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Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

Host colonization by Gram-negative pathogens often involves delivery of bacterial proteins called “effectors” into the host cell. The pneumonia-causing pathogen Legionella pneumophila delivers more than 330 effectors into the host cell via its type IVB Dot/Icm secretion system. The collective functions of these proteins are the establishment of a replicative niche from which Legionella can recruit cellular materials to grow while evading lysosomal fusion inhibiting its growth. Using a combination of structural, biochemical, and in vivo approaches, we show that one of these translocated effector proteins, Ceg4, is a phosphotyrosine phosphatase harboring a haloacid dehalogenase–hydrolase domain. Ceg4 could dephosphorylate a broad range of phosphotyrosine-containing peptides in vitro and attenuated activation of MAPK-controlled pathways in both yeast and human cells. Our findings indicate that L. pneumophila's infectious program includes manipulation of phosphorylation cascades in key host pathways. The structural and functional features of the Ceg4 effector unraveled here provide first insight into its function as a phosphotyrosine phosphatase, paving the way to further studies into L. pneumophila pathogenicity.

G protein-coupled receptors (GPCRs) must discriminate between hundreds of related signal molecules. In order to better understand how GPCR specificity can arise from a common promiscuous ancestor, we used laboratory evolution to invert the specificity of the Saccharomyces cerevisiae mating receptor Ste2. This GPCR normally responds weakly to the pheromone of the related species Kluyveromyces lactis, though we previously showed that mutation N216S is sufficient to make this receptor promiscuous. Here, we found that three additional substitutions, A265T, Y266F and P290Q, can act together to confer a novel specificity for K. lactis pheromone. Unlike wild-type Ste2, this new variant does not rely on differences in binding affinity to discriminate against its non-preferred ligand. Instead, the mutation P290Q is critical for suppressing the efficacy of the native pheromone. These two alternative methods of ligand discrimination were mapped to specific amino acid positions on the peptide pheromones. Our work demonstrates that changes in ligand efficacy can drive changes in GPCR specificity, thus obviating the need for extensive binding pocket re-modeling.

Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae. This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to identify potential host targets of P. syringae T3SEs. We focused on the acetyltransferase, HopZ1a, which interacts with plant tubulin and alters microtubule networks. To uncover putative HopZ1a host targets, we identified yeast genes with genetic interaction profiles most similar (i.e., congruent) to the PGA profile of HopZ1a and performed a functional enrichment analysis of these HopZ1a-congruent genes. We compared the congruence analyses above to previously described HopZ physical interaction datasets and identified kinesins as potential HopZ1a targets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1, illustrating the utility of our T3SE-expressing yeast library to characterize T3SE functions.

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as promiscuous individual T3SEs that can have multiple host targets. To overcome these challenges, we conducted heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. We screened 75 T3SEs from the plant pathogen Pseudomonas syringae and identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media, including the acetyltransferase HopZ1a. We focused our further analysis on HopZ1a, which interacts with plant tubulin and alters microtubule networks. We first performed a Pathogenic Genetic Array (PGA) screen of HopZ1a against ~4400 yeast carrying non-essential mutations and found 95 and 10 deletion mutants which reduced or enhanced HopZ1a toxicity, respectively. To uncover putative HopZ1a host targets, we interrogated both the genetic- and physical-interaction profiles of HopZ1a by identifying yeast genes with PGA profiles most similar (i.e. congruent) to that of HopZ1a, performing a functional enrichment analysis of these HopZ1a-congruent genes, and by analyzing previously described HopZ physical interaction datasets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1.ARTICLE SUMMARYBacterial pathogens utilize secretion systems to directly deliver effector proteins into host cells, with the ultimate goal of promoting pathogen fitness. Despite the central role that effectors play in infection, the molecular function and host targets of most effectors remain uncharacterized. We used yeast genomics and protein interaction data to identify putative virulence targets of the effector HopZ1a from the plant pathogen Pseudomonas syringae. HopZ1a is an acetyltransferase that induces plant microtubule destruction. We showed that HopZ1a acetylated plant kinesin proteins known to regulate microtubule networks. Our study emphasizes the power of yeast functional genomic screens to characterize effector functions.

G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, and direct measurement of GPCR-mediated signaling is useful for high-throughput mutational studies. However, this is particularly difficult for the light-activated GPCR rhodopsin... G protein-coupled receptors (GPCRs) are crucial sensors of extracellular signals in eukaryotes, with multiple GPCR mutations linked to human diseases. With the growing number of sequenced human genomes, determining the pathogenicity of a mutation is challenging, but can be aided by a direct measurement of GPCR-mediated signaling. This is particularly difficult for the visual pigment rhodopsin—a GPCR activated by light—for which hundreds of mutations have been linked to inherited degenerative retinal diseases such as retinitis pigmentosa. In this study, we successfully engineered, for the first time, activation by human rhodopsin of the yeast mating pathway, resulting in signaling via a fluorescent reporter. We combine this novel assay for rhodopsin light-dependent activation with studies of subcellular localization, and the upregulation of the unfolded protein response in response to misfolded rhodopsin protein. We use these assays to characterize a panel of rhodopsin mutations with known molecular phenotypes, finding that rhodopsin maintains a similar molecular phenotype in yeast, with some interesting differences. Furthermore, we compare our assays in yeast with clinical phenotypes from patients with novel disease-linked mutations. We demonstrate that our engineered yeast strain can be useful in rhodopsin mutant classification, and in helping to determine the molecular mechanisms underlying their pathogenicity. This approach may also be applied to better understand the clinical relevance of other human GPCR mutations, furthering the use of yeast as a tool for investigating molecular mechanisms relevant to human disease.

The ability to sense and process cues about changing environments is fundamental to life. Cells have evolved elaborate signaling pathways in order to respond to both internal and external stimuli appropriately. These pathways combine protein receptors, signal transducers, and effector genes in highly connected networks. The numerous interactions found between signaling proteins are essential to maintain strict regulation and produce a suitable cellular response. As a result, a signaling protein's activity in isolation can differ greatly from its activity in a native context. This is an important consideration when studying or engineering signaling pathways. Fortunately, the difficulty of studying network interactions is fading thanks to advances in library construction and cell sorting. In this chapter, we describe two methods for generating libraries of mutant proteins that exhibit altered network interactions: whole-gene point mutagenesis and domain shuffling. We then provide a protocol for using fluorescence-activated cell sorting to isolate interesting variants in live cells by focusing on the unicellular eukaryotic model organism Saccharomyces cerevisiae, using as an example recent work that we have done on its G protein-coupled receptor Ste2.

In a seminal paper entitled "Evolution and Tinkering," Francois Jacob affirmed that: "Novelties come from previously unseen association of old material. To create is to recombine" [Jacob F. (1977) Science 196:1161-1166]. In the 35 years that have passed since Jacob's insight, we have amassed enough data to actually shed light on many of the molecular mechanisms that enable evolution to create novelty by simply recombining what existed already. In this review, we will succinctly discuss the role that the recombination of protein domains has in the evolution of signaling networks, drawing from examples provided by diverse disciplines, including bioinformatics, systems and synthetic biology, and laboratory evolution.

The cellular concentrations of key components of signaling networks are tightly regulated, as deviations from their optimal ranges can have negative effects on signaling function. For example, overexpression of the yeast mating pathway mitogen-activated protein kinase (MAPK) Fus3 decreases pathway output, in part by sequestering individual components away from functional multiprotein complexes. Using a synthetic biology approach, we investigated potential mechanisms by which selection could compensate for a decrease in signaling activity caused by overexpression of Fus3. We overexpressed a library of random mutants of Fus3 and used cell sorting to select variants that rescued mating pathway activity. Our results uncovered that one remarkable way in which selection can compensate for protein overexpression is by introducing premature stop codons at permitted positions. Because of the low efficiency with which premature stop codons are read through, the resulting cellular concentration of active Fus3 returns to values within the range required for proper signaling. Our results underscore the importance of interpreting genotypic variation at the systems rather than at the individual gene level, as mutations can have opposite effects on protein and network function.

Virulence factors from two bacteria are used to reprogram intracellular signalling in yeast and immune T cells, illustrating how pathogens can provide a toolkit to engineer cells for biotechnological or therapeutic applications. Effector proteins developed by bacterial pathogens to subvert host systems have potential uses in cell engineering for biotechnological or therapeutic applications. For example, many proteins from pathogens with the type III secretion system interfere with mitogen-activated protein kinase (MAPK) pathways. MAPK pathways are central to many eukaryotic responses, so agents capable of modulating them could have many uses — in biotechnological cells such as yeast, in immune cells engineered for adoptive immunotherapy, or to suppress cell growth associated with cancer, for instance. As a proof of principle, Wendell Lim and colleagues use two bacterial virulence factors, OspF from Shigella flexneri and YopH from Yersinia pestis, to modulate MAPK signalling in yeast and human immune cells. Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signalling pathways, provide a mechanism to evade immune responses during infection1,2. Although these effectors contribute to pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behaviour. Here we exploit two effector proteins, the Shigella flexneri OspF protein3 and Yersinia pestis YopH protein4, to rewire kinase-mediated responses systematically both in yeast and mammalian immune cells. Bacterial effector proteins can be directed to inhibit specific mitogen-activated protein kinase pathways selectively in yeast by artificially targeting them to pathway-specific complexes. Moreover, we show that unique properties of the effectors generate new pathway behaviours: OspF, which irreversibly inactivates mitogen-activated protein kinases4, was used to construct a synthetic feedback circuit that shows novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to tune the T-cell response amplitude precisely, or as an inducible pause switch that can temporarily disable T-cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications.

This is an exciting time to be an evolutionary biologist. Indeed, it is difficult to keep up with all the studies that fall under the broad category of “Evolution” since they span species, traits, and scales of organization. This special issue gives a flavor of exciting new approaches in evolutionary biology, but also emphasizes universal themes. The reviews contained here discuss important aspects of molecular evolution at multiple scales, from individual proteins to complex regulatory networks, as well as from unicellular organisms to macroscopic traits in animals. Though the model systems are diverse, the issues addressed are fundamental: the origin of evolutionary novelties, and the forces that drive them to fixation. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 465–467, 2014. © 2014 Wiley Periodicals, Inc.

The living cell is an incredibly complex entity, and the goal of predictively and quantitatively understanding its function is one of the next great challenges in biology. Much of what we know about the cell concerns its constituent parts, but to a great extent, we have yet to decode how these parts are organized to yield complex physiological function. Classically, we have learned about the organization of cellular networks by perturbing them through genetic or chemical means. The emerging discipline of synthetic biology offers an additional, powerful way to study systems. By rearranging the parts that comprise existing networks, we can gain valuable insight into the hierarchical logic of the networks and identify the modular building blocks that evolution uses to generate innovative function. Additionally, by building minimal “toy” networks, one can systematically explore the relationship between network space (linkages and parameters) and functional space (the system's physiological behavior). Here, we outline recent work that uses synthetic biology approaches to investigate the organization and function of cellular networks, and describe a vision for a synthetic biology toolkit that could be used to interrogate the design principles of diverse systems.

When generating novel tailor-made proteins, protein engineers routinely apply the principles of 'Darwinian' evolution. However, laboratory evolution of proteins also has the potential to test evolutionary theories and reproduce evolutionary scenarios, thus reconstructing putative protein intermediates and providing a glimpse of 'protein fossils'. This commentary describes research at the interface of applied and fundamental molecular evolution, and provides a personal view of how synergy between fundamental and applied experiments indicates novel and more efficient ways of generating new proteins in the laboratory.

New protein folds have emerged throughout evolution, but it remains unclear how a protein fold can evolve while maintaining its function, particularly when fold changes require several sequential gene rearrangements. Here, we explored hypothetical evolutionary pathways linking different topological families of the DNA-methyltransferase superfamily. These pathways entail successive gene rearrangements through a series of intermediates, all of which should be sufficiently active to maintain the organism's fitness. By means of directed evolution, and starting from HaeIII methyltransferase (M.HaeIII), we selected all the required intermediates along these paths (a duplicated fused gene and duplicates partially truncated at their 5' or 3' coding regions) that maintained function in vivo. These intermediates led to new functional genes that resembled natural methyltransferases from three known classes or that belonged to a new class first seen in our evolution experiments and subsequently identified in natural genomes. Our findings show that new protein topologies can evolve gradually through multistep gene rearrangements and provide new insights regarding these processes.

In recent years, the simple picture of a viral fusion protein interacting with the cell and/or viral membranes by means of only two localized segments (i.e. the fusion peptide and the transmembrane domain) has given way to a more complex picture in which multiple regions from the viral proteins interact with membranes. Indeed, possible roles in membrane binding and/or destabilization have been postulated for the N-terminal heptad repeats, pre-transmembrane segments, and other internal regions of fusion proteins from distant viruses (such as orthomyxo-, retro-, paramyxo-, or flaviviruses). This review focuses on the experimental evidence and functional models postulated so far about the role of these regions in the process of virus-induced membrane fusion.

T20, a synthetic peptide corresponding to a C-terminal segment of the envelope glycoprotein (gp41) of human and simian immunodeficiency viruses, is a potent inhibitor of viral infection. We report here that C-terminal octylation of simian immunodeficiency virus gp41-derived T20 induces a significant increase in its inhibitory potency. Furthermore, when C-terminally octylated, an otherwise inactive mutant in which the C-terminal residues GNWF were replaced by ANAA has potency similar to that of the wild type T20. This effect cannot be explained by a trivial inhibitory effect of the octyl group added to the peptides, because the N-terminally octylated peptides have the same activity as the non-octylated parent peptides. The effects caused by octylation on the oligomerization, secondary structure, and membrane-interaction properties of the peptides were investigated. Our results shed light on the mechanism of inhibition by T20 and provide experimental support for the existence of a pre-hairpin intermediate.

gp41 is the protein responsible for the process of membrane fusion that allows primate lentiviruses (HIV and SIV) to enter into their host cells. gp41 ectodomain contains an N-terminal and a C-terminal heptad repeat region (NHR and CHR) connected by an immunodominant loop. In the absence of membranes, the NHR and CHR segments fold into a protease-resistant core with a trimeric helical hairpin structure. However, when the immunodominant loop is not present (either in a complex formed by HIV-1 gp41-derived NHR and CHR peptides or by mild treatment with protease of recombinant constructs of HIV-1 gp41 ectodomain, which also lack the N-terminal fusion peptide and the C-terminal Trp-rich region) membrane binding induces a conformational change in the gp41 core structure. Here, we further investigated whether covalently linking the NHR and CHR segments by the immunodominant loop affects this conformational change. Specifically, we analyzed a construct corresponding to a fragment of SIVmac239 gp41ectodomain (residues 27-149, named e-gp41) by means of surface plasmon resonance, Trp and rhodamine fluorescence, ATR-FTIR spectroscopy, and differential scanning calorimetry. Our results suggest that the presence of the loop stabilizes the trimeric helical hairpin both when e-gp41 is in aqueous solution and when it is bound to the membrane surface. Bearing in mind possible differences between HIV-1 and SIV gp41, and considering that the gp41 ectodomain constructs analyzed to date lack the N-terminal fusion peptide and the C-terminal Trp-rich region, we discuss our observations in relation to the mechanism of virus-induced membrane fusion.

Signaling scaffolds are proteins that interact via modular domains with multiple partners, regulating signaling networks in space and time and providing an ideal platform from which to alter signaling functions. However, to better exploit scaffolds for signaling engineering, it is necessary to understand the full extent of their modularity. We used a directed evolution approach to identify, from a large library of randomly shuffled protein interaction domains, variants capable of rescuing the signaling defect of a yeast strain in which Ste5, the scaffold in the mating pathway, had been deleted. After a single round of selection, we identified multiple synthetic scaffold variants with diverse domain architectures, able to mediate mating pathway activation in a pheromone-dependent manner. The facility with which this signaling network accommodates changes in scaffold architecture suggests that the mating signaling complex does not possess a single, precisely defined geometry into which the scaffold has to fit. These relaxed geometric constraints may facilitate the evolution of signaling networks, as well as their engineering for applications in synthetic biology.

The N-terminal fusion peptide of Sendai virus F1 envelope glycoprotein is a stretch of 14 amino acids, most of which are hydrophobic. Following this region, we detected a segment of 11 residues that are strikingly similar to the N-terminal fusion peptide. We found that, when anchored to the membrane by palmitoylation of its N-terminus, this segment (WT-palm-19–33) induces membrane fusion of large unilamellar liposomes to almost the same extent as a segment that includes the N-terminal fusion peptide. The activity of WT-palm-19–33 was dependent on its specific sequence, as a palmitoylated peptide with the same amino-acid composition but a scrambled sequence was inactive. Interestingly, two mutations (G7A and G12A) known to increase F1- induced cell-cell fusion, also increased the homology between the N-terminal fusion peptide and WT-palm-19–33. The role of the amino-acid sequence on the fusogenicity, secondary structure, and mechanism of membrane fusion was analyzed by comparing a peptide comprising both homologous segments (WT 1–33), a G12A mutant (G12A 1–33), a G7A–G12A double mutant (G7A–G12A 1–33), and a peptide with a scrambled sequence (SC 1–33). Based on these experiments, we postulate that replacement of Gly 7 and Gly12 by Ala increases the α helical content of the N-terminal region, with a concomitant increase in its fusogenic activity. Furthermore, the dissimilar abilities of the different peptides to induce membrane negative curvature as well as to promote isotropic 31P NMR signals, suggest that these mutations might also alter the extent of membrane penetration of the 33-residue peptide. Interestingly, our results serve to explain the effect of the G7A and G12A mutations on the fusogenic activity of the parent F1 protein in vivo.

DP178, a synthetic peptide corresponding to a segment of the transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus, type 1 (HIV-1), is a potent inhibitor of viral infection and virus-mediated cell-cell fusion. Nevertheless, DP178 does not contain gp41 coiled-coil cavity binding residues postulated to be essential for inhibiting HIV-1 entry. We find that DP178 inhibits phospholipid redistribution mediated by the HIV-1 envelope glycoprotein at a concentration 8 times greater than that of solute redistribution (the IC50 values are 43 and 335 nm, respectively). In contrast, C34, a synthetic peptide which overlaps with DP178 but contains the cavity binding residues, did not show this phenomenon (11 and 25 nm, respectively). The ability of DP178 to inhibit membrane fusion at a post-lipid mixing stage correlates with its ability to bind and oligomerize on the surface of membranes. Furthermore, our results are consistent with a model in which DP178 inhibits the formation of gp41 viral hairpin structure at low affinity, whereas C34 inhibits its formation at high affinity: the failure to form the viral hairpin prevents both lipid and solute from redistributing between cells. However, our data also suggest an additional membrane-bound inhibitory site for DP178 in the ectodomain of gp41 within a region immediately adjacent to the membrane-spanning domain. By binding to this higher affinity site, DP178 inhibits the recruitment of several gp41-membrane complexes, thus inhibiting fusion pore formation.

The human immunodeficiency virus type 1 gp41 ectodomain forms a three-hairpin protease-resistant core in the absence of membranes, namely, the putative gp41 fusion-active state. Here, we show that recombinant proteins corresponding to the ectodomain of gp41, but lacking the fusion peptide, bind membranes and consequently undergo a major conformational change. As a result, the protease-resistant core becomes susceptible to proteolytic digestion. Accordingly, synthetic peptides corresponding to the segments that construct this core bind the membrane. It is remarkable that the hetero-oligomer formed by these peptides dissociates upon binding to the membrane. These results are consistent with a model in which, after the three-hairpin conformation is formed, membrane binding induces opening of the gp41 core complex. We speculate that binding of the segments that constructed the core to the viral and cellular membranes could bring the membranes closer together and facilitate their merging.

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses.

The last three C-terminal residues (129–131) of intestinal fatty acid-binding protein (IFABP) participate in four main-chain hydrogen bonds and two electrostatic interactions to sequentially distant backbone and side-chain atoms. To assess if these interactions are involved in the final adjustment of the tertiary structure during folding, we engineered an IFABP variant truncated at residue 128. An additional mutation, Trp-6→Phe, was introduced to simplify the conformational analysis by optical methods. Although the changes were limited to a small region of the protein surface, they resulted in an IFABP with altered secondary and tertiary structure. Truncated IFABP retains some cooperativity, is monomeric, highly compact, and has the molecular dimensions and shape of the native protein. Our results indicated that residues 129–131 are part of a crucial conformational determinant in which several long-range interactions, essential for the acquisition of the native state, are established. This work suggests that carefully controlled truncation can populate equilibrium non-native states under physiological conditions. These non-native states hold a great promise as experimental models for protein folding.

A synthetic heptad repeat, SV-473, derived from Sendai virus fusion protein is a potent inhibitor of virus-cell fusion. In order to understand the mechanism of the inhibitory effect, we synthesized and fluorescently labeled SV-465, an extended version of SV-473 by one more heptad, its mutant peptide A17,24-SV-465, in which two heptadic leucines were substituted with two alanines, and its enatiomer D-SV-465, composed entirely of Damino acids. Similar mutations in the homologous fusion protein of the Newcastle disease virus drastically reduced its activity. The data revealed that SV-465, but not A17,24-SV-465 or its enantiomer, is highly active in inhibiting Sendai virus-induced hemolysis of red blood cells. None of the peptides interfere with the binding of virions to the target red blood cells as demonstrated by hemagglutinin assay. Fluorescence and circular dichroism (CD) spectroscopy indicated that: (i) only SV-465 could self-assemble in aqueous environment; (ii) only SV-465 could co-assemble with two other biologically active heptad repeats derived from Sendai virus fusion protein; (iii) SV-465 has a higher helical content than A17,24-SV-465 in solution, and (iv) all the peptides bind strongly to zwitterionic and negatively charged phospholipids. Polarized attenuated total reflection infrared spectroscopy revealed that they bound as monomers onto the surface of zwitterionic membranes with predominantly alpha-helical structures. The functional role of the amino acid 465-497 domain in Sendai virus-mediated membrane fusion is discussed in light of these findings.

In a seminal paper entitled "Evolution and Tinkering," François Jacob affirmed that: "Novelties come from previously unseen association of old material. To create is to recombine" [Jacob F. (1977) Science 196:1161-1166]. In the 35 years that have passed since Jacob's insight, we have amassed enough data to actually shed light on many of the molecular mechanisms that enable evolution to create novelty by simply recombining what existed already. In this review, we will succinctly discuss the role that the recombination of protein domains has in the evolution of signaling networks, drawing from examples provided by diverse disciplines, including bioinformatics, systems and synthetic biology, and laboratory evolution.

Signaling networks process vast amounts of environmental information to generate specific cellular responses. As cellular environments change, signaling networks adapt accordingly. Here, I will discuss how the integration of synthetic biology and directed evolution approaches is shedding light on the molecular mechanisms that guide the evolution of signaling networks. In particular, I will review studies that demonstrate how different types of mutations, from the replacement of individual amino acids to the shuffling of modular domains, lead to markedly different evolutionary trajectories and consequently to diverse network rewiring. Moreover, I will argue that intrinsic evolutionary properties of signaling proteins, such as the robustness of wild type functions, the promiscuous nature of evolutionary intermediates, and the modular decoupling between binding and catalysis, play important roles in the evolution of signaling networks. Finally, I will argue that rapid advances in our ability to synthesize DNA will radically alter how we study signaling network evolution at the genome-wide level.

Domain Swaps to Phenotype Shifts For natural selection there must be mechanisms that create phenotypic diversity, presumably from relatively simple molecular changes in an organism. Peisajovich et al. (p. 368 ) tested the extent to which changes in phenotype can occur by systematic swapping of protein domains in the components of the biochemical signaling pathway that controls mating in yeast. Such changes decreased or increased responsiveness to yeast mating pheromone, and some translated into changes in mating efficiency. The authors propose that shuffling of modular protein domains may be an important source of phenotypic diversity in evolution and may also be a useful strategy for the engineering of biological systems.

Entry of HIV into its target cell requires the fusion of the virion envelope and the cell plasma membrane. This process is catalyzed by the viral envelope glycoproteins gp120, which mediates the binding to specific receptors on the target cell surface, and gp41, responsible for the actual membrane merging. This chapter will focus on the conformational changes that gp41 undergoes during the fusion process, on the mechanism by which these conformational changes are linked to membrane fusion, and on the design of inhibitors able to block different stages of this process.

T20, a synthetic peptide corresponding to a C-terminal segment of the envelope glycoprotein (gp41) of human and simian immunodeficiency viruses, is a potent inhibitor of viral infection. We report here that C-terminal octylation of simian immunodeficiency virus gp41-derived T20 induces a significant increase in its inhibitory potency. Furthermore, when C-terminally octylated, an otherwise inactive mutant in which the C-terminal residues GNWF were replaced by ANAA has potency similar to that of the wild type T20. This effect cannot be explained by a trivial inhibitory effect of the octyl group added to the peptides, because the N-terminally octylated peptides have the same activity as the non-octylated parent peptides. The effects caused by octylation on the oligomerization, secondary structure, and membrane-interaction properties of the peptides were investigated. Our results shed light on the mechanism of inhibition by T20 and provide experimental support for the existence of a pre-hairpin intermediate.

Publisher Summary The use of liposomes to identify and characterize viral fusogenic peptides has enabled rapid advances in the field of virus-mediated membrane fusion. The development of sensitive and concomitantly versatile fluorescence-based assays has facilitated the characterization of the different stages involved in the process of membrane fusion. Currently, most liposome-based in vitro studies of virus-mediated membrane fusion focuses on the role of short fragments derived from large viral proteins. This chapter determines the ability of virus-derived peptide to cause fusion of liposomes. Like other “membrane-active” peptides—such as antimicrobial peptides or some signal peptides—most fusion peptides destabilize the membrane structure. Virus-induced membrane fusion proceeds through an intermediate in which only fusion between the outer monolayers occurs. An assay originally developed to study membrane asymmetry can be useful to detect such intermediates.

The N-terminal fusion peptide of Sendai virus F1 envelope glycoprotein is a stretch of 14 amino acids, most of which are hydrophobic. Following this region, we detected a segment of 11 residues that are strikingly similar to the N-terminal fusion peptide. We found that, when anchored to the membrane by palmitoylation of its N-terminus, this segment (WT-palm-19-33) induces membrane fusion of large unilamellar liposomes to almost the same extent as a segment that includes the N-terminal fusion peptide. The activity of WT-palm-19-33 was dependent on its specific sequence, as a palmitoylated peptide with the same amino-acid composition but a scrambled sequence was inactive. Interestingly, two mutations (G7A and G12A) known to increase F1- induced cell-cell fusion, also increased the homology between the N-terminal fusion peptide and WT-palm-19-33. The role of the amino-acid sequence on the fusogenicity, secondary structure, and mechanism of membrane fusion was analyzed by comparing a peptide comprising both homologous segments (WT 1-33), a G12A mutant (G12A 1-33), a G7A-G12A double mutant (G7A-G12A 1-33), and a peptide with a scrambled sequence (SC 1-33). Based on these experiments, we postulate that replacement of Gly 7 and Gly12 by Ala increases the alpha helical content of the N-terminal region, with a concomitant increase in its fusogenic activity. Furthermore, the dissimilar abilities of the different peptides to induce membrane negative curvature as well as to promote isotropic 31P NMR signals, suggest that these mutations might also alter the extent of membrane penetration of the 33-residue peptide. Interestingly, our results serve to explain the effect of the G7A and G12A mutations on the fusogenic activity of the parent F1 protein in vivo.

The broad tolerance of domain-rearranging mutations by a yeast signaling network suggests that signaling complexes have loose spatial constraints, making manipulation and perhaps evolution easier., The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering., Author Summary Cells use complex protein interaction networks to sense and process external signals. Proteins involved in signaling are often composed of multiple functional units called domains. Because domains are modular, mutations that rearrange domains among proteins have the potential to result in the creation of novel proteins with altered functions. At an evolutionary timescale, domain rearrangements contribute to the functional diversification of signaling networks; at the shorter timescale of the life of an individual, domain rearrangements can impair cellular functions and lead to disease. Here, we investigated how domain-rearranging mutations alter the function of signaling networks, in particular when these mutations disrupt pre-existing proteins. We used as a model system the yeast mating signaling pathway, which shares many properties with more complex pathways active in human cells. Our results demonstrate that signaling networks are often robust to domain rearrangements that disrupt pre-existing genes. In addition, our experiments suggest a possible mechanism to explain this robustness: rather than being a rigid multi-protein machine, the yeast mating signaling complex is a dynamic ensemble with loose spatial constraints. Because of this, the changes in protein interaction partners caused by domain-rearrangement mutations can be accommodated without disrupting network function.

The viral envelope glycoprotein gp41 mediates membrane fusion in HIV/SIV infection. gp41 ectodomain (e-gp41, residues 27-149), which was shown to interact with phospholipid membranes, exists in an equilibrium between the monomeric and trimeric states. Here, we analyzed, by intrinsic Trp fluorescence and resonance energy transfer, whether SIV e-gp41-membrane interaction depends on the gp41 oligomeric state. We found that both gp41 monomers and trimers bind membranes, with the monomers' full binding being reached at substantially lower lipid to protein ratios. Furthermore, the different characteristics of the Trp fluorescence of monomers and trimers enabled us to detect binding of each form at concentrations at which both species were present. CD spectroscopy revealed that the secondary structure of gp41 monomers does not change upon membrane binding, suggesting that membrane-bound monomeric-gp41 is a possible target for DP-178, a potent peptide inhibitor of HIV infection. The consequences of the interaction between monomeric and trimeric gp41 with membranes in HIV/SIV infection, its inhibition, and its associated neuropathologies are discussed.

Viral glycoproteins catalyze the fusion between viral and cellular membranes. The fusion protein (F(1)) of Sendai virus has two fusion peptides. One is located at its N-terminus and the other, highly homologous to the HIV-1 and RSV fusion peptides, in the interior of the F(1) protein. A synthetic peptide corresponding to the internal fusogenic domain, namely, SV-201, was found to inhibit virus-cell fusion without interfering with the binding of the virus to the target cells, thus highlighting the importance of this region in Sendai virus-induced membrane fusion. However, its detailed mechanism of inhibition remains unknown. Here, we synthesized a shorter version of SV-201, namely, SV-208, an elongated one, SV-197, and two mutants of SV-201, and compared them functionally and structurally with SV-201. In contrast to SV-201, SV-208 and the two mutants do not inhibit virus-cell fusion. The differences in the oligomerization state of these peptides in aqueous solution and within the membrane, and in their ability to bind to Sendai virions, enabled us to postulate a possible mechanism of viral entry inhibition: SV-201 binds to its target in Sendai virions before the F(1) internal fusion peptide binds to the membrane, therefore blocking the F(1) conformational change required for fusion. In addition, we further characterized the fusogenic activity of the internal fusion peptide, compared to the N-terminal one, and determined its structure in the membrane-bound state by means of fluorescence, CD, and ATR-FTIR spectroscopy. Remarkably, we found that SV-201 and its elongated form, SV-197, are highly potent in inducing fusion of the highly stable large unilamellar vesicles composed of egg phosphatidylcholine, a property found only in an extended version of the HIV-1 fusion peptide. The inhibitory activity of SV-201 and its fusogenic ability are discussed in terms of the "umbrella" model of Sendai virus-induced membrane fusion.

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.