The Role of Ancestral Duplicated Genes in Adaptation to Growth on Lactate, a Non-Fermentable Carbon Source for the Yeast Saccharomyces cerevisiae.

The cell central metabolism has been shaped throughout evolutionary times when facing challenges from the availability of resources. In the budding yeast, Saccharomyces cerevisiae, a set of duplicated genes originating from an ancestral whole-genome and several coetaneous small-scale duplication events drive energy transfer through glucose metabolism as the main carbon source either by fermentation or respiration. These duplicates (~a third of the genome) have been dated back to approximately 100 MY, allowing for enough evolutionary time to diverge in both sequence and function. Gene duplication has been proposed as a molecular mechanism of biological innovation, maintaining balance between mutational robustness and evolvability of the system. However, some questions concerning the molecular mechanisms behind duplicated genes transcriptional plasticity and functional divergence remain unresolved. In this work we challenged S. cerevisiae to the use of lactic acid/lactate as the sole carbon source and performed a small adaptive laboratory evolution to this non-fermentative carbon source, determining phenotypic and transcriptomic changes. We observed growth adaptation to acidic stress, by reduction of growth rate and increase in biomass production, while the transcriptomic response was mainly driven by repression of the whole-genome duplicates, those implied in glycolysis and overexpression of ROS response. The contribution of several duplicated pairs to this carbon source switch and acidic stress is also discussed.

Exploring molecular evolution of Rubisco in C3 and CAM Orchidaceae and Bromeliaceae.

BACKGROUND: The CO2-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence. RESULTS: We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C3 orchids and between strong CAM and C3 bromeliads suggested Rubisco had evolved in response to differing CO2 concentration. CONCLUSIONS: The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ13C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.

Inferring the regulatory network of the miRNA-mediated response to biotic and abiotic stress in melon.

BACKGROUND: MiRNAs have emerged as key regulators of stress response in plants, suggesting their potential as candidates for knock-in/out to improve stress tolerance in agricultural crops. Although diverse assays have been performed, systematic and detailed studies of miRNA expression and function during exposure to multiple environments in crops are limited. RESULTS: Here, we present such pioneering analysis in melon plants in response to seven biotic and abiotic stress conditions. Deep-sequencing and computational approaches have identified twenty-four known miRNAs whose expression was significantly altered under at least one stress condition, observing that down-regulation was preponderant. Additionally, miRNA function was characterized by high scale degradome assays and quantitative RNA measurements over the intended target mRNAs, providing mechanistic insight. Clustering analysis provided evidence that eight miRNAs showed a broad response range under the stress conditions analyzed, whereas another eight miRNAs displayed a narrow response range. Transcription factors were predominantly targeted by stress-responsive miRNAs in melon. Furthermore, our results show that the miRNAs that are down-regulated upon stress predominantly have as targets genes that are known to participate in the stress response by the plant, whereas the miRNAs that are up-regulated control genes linked to development. CONCLUSION: Altogether, this high-resolution analysis of miRNA-target interactions, combining experimental and computational work, Illustrates the close interplay between miRNAs and the response to diverse environmental conditions, in melon.

The origins of mutational robustness.

Biological systems are resistant to genetic changes; a property known as mutational robustness, the origin of which remains an open question. In recent years, researchers have explored emergent properties of biological systems and mechanisms of genetic redundancy to reveal how mutational robustness emerges and persists. Several mechanisms have been proposed to explain the origin of mutational robustness, including molecular chaperones and gene duplication. The latter has received much attention, but its role in robustness remains controversial. Here, I examine recent findings linking genetic redundancy through gene duplication and mutational robustness. Experimental evolution and genome resequencing have made it possible to test the role of gene duplication in tolerating mutations at both the coding and regulatory levels. This evidence as well as previous findings on regulatory reprogramming of duplicates support the role of gene duplication in the origin of robustness.

Arabidopsis Heat Stress-Induced Proteins Are Enriched in Electrostatically Charged Amino Acids and Intrinsically Disordered Regions.

Comparison of the proteins of thermophilic, mesophilic, and psychrophilic prokaryotes has revealed several features characteristic to proteins adapted to high temperatures, which increase their thermostability. These characteristics include a profusion of disulfide bonds, salt bridges, hydrogen bonds, and hydrophobic interactions, and a depletion in intrinsically disordered regions. It is unclear, however, whether such differences can also be observed in eukaryotic proteins or when comparing proteins that are adapted to temperatures that are more subtly different. When an organism is exposed to high temperatures, a subset of its proteins is overexpressed (heat-induced proteins), whereas others are either repressed (heat-repressed proteins) or remain unaffected. Here, we determine the expression levels of all genes in the eukaryotic model system Arabidopsis thaliana at 22 and 37 °C, and compare both the amino acid compositions and levels of intrinsic disorder of heat-induced and heat-repressed proteins. We show that, compared to heat-repressed proteins, heat-induced proteins are enriched in electrostatically charged amino acids and depleted in polar amino acids, mirroring thermophile proteins. However, in contrast with thermophile proteins, heat-induced proteins are enriched in intrinsically disordered regions, and depleted in hydrophobic amino acids. Our results indicate that temperature adaptation at the level of amino acid composition and intrinsic disorder can be observed not only in proteins of thermophilic organisms, but also in eukaryotic heat-induced proteins; the underlying adaptation pathways, however, are similar but not the same.

Glycerol stress in Saccharomyces cerevisiae: Cellular responses and evolved adaptations.

Glycerol synthesis is key to central metabolism and stress biology in Saccharomyces cerevisiae, yet the cellular adjustments needed to respond and adapt to glycerol stress are little understood. Here, we determined impacts of acute and chronic exposures to glycerol stress in S. cerevisiae. Glycerol stress can result from an increase of glycerol concentration in the medium due to the S. cerevisiae fermenting activity or other metabolic activities. Acute glycerol-stress led to a 50% decline in growth rate and altered transcription of more than 40% of genes. The increased genetic diversity in S. cerevisiae population, which had evolved in the standard nutrient medium for hundreds of generations, led to an increase in growth rate and altered transcriptome when such population was transferred to stressful media containing a high concentration of glycerol; 0.41 M (0.990 water activity). Evolution of S. cerevisiae populations during a 10-day period in the glycerol-containing medium led to transcriptome changes and readjustments to improve control of glycerol flux across the membrane, regulation of cell cycle, and more robust stress response; and a remarkable increase of growth rate under glycerol stress. Most of the observed regulatory changes arose in duplicated genes. These findings elucidate the physiological mechanisms, which underlie glycerol-stress response, and longer-term adaptations, in S. cerevisiae; they also have implications for enigmatic aspects of the ecology of this otherwise well-characterized yeast.

Preservation of genetic and regulatory robustness in ancient gene duplicates of Saccharomyces cerevisiae.

Biological systems remain robust against certain genetic and environmental challenges. Robustness allows the exploration of ecological adaptations. It is unclear what factors contribute to increasing robustness. Gene duplication has been considered to increase genetic robustness through functional redundancy, accelerating the evolution of novel functions. However, recent findings have questioned the link between duplication and robustness. In particular, it remains elusive whether ancient duplicates still bear potential for innovation through preserved redundancy and robustness. Here we have investigated this question by evolving the yeast Saccharomyces cerevisiae for 2200 generations under conditions allowing the accumulation of deleterious mutations, and we put mechanisms of mutational robustness to a test. S. cerevisiae declined in fitness along the evolution experiment, but this decline decelerated in later passages, suggesting functional compensation of mutated genes. We resequenced 28 genomes from experimentally evolved S. cerevisiae lines and found more mutations in duplicates--mainly small-scale duplicates--than in singletons. Genetically interacting duplicates evolved similarly and fixed more amino acid-replacing mutations than expected. Regulatory robustness of the duplicates was supported by a larger enrichment for mutations at the promoters of duplicates than at those of singletons. Analyses of yeast gene expression conditions showed a larger variation in the duplicates' expression than that of singletons under a range of stress conditions, sparking the idea that regulatory robustness allowed a wider range of phenotypic responses to environmental stresses, hence faster adaptations. Our data support the persistence of genetic and regulatory robustness in ancient duplicates and its role in adaptations to stresses.

Fitness Trade-Offs Determine the Role of the Molecular Chaperonin GroEL in Buffering Mutations.

Molecular chaperones fold many proteins and their mutated versions in a cell and can sometimes buffer the phenotypic effect of mutations that affect protein folding. Unanswered questions about this buffering include the nature of its mechanism, its influence on the genetic variation of a population, the fitness trade-offs constraining this mechanism, and its role in expediting evolution. Answering these questions is fundamental to understand the contribution of buffering to increase genetic variation and ecological diversification. Here, we performed experimental evolution, genome resequencing, and computational analyses to determine the trade-offs and evolutionary trajectories of Escherichia coli expressing high levels of the essential chaperonin GroEL. GroEL is abundantly present in bacteria, particularly in bacteria with large loads of deleterious mutations, suggesting its role in mutational buffering. We show that groEL overexpression is costly to large populations evolving in the laboratory, leading to groE expression decline within 66 generations. In contrast, populations evolving under the strong genetic drift characteristic of endosymbiotic bacteria avoid extinction or can be rescued in the presence of abundant GroEL. Genomes resequenced from cells evolved under strong genetic drift exhibited significantly higher tolerance to deleterious mutations at high GroEL levels than at native levels, revealing that GroEL is buffering mutations in these cells. GroEL buffered mutations in a highly diverse set of proteins that interact with the environment, including substrate and ion membrane transporters, hinting at its role in ecological diversification. Our results reveal the fitness trade-offs of mutational buffering and how genetic variation is maintained in populations.

Evolution by gene duplication of Medicago truncatula PISTILLATA-like transcription factors.

PISTILLATA (PI) is a member of the B-function MADS-box gene family, which controls the identity of both petals and stamens in Arabidopsis thaliana. In Medicago truncatula (Mt), there are two PI-like paralogs, known as MtPI and MtNGL9. These genes differ in their expression patterns, but it is not known whether their functions have also diverged. Describing the evolution of certain duplicated genes, such as transcription factors, remains a challenge owing to the complex expression patterns and functional divergence between the gene copies. Here, we report a number of functional studies, including analyses of gene expression, protein-protein interactions, and reverse genetic approaches designed to demonstrate the respective contributions of each M. truncatula PI-like paralog to the B-function in this species. Also, we have integrated molecular evolution approaches to determine the mode of evolution of Mt PI-like genes after duplication. Our results demonstrate that MtPI functions as a master regulator of B-function in M. truncatula, maintaining the overall ancestral function, while MtNGL9 does not seem to have a role in this regard, suggesting that the pseudogenization could be the functional evolutionary fate for this gene. However, we provide evidence that purifying selection is the primary evolutionary force acting on this paralog, pinpointing the conservation of its biochemical function and, alternatively, the acquisition of a new role for this gene.

The roles of whole-genome and small-scale duplications in the functional specialization of Saccharomyces cerevisiae genes.

Researchers have long been enthralled with the idea that gene duplication can generate novel functions, crediting this process with great evolutionary importance. Empirical data shows that whole-genome duplications (WGDs) are more likely to be retained than small-scale duplications (SSDs), though their relative contribution to the functional fate of duplicates remains unexplored. Using the map of genetic interactions and the re-sequencing of 27 Saccharomyces cerevisiae genomes evolving for 2,200 generations we show that SSD-duplicates lead to neo-functionalization while WGD-duplicates partition ancestral functions. This conclusion is supported by: (a) SSD-duplicates establish more genetic interactions than singletons and WGD-duplicates; (b) SSD-duplicates copies share more interaction-partners than WGD-duplicates copies; (c) WGD-duplicates interaction partners are more functionally related than SSD-duplicates partners; (d) SSD-duplicates gene copies are more functionally divergent from one another, while keeping more overlapping functions, and diverge in their sub-cellular locations more than WGD-duplicates copies; and (e) SSD-duplicates complement their functions to a greater extent than WGD-duplicates. We propose a novel model that uncovers the complexity of evolution after gene duplication.

Molecular evolutionary mechanisms driving functional diversification of the HSP90A family of heat shock proteins in eukaryotes.

The ubiquitous and conserved cytosolic heat-shock proteins 90 (HSP90A) perform essential functions in the cell. To understand the evolutionary origin of HSP90A functional diversification, we analyzed the distribution of HSP90A family from 54 species representing the main eukaryotic lineages. Three independent HSP90A duplications led to the paralog subfamilies HSP90AA (heat-stress inducible) and HSP90AB (constitutive) and trace back to key time points during vertebrate, seed plant, and yeast evolution. HSP90AA and HSP90AB present divergent selection pressures, positive selection (PS), and signatures of functional divergence (FD) after duplication. The differential evolutionary patterns support different mechanisms for HSP90A functional diversification in vertebrates and seed plants. Mapping of PS and FD residues onto the HSP90A structure suggests the acquisition of novel and/or specialized client protein and/or cochaperone binding functions. We propose these residues as targets for further experimental studies of HSP90A proteins, reported to be capacitors of rapid evolutionary change, and targets for anticancer therapeutics.

Molecular evolution of viral multifunctional proteins: the case of potyvirus HC-Pro.

Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlapping.

The evolution of protein moonlighting: adaptive traps and promiscuity in the chaperonins.

Moonlighting proteins exhibit functions that are alternative to their main role in the cell. Heat-shock proteins, also known as molecular chaperones, are now recognized for their wide range of activities in and/or outside the cell, being prominent examples of moonlighting proteins. Chaperonins are highly conserved molecular chaperones that fold other proteins into their native conformation allowing them to carry out essential functions in the cell. Activities alternative to folding have been reported for the chaperonin (Cpn) 60 protein. Preservation of various alternative functions in one protein conflicts with the optimization of each of the functions. What evolutionary mechanisms have allowed the persistence of moonlighting proteins, and in particular the chaperonins, remains a mystery. In the present article, I argue that mechanisms that increase the resistance of phenotypes to genetic and environmental perturbations enable the persistence of a reservoir of genetic variants, each potentially codifying for a distinct function. Gene duplication is one such mechanism that has characterized the expansion and has been concomitant with the emergence of novel functions in these protein families. Indeed, Cpn60 performs a large list of folding-independent functions, including roles in the transmission of viruses from insects to plants and stimulation of the immune system, among others. In addition to the innovation promoted by gene duplication, I discuss that the Cpn60 protein comprises a hidden amino acid combinatorial code that may well be responsible for its ability to develop novel functions while maintaining an optimized folding ability. The present review points to a complex model of evolution of protein moonlighting.

Coevolution analyses illuminate the dependencies between amino acid sites in the chaperonin system GroES-L.

BACKGROUND: GroESL is a heat-shock protein ubiquitous in bacteria and eukaryotic organelles. This evolutionarily conserved protein is involved in the folding of a wide variety of other proteins in the cytosol, being essential to the cell. The folding activity proceeds through strong conformational changes mediated by the co-chaperonin GroES and ATP. Functions alternative to folding have been previously described for GroEL in different bacterial groups, supporting enormous functional and structural plasticity for this molecule and the existence of a hidden combinatorial code in the protein sequence enabling such functions. Describing this plasticity can shed light on the functional diversity of GroEL. We hypothesize that different overlapping sets of amino acids coevolve within GroEL, GroES and between both these proteins. Shifts in these coevolutionary relationships may inevitably lead to evolution of alternative functions. RESULTS: We conducted the first coevolution analyses in an extensive bacterial phylogeny, revealing complex networks of evolutionary dependencies between residues in GroESL. These networks differed among bacterial groups and involved amino acid sites with functional importance and others with previously unsuspected functional potential. Coevolutionary networks formed statistically independent units among bacterial groups and map to structurally continuous regions in the protein, suggesting their functional link. Sites involved in coevolution fell within narrow structural regions, supporting dynamic combinatorial functional links involving similar protein domains. Moreover, coevolving sites within a bacterial group mapped to regions previously identified as involved in folding-unrelated functions, and thus, coevolution may mediate alternative functions. CONCLUSIONS: Our results highlight the evolutionary plasticity of GroEL across the entire bacterial phylogeny. Evidence on the functional importance of coevolving sites illuminates the as yet unappreciated functional diversity of proteins.

Evolutionary dynamics and functional specialization of plant paralogs formed by whole and small-scale genome duplications.

Gene duplicates are a major source of evolutionary novelties in the form of new or specialized functions and play a key role in speciation. Gene duplicates are generated through whole genome duplications (WGD) or small-scale genome duplications (SSD). Although WGD preserves the stoichiometric relationships between duplicates, those arising from SSD are usually unbalanced and are expected to follow different evolutionary dynamics than those formed by WGD. To dissect the role of the mechanism of duplication in these differential dynamics and determine whether this role was shared across species, we performed a genome wide evolutionary analysis of gene duplications arising from the most recent WGD events and contemporary episodes of SSD in four model species representing distinct plant evolutionary lineages. We found an excess of relaxed purifying selection after duplication in SSD paralogs compared with WGD, most of which may have been the result of functional divergence events between gene copies as estimated by measures of genetic distances. These differences were significant in three angiosperm genomes but not in the moss species Physcomitrella patens. Although the comparison of models of evolution does not attribute a relevant role to the mechanism of duplication in the evolution duplicates, distribution of retained genes among Gene Ontology functional categories support the conclusion that evolution of gene duplicates depends on its origin of duplication (WGD and SSD) but, most importantly, on the species. Similar lineage-specific biases were also observed in protein network connectivity, translational efficiency, and selective constraints acting on synonymous codon usage. Although the mechanism of duplication may determine gene retention, our results attribute a dominant role to the species in determining the ultimate pattern of duplicate gene retention and reveal an unanticipated complexity in the evolutionary dynamics and functional specialization of duplicated genes in plants.

Two chaperonin systems in bacterial genomes with distinct ecological roles.

Bacterial chaperonins are essential to cell viability and have a role in endosymbiosis, which leads to increased biological complexity. However, the extent to which chaperonins promote ecological innovation is unknown. We screened 622 bacterial genomes for genes encoding chaperonins, and found archaeal-like chaperonins in bacteria that inhabit archaeal ecological niches. We found that chaperonins encoded in pathogenic bacteria are the most functionally divergent. We identified the molecular basis of the dramatic structural changes in mitochondrial GROEL, a highly derived chaperonin gene. Our analysis suggests that chaperonins are important capacitors of evolutionary and ecological change.

Describing the structural robustness landscape of bacterial small RNAs.

BACKGROUND: The potential role of RNA molecules as gene expression regulators has led to a new perspective on the intracellular control and genome organization. Because secondary structures are crucial for their regulatory role, we sought to investigate their robustness to mutations and environmental changes. RESULTS: Here, we dissected the structural robustness landscape of the small non-coding RNAs (sncRNAs) encoded in the genome of the bacterium Escherichia coli. We found that bacterial sncRNAs are not significantly robust to both mutational and environmental perturbations when compared against artificial, unbiased sequences. However, we found that, on average, bacterial sncRNAs tend to be significantly plastic, and that mutational and environmental robustness strongly correlate. We further found that, on average, epistasis in bacterial sncRNAs is significantly antagonistic, and positively correlates with plasticity. Moreover, the evolution of robustness is likely dependent upon the environmental stability of the cell, with more fluctuating environments leading to the emergence and fixation of more robust molecules. Mutational robustness also appears to be correlated with structural functionality and complexity. CONCLUSION: Our study provides a deep characterization of the structural robustness landscape of bacterial sncRNAs, suggesting that evolvability could be evolved as a consequence of selection for more plastic molecules. It also supports that environmental fluctuations could promote mutational robustness. As a result, plasticity emerges to link robustness, functionality and evolvability.

Functional diversification of the twin-arginine translocation pathway mediates the emergence of novel ecological adaptations.

Microorganisms occupy a myriad of ecological niches that show an astonishing diversity. The molecular mechanisms underlying microbes' ecological diversity remain a fundamental conundrum in evolutionary biology. Evidence points to that the secretion of a particular set of proteins mediates microbes' interaction with the environment. Several systems are involved in this secretion, including the Sec secretion system and the Tat pathway. Shifts in the functions of proteins from the secretion systems may condition the set of secreted proteins and can, therefore, mediate adaptations to new ecological niches. In this manuscript, we have investigated processes of functional divergence (FD)-a term used here to refer to the emergence of novel functions by the modification of ancestral ones-of Tat pathway proteins using a large set of microbes with different lifestyles. The application of a novel approach to identify FD allowed us to distinguish molecular changes in the three Tat proteins among different groups of archaea and bacteria. We found these changes as well as the composition of secreted proteins to be correlated with differences in microbe's lifestyles. We identified major signatures of FD in halophilic and thermophilic archaea as well as in pathogenic bacteria. The location of amino acids affected by FD in functionally important domains of Tat proteins made it possible to find the link between the molecular changes in Tat, the set of secreted proteins and the environmental features of the microbes. We present evidence that links specific molecular changes in secretion mediating proteins of microbes to their ecological adaptations.

Structural calibration of the rates of amino acid evolution in a search for Darwin in drifting biological systems.

In the last two decades, many reports of proteins under positive selection have brought the neutral theory into question. However, the methods used to detect selection have ignored the evolvability of amino acids within proteins, which is fundamental to distinguishing positive selection from the relaxed constraints caused by genetic drift. Disentangling these two counterbalancing forces is essential to test the neutral theory. Here, we calibrate rates of amino acid divergence by using structural information from the full set of crystallized proteins in bacteria. In agreement with previous reports, we show that rates of amino acid evolution correlate negatively with the number of per-amino acid atomic interactions. Calibration of the rates of evolution allows identifying signatures of selection in biological systems that evolve under strong genetic drift, such as endosymbiotic bacteria. Application of this method identifies different rates and dynamics of evolution for highly connected amino acids in the structure compared with sparsely connected ones. We also unearth patterns of Darwinian selection in fundamental cellular proteins in endosymbiotic bacteria including the cochaperonin GroES, ribosomal proteins, proteins involved in cell cycle control, DNA-binding proteins, and proteins involved in DNA replication and repair. This is, to our knowledge, the first attempt to distinguish adaptive evolution from relaxed constraints in biological systems under genetic drift.

Protein coadaptation and the design of novel approaches to identify protein-protein interactions.

Proteins rarely function in isolation but they form part of complex networks of interactions with other proteins within or among cells. The importance of a particular protein for cell viability is directly dependent upon the number of interactions where it participates and the function it performs: the larger the number of interactions of a protein the greater its functional importance is for the cell. With the advent of genome sequencing and "omics" technologies it became feasible conducting large-scale searches for protein interacting partners. Unfortunately, the accuracy of such analyses has been underwhelming owing to methodological limitations and to the inherent complexity of protein interactions. In addition to these experimental approaches, many computational methods have been developed to identify protein-protein interactions by assuming that interacting proteins coevolve resulting from the coadaptation dynamics between the amino acids of their interacting faces. We review the main technological advances made in the field of interactomics and discuss the feasibility of computational methods to identify protein-protein interactions based on the estimation of coevolution. As proof-of-concept, we present a classical case study: the interactions of cell surface proteins (receptors) and their ligands. Finally, we take this discussion one step forward to include interactions between organisms and species to understand the generation of biological complexity. Development of technologies for accurate detection of protein-protein interactions may shed light on processes that go from the fine-tuning of pathways and metabolic networks to the emergence of biological complexity.