunctionality is the Large T protein of the mammalian SV40 virus, which contains at least seven different sites performing distinct functions.’     These are: Cul7 binding, pocket protein binding, DNA binding and initiation of viral replication, helicase, ATPase, p53/p300 binding, and host range determination. In addition, Large T has a nuclear localization signal, numerous phosphorylation sites, and an acetylation site. (See the ftp site for Searching for Molecular Solutions, specifically SMS-Cited Notes-Ch.9).

This [the evolution of multifunctionality / moonlighting] has been considered in some depths by those interested in the field.’      See Jeffery 1999, and also Sriram et al. 2005, who consider this question and note earlier opinions.

Also, it has been noted that a conflict would exist between a protein with multiple functions and optimization of each function.’      See Fares 2014.

‘….mutations which themselves minimize such ‘adaptive conflict’ may be key innovators…..’      See Copley 2014.

 

Next Post: January 2017.

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