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1.  Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles 
The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods.
doi:10.1021/ct400486p
PMCID: PMC3827836  PMID: 24250278
2.  An intrinsically disordered yeast prion arrests the cell cycle by sequestering a spindle pole body component 
The Journal of Cell Biology  2012;197(3):369-379.
Promiscuous interactions of the intrinsically disordered Rnq1 prion protein with the spindle pole body component Spc42 result in Spc42’s sequestration in insoluble bodies and cell cycle arrest.
Intrinsically disordered proteins play causative roles in many human diseases. Their overexpression is toxic in many organisms, but the causes of toxicity are opaque. In this paper, we exploit yeast technologies to determine the root of toxicity for one such protein, the yeast prion Rnq1. This protein is profoundly toxic when overexpressed but only in cells carrying the endogenous Rnq1 protein in its [RNQ+] prion (amyloid) conformation. Surprisingly, toxicity was not caused by general proteotoxic stress. Rather, it involved a highly specific mitotic arrest mediated by the Mad2 cell cycle checkpoint. Monopolar spindles accumulated as a result of defective duplication of the yeast centrosome (spindle pole body [SPB]). This arose from selective Rnq1-mediated sequestration of the core SPB component Spc42 in the insoluble protein deposit (IPOD). Rnq1 does not normally participate in spindle pole dynamics, but it does assemble at the IPOD when aggregated. Our work illustrates how the promiscuous interactions of an intrinsically disordered protein can produce highly specific cellular toxicities through illicit, yet highly specific, interactions with the proteome.
doi:10.1083/jcb.201108146
PMCID: PMC3341155  PMID: 22529103
3.  The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains 
PLoS Computational Biology  2012;8(11):e1002749.
In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested.
Author Summary
Proteins that are capable of binding to many different ligands are said to have broad specificity. This is sometimes also referred to as promiscuity. Whether a protein is promiscuous or not can sometimes be readily explained by the structure of the protein and the ligand in terms of electrostatic and steric effects. Sometimes however, this simple interpretation can struggle to explain the experimentally observed data. A prominent case in point is the PDZ domains. These small protein domains bind to unstructured regions of other proteins and are involved in many signaling pathways. Some PDZ domains appear to be more promiscuous than others, but this has been difficult to explain purely on the basis of the composition of residues in the binding groove. In this work we examine the dynamics and conformational flexibility of five key PDZ domains and demonstrate that despite similar folds, these proteins can exhibit quite different dynamics. Furthermore the difference in the dynamic behavior appears to correlate with the observed promiscuity. Our findings suggest that knowledge of the dynamic behavior of the PDZs can be used to rationalize the extent of expected promiscuity. Such knowledge will be critical for drug design against PDZ domains.
doi:10.1371/journal.pcbi.1002749
PMCID: PMC3486844  PMID: 23133356
4.  Three serendipitous pathways in E. coli can bypass a block in pyridoxal-5′-phosphate synthesis 
Overexpression of seven different genes restores growth of a ΔpdxB strain of E. coli, which cannot make pyridoxal phosphate (PLP), on M9/glucose.None of the enzymes encoded by these genes has a promiscuous 4-phosphoerythronate dehydrogenase activity that can replace the activity of PdxB.Overexpression of these genes restores PLP synthesis by three different serendipitous pathways that feed into the normal PLP synthesis pathway downstream of the blocked step.Reactions in one of these pathways are catalyzed by low-level activities of enzymes of unknown function and a promiscuous activity of an enzyme that normally has a role in another pathway; one reaction appears to be non-enzymatic.
Most metabolic enzymes are prodigious catalysts that have evolved to accelerate chemical reactions with high efficiency and specificity. However, many enzymes have inefficient promiscuous activities, as well, as a result of the assemblage of highly reactive catalytic residues and cofactors in active sites. Although promiscuous activities are generally orders of magnitude less efficient than well-evolved activities (O'Brien and Herschlag, 1998, 2001; Wang et al, 2003; Taylor Ringia et al, 2004), they often enhance reaction rates by orders of magnitude relative to those of uncatalyzed reactions (O'Brien and Herschlag, 1998, 2001). Thus, promiscuous activities provide a reservoir of novel catalytic activities that can be recruited to serve new functions.
The evolutionary potential of promiscuous enzymes extends beyond the recruitment of single enzymes to serve new functions. Microbes contain hundreds of enzymes—E. coli contains about 1700 (Freilich et al, 2005)—raising the possibility that promiscuous enzymes can be patched together to generate ‘serendipitous' pathways that are not part of normal metabolism. We distinguish serendipitous pathways from latent or cryptic pathways, which are bona fide pathways involving dedicated enzymes that are produced only under particular environmental circumstances. In contrast, serendipitous pathways are patched together from enzymes that normally serve other functions and are not regulated in a coordinated manner in response to the need to synthesize or degrade a metabolite.
In this study, we describe the discovery of three serendipitous pathways that allow synthesis of pyridoxal phosphate (PLP) in a strain of E. coli that lacks 4-phosphoerythronate dehydrogenase (PdxB) when one of the seven different genes is overexpressed. These genes were identified in a multicopy suppression experiment in which a library of E. coli genes (from the ASKA collection) was introduced into a ΔpdxB strain of E. coli that is unable to synthesize PLP. Surprisingly, none of the enzymes encoded by these genes has a promiscuous 4-phosphoerythronate (4PE) dehydrogenase activity that can substitute for the missing PdxB. Rather, overproduction of these enzymes appears to facilitate at least three serendipitous pathways that draw material from other metabolic pathways and feed into the normal PLP synthesis pathway downstream of the blocked step (Figure 1).
We have characterized one of these pathways in detail (Figure 3). The first step, dephosphorylation of 3-phosphohydroxypyruvate, is catalyzed by YeaB, a predicted NUDIX hydrolase of unknown function. Although catalysis is inefficient (kcat=5.7×10−5 s−1 and kcat/KM>0.028 M−1 s−1), the enzymatic rate is 4×107-fold faster than the rate of the uncatalyzed reaction, and is sufficient to support PLP synthesis when YeaB is overproduced. The second step in the pathway is decarboxylation of 3-hydroxypyruvate (3HP). Although we found two enzymes (1-deoxyxylulose-5-phosphate synthase and the catalytic domain of α-ketoglutarate dehydrogenase) that catalyze this reaction with low but respectable activity in vitro, their involvement in pathway 1 was ruled out by genetic methods. Surprisingly, the non-enzymatic rate of decarboxylation of 3HP appears to be sufficient to support PLP synthesis. The third step in the pathway, condensation of glycolaldehyde and glycine to form 4-hydroxy-L-threonine, is catalyzed by LtaE, a low-specificity threonine aldolase whose physiological role is not known. The final step, phosphorylation of 4-hydroxy-L-threonine, is catalyzed by homoserine kinase (ThrB), which is required for synthesis of threonine. The promiscuous phosphorylation of 4-hydroxy-L-threonine is 80-fold slower than the physiological phosphorylation of homoserine. The involvement of LtaE and ThrB in pathway 1 was confirmed by genetic experiments showing that overexpression of yeaB no longer restores growth of ΔpdxB strains lacking either ltaE or thrB.
Although pathway 1 is inefficient, it provides the ΔpdxB strain with the ability to grow under conditions in which survival is otherwise impossible. In general, serendipitous assembly of an inefficient pathway from promiscuous activities of available enzymes will be important whenever the pathway provides increased fitness. This might occur when a critical metabolite is no longer available from the environment, and survival depends on assembly of a new biosynthetic pathway. A second circumstance in which an inefficient serendipitous pathway might improve fitness is the appearance of a novel compound in the environment that can be exploited as a source of carbon, nitrogen or phosphorous. Finally, chemotherapeutic agents that block metabolic pathways in bacteria or cancer cells could provide selective pressure for assembly of serendipitous pathways that allow synthesis of the end product of the blocked pathway and thus a previously unappreciated source of drug resistance. In all of these cases, even an inefficient pathway can provide a selective advantage over other cells in a particular environmental niche, allowing survival and subsequent mutations that elevate the efficiency of the pathway.
Our work is consistent with the hypothesis that the recognized metabolic network of E. coli is underlain by a denser network of reactions due to promiscuous enzymes that use and generate recognized metabolites, but also unusual metabolites that normally have no physiological role. The findings reported here highlight the abundance of cryptic capabilities in the E. coli proteome that can be drawn on to generate novel pathways. Such pathways could provide a starting place for assembly of more efficient pathways, both in nature and in the hands of metabolic engineers.
Bacterial genomes encode hundreds to thousands of enzymes, most of which are specialized for particular functions. However, most enzymes have inefficient promiscuous activities, as well, that generally serve no purpose. Promiscuous reactions can be patched together to form multistep metabolic pathways. Mutations that increase expression or activity of enzymes in such serendipitous pathways can elevate flux through the pathway to a physiologically significant level. In this study, we describe the discovery of three serendipitous pathways that allow synthesis of pyridoxal-5′-phosphate (PLP) in a strain of E. coli that lacks 4-phosphoerythronate (4PE) dehydrogenase (PdxB) when one of seven different genes is overexpressed. We have characterized one of these pathways in detail. This pathway diverts material from serine biosynthesis and generates an intermediate in the normal PLP synthesis pathway downstream of the block caused by lack of PdxB. Steps in the pathway are catalyzed by a protein of unknown function, a broad-specificity enzyme whose physiological role is unknown, and a promiscuous activity of an enzyme that normally serves another function. One step in the pathway may be non-enzymatic.
doi:10.1038/msb.2010.88
PMCID: PMC3010111  PMID: 21119630
metabolic bypass; multicopy suppression; promiscuity; pyridoxal-5′-phosphate; serendipitous pathway
5.  The Role of Abcb5 Alleles in Susceptibility to Haloperidol-Induced Toxicity in Mice and Humans 
PLoS Medicine  2015;12(2):e1001782.
Background
We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study.
Methods and Findings
A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered.
Conclusions
ABCB5 alleles alter susceptibility to HIT in mouse and humans. This discovery leads to a new model that (at least in part) explains inter-individual differences in susceptibility to a drug-induced CNS toxicity.
Gary Peltz and colleagues examine the role of ABCB5 alleles in haloperidol-induced toxicity in a murine genetic model and humans treated with haloperidol.
Editors' Summary
Background
The brain is the control center of the human body. This complex organ controls thoughts, memory, speech, and movement, it is the seat of intelligence, and it regulates the function of many organs. The brain comprises many different parts, all of which work together but all of which have their own special functions. For example, the forebrain is involved in intellectual activities such as thinking whereas the hindbrain controls the body’s vital functions and movements. Messages are passed between the various regions of the brain and to other parts of the body by specialized cells called neurons, which release and receive signal molecules known as neurotransmitters. Like all the organs in the body, blood vessels supply the brain with the oxygen, water, and nutrients it needs to function. Importantly, however, the brain is protected from infectious agents and other potentially dangerous substances circulating in the blood by the “blood-brain barrier,” a highly selective permeability barrier that is formed by the cells lining the fine blood vessels (capillaries) within the brain.
Why Was This Study Done?
Although drugs have been developed to treat various brain disorders, more active and less toxic drugs are needed to improve the treatment of many if not most of these conditions. Unfortunately, relatively little is known about how the blood-brain barrier regulates the entry of drugs into the brain or about the genetic factors that affect the brain’s susceptibility to drug-induced toxicities. It is not known, for example, why about half of patients given haloperidol—a drug used to treat psychotic disorders (conditions that affect how people think, feel, or behave)—develop tremors and other symptoms caused by alterations in the brain region that controls voluntary movements. Here, to improve our understanding of how drugs enter the brain and impact its function, the researchers investigate the genetic factors that affect haloperidol-induced toxicity by genetically analyzing several inbred mouse strains (every individual in an inbred mouse strain is genetically identical) with different susceptibilities to haloperidol-induced toxicity and by undertaking a human genetic association study (a study that looks for non-chance associations between specific traits and genetic variants).
What Did the Researchers Do and Find?
The researchers used a database of genetic variants called single nucleotide polymorphisms (SNPs) and a computational genetic mapping approach to show first that variations within the gene encoding Abcb5 affected susceptibility to haloperidol-induced toxicity (indicated by changes in the length of time taken by mice to move their paws when placed on an inclined wire-mesh screen) among inbred mouse strains. Abcb5 is an ATP-binding cassette transporter, a type of protein that moves molecules across cell membranes. The researchers next showed that Abcb5 is expressed in brain capillaries, which is the location of the blood-brain barrier. Abcb5 was also expressed in cerebellar Purkinje cells, which help to control motor (intentional) movements. They also measured the measured the effect of haloperidol and the haloperidol concentration in brain tissue sections in mice that were genetically engineered to make no Abcb5 (Abcb5 knockout mice). Finally, the researchers investigated whether specific alleles (alternative versions) of ABCB5 are associated with haloperidol-induced toxicity in people. Among a group of 85 patients treated with haloperidol for a psychotic illness, one specific ABCB5 allele was associated with haloperidol-induced toxicity during the first few days of treatment.
What Do These Findings Mean?
These findings indicate that Abcb5 is a component of the blood-brain barrier in mice and suggest that genetic variants in the gene encoding this protein underlie, at least in part, the differences in susceptibility to haloperidol-induced toxicity seen among inbred mice strains. Moreover, the human genetic association study indicates that a specific ABCB5 allele also affects the susceptibility of people to haloperidol-induced toxicity. The researchers note that other ABCB5 alleles or other genetic factors that affect haloperidol-induced toxicity in people might emerge if larger groups of patients were studied. However, based on their findings, the researchers propose a new model for the genetic mechanisms that underlie inter-individual and cell type-specific differences in susceptibility to haloperidol-induced brain toxicity. If confirmed in future studies, this model might facilitate the development of more effective and less toxic drugs to treat a range of brain disorders.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001782.
The US National Institute of Neurological Disorders and Stroke provides information about a wide range of brain diseases (in English and Spanish); its fact sheet “Brain Basics: Know Your Brain” is a simple introduction to the human brain; its “Blueprint Neurotherapeutics Network” was established to develop new drugs for disorders affecting the brain and other parts of the nervous system
MedlinePlus provides links to additional resources about brain diseases and their treatment (in English and Spanish)
Wikipedia provides information about haloperidol, about ATP-binding cassette transporters and about genetic association (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001782
PMCID: PMC4315575  PMID: 25647612
6.  GroEL dependency affects codon usage—support for a critical role of misfolding in gene evolution 
Integrating genome-scale sequence, expression, structural and protein interaction data from E. coli we establish an interaction between chaperone (GroEL) dependency and optimal codon usage.Highly expressed sporadic substrates of GroEL employ more optimal codons than expected, show enrichment for optimal codons at structurally sensitive sites and greater conservation of codon optimality under conditions of relaxed purifying selection.We suggest that highly expressed genes cannot routinely utilize GroEL for error control so that codon usage has evolved to provide complementary error limitation, whereas obligate GroEL substrates experience relaxed selection on codon usage.Our results support a critical role of misfolding prevention in gene evolution.
Errors during gene expression are relatively commonplace, which has prompted speculations that many features of gene and genome anatomy and organization have evolved to reduce or mitigate such errors. One type of error that can be particularly costly occurs when the polypeptide chain that emerges from the ribosome fails to fold into its native structure. Some aberrantly folded proteins, exposing hydrophobic residues that would normally be buried, may begin to promiscuously interact with other proteins, become toxic to the cell and thus pose a substantial fitness concern (Gregersen et al, 2006).
In trans, molecular chaperones have long been recognized to play crucial roles in misfolding prevention and remedy. In cis, it has recently been suggested that the use of optimal codons limits mistranslation-induced protein misfolding (Drummond and Wilke, 2008). Evidence for the latter is centred on the argument that synonymous codons differ in their propensity to cause mistranslation. Translationally optimal codons, typically represented by more abundant cognate tRNAs (Duret, 2000), are thought less likely to cause ribosomal stalling and/or incorporation of the wrong amino acid.
Here, we suggest that the role, if any, of error limitation in cis can be revealed by studying its interaction with well-established error management systems in trans (chaperones). If codon usage does indeed play a tangible role in misfolding prevention, we would expect selection on codon identity to vary with the degree to which a protein can rely on other error control mechanisms, namely chaperones. We use the E. coli chaperonin GroEL as a model system to explore whether there is any interaction between optimal codon usage and chaperone dependency.
Kerner et al (2005) had previously determined GroEL substrates on a genome-wide scale. Based on enrichment in GroEL complexes the authors assigned ∼250 proteins to three classes reflecting GroEL dependency: class-I proteins, only a small fraction of which (<1%) associates with GroEL and which spontaneously regain some activity; class-II proteins, which only exhibit spontaneous refolding at more permissive temperatures and class-III proteins, which are obligate substrates of GroEL and largely fail to refold even under more benign conditions. Notably, although on average less abundant than class-I/II proteins (‘sporadic clients'), class-III proteins (‘obligate clients') occupy ∼80% of GroEL's capacity in vivo. Consequently, a higher proportion (∼100% versus ∼20% for class-II and ∼1% for class-I) of these proteins is routinely processed by the GroEL system.
We demonstrate that sporadic but not obligate clients of GroEL exhibit enhanced codon adaptation, carefully controlling for possible confounding factors, notably expression level and protein length (Figure 1). We also point out that genes that recently entered the E. coli genome via horizontal gene transfer will distort equilibrium analyses of codon usage in bacteria and should thus be routinely eliminated from analysis.
Building on earlier work by Zhou et al (2009), we further show that sporadic substrates are conspicuously enriched for optimal codons at structurally sensitive sites, consistent with more severe fitness implications of codon choice for these proteins.
Lastly, we reveal that codon optimality in sporadic clients is more highly conserved in S. dysenteriae. S. dysenteriae is an E. coli clone that has diverged relatively recently from the E. coli K12 strain and has adopted an intracellular lifestyle (Balbi et al, 2009). Concomitant with that lifestyle, Shigella has experienced a lower effective population size and therefore reduced efficiency of purifying selection. This has generated conditions where, overall, codon optimality has started to decay. However, when we followed the fate of ancestrally optimal codons at buried sites in the S. dysenteriae and E. coli K12 genomes, we found that a lower fraction of buried sites has lost codon optimality in sporadic substrates (Figure 4), again consistent with greater structural importance of codon choice in these substrates.
Based on the these findings, we suggest the following explanation: As mentioned above, class-III substrates are defined not only by GroEL being critical for proper folding, but also by occupying most of GroEL's capacity (∼80%). With a high proportion of class-III protein passaged through the GroEL system, mistranslation errors in these proteins weigh less severely as GroEL can remedy at least some misfolding that ensues. In contrast, class-I and II genes are more highly expressed and cannot routinely rely on GroEL to rectify folding errors. Yet class-I/II proteins are clearly liable to misfold as testified by their sporadic association with GroEL. We argue that augmenting GroEL's capacity to address the misfolding propensity of these genes would be prohibitively costly to the organism and that, as an alternative strategy, these genes employ optimal codons to reduce the rate of misfolding error.
Our findings (a) reveal a cis–trans interaction between codon usage and chaperones in providing an integrated error management system, (b) provide independent evidence for a role of misfolding in shaping gene evolution and (c) suggest that the burden of deleterious mutations in long-term bottlenecking populations like that of the insect endosymbiont Buchnera not only comprises unfavourable amino-acid (Moran, 1996) but also synonymous substitutions.
It has recently been suggested that the use of optimal codons limits mistranslation-induced protein misfolding, yet evidence for this remains largely circumstantial. In contrast, molecular chaperones have long been recognized to play crucial roles in misfolding prevention and remedy. We propose that putative error limitation in cis can be elucidated by examining the interaction between codon usage and chaperoning processes. Using Escherichia coli as a model system, we find that codon optimality covaries with dependency on the chaperonin GroEL. Sporadic but not obligate substrates of GroEL exhibit higher average codon adaptation and are conspicuously enriched for optimal codons at structurally sensitive sites. Further, codon optimality of sporadic clients is more conserved in the E. coli clone Shigella dysenteriae. We suggest that highly expressed genes cannot routinely use GroEL for error control so that codon usage has evolved to provide complementary error limitation. These findings provide independent evidence for a role of misfolding in shaping gene evolution and highlight the need to co-characterize adaptations in cis and trans to unravel the workings of integrated molecular systems.
doi:10.1038/msb.2009.94
PMCID: PMC2824523  PMID: 20087338
codon bias; GroEL; misfolding
7.  Evolution of Minimal Specificity and Promiscuity in Steroid Hormone Receptors 
PLoS Genetics  2012;8(11):e1003072.
Most proteins are regulated by physical interactions with other molecules; some are highly specific, but others interact with many partners. Despite much speculation, we know little about how and why specificity/promiscuity evolves in natural proteins. It is widely assumed that specific proteins evolved from more promiscuous ancient forms and that most proteins' specificity has been tuned to an optimal state by selection. Here we use ancestral protein reconstruction to trace the evolutionary history of ligand recognition in the steroid hormone receptors (SRs), a family of hormone-regulated animal transcription factors. We resurrected the deepest ancestral proteins in the SR family and characterized the structure-activity relationships by which they distinguished among ligands. We found that that the most ancient split in SR evolution involved a discrete switch from an ancient receptor for aromatized estrogens—including xenobiotics—to a derived receptor that recognized non-aromatized progestagens and corticosteroids. The family's history, viewed in relation to the evolution of their ligands, suggests that SRs evolved according to a principle of minimal specificity: at each point in time, receptors evolved ligand recognition criteria that were just specific enough to parse the set of endogenous substances to which they were exposed. By studying the atomic structures of resurrected SR proteins, we found that their promiscuity evolved because the ancestral binding cavity was larger than the primary ligand and contained excess hydrogen bonding capacity, allowing adventitious recognition of larger molecules with additional functional groups. Our findings provide an historical explanation for the sensitivity of modern SRs to natural and synthetic ligands—including endocrine-disrupting drugs and pollutants—and show that knowledge of history can contribute to ligand prediction. They suggest that SR promiscuity may reflect the limited power of selection within real biological systems to discriminate between perfect and “good enough.”
Author Summary
The functions of most proteins are defined by their interactions with other biological substances, such as DNA, nutrients, hormones, or other proteins. Some proteins are highly specific, but others are more promiscuous and can interact with a variety of natural substances, as well as drugs and pollutants. Understanding molecular interactions is a key goal in pharmacology and toxicology, but there are few general principles to help explain or predict protein specificity. Because every biological entity is the result of evolution, understanding a protein's history might help explain why it interacts with the substances to which it is sensitive. In this paper, we used ancestral protein reconstruction to experimentally trace how specificity evolved in an ancient group of proteins, the steroid hormone receptors (SRs), a family of proteins that regulate reproduction and other biological processes in animals. We show that SRs evolved according to a principle of minimal specificity: at each point in time, these proteins evolved to be specific enough to distinguish among the substances to which they were naturally exposed, but not more so. Our findings provide an historical explanation for modern SRs' diverse sensitivities to natural and man-made substances; they show that knowledge of history can contribute to predicting the ligands to which a modern protein will respond and indicate that promiscuity reflects the limited power of natural selection to discriminate between perfect and “good enough.”
doi:10.1371/journal.pgen.1003072
PMCID: PMC3499368  PMID: 23166518
8.  Acute Human Lethal Toxicity of Agricultural Pesticides: A Prospective Cohort Study 
PLoS Medicine  2010;7(10):e1000357.
In a prospective cohort study of patients presenting with pesticide self-poisoning, Andrew Dawson and colleagues investigate the relative human toxicity of agricultural pesticides and contrast it with WHO toxicity classifications, which are based on toxicity in rats.
Background
Agricultural pesticide poisoning is a major public health problem in the developing world, killing at least 250,000–370,000 people each year. Targeted pesticide restrictions in Sri Lanka over the last 20 years have reduced pesticide deaths by 50% without decreasing agricultural output. However, regulatory decisions have thus far not been based on the human toxicity of formulated agricultural pesticides but on the surrogate of rat toxicity using pure unformulated pesticides. We aimed to determine the relative human toxicity of formulated agricultural pesticides to improve the effectiveness of regulatory policy.
Methods and Findings
We examined the case fatality of different agricultural pesticides in a prospective cohort of patients presenting with pesticide self-poisoning to two clinical trial centers from April 2002 to November 2008. Identification of the pesticide ingested was based on history or positive identification of the container. A single pesticide was ingested by 9,302 patients. A specific pesticide was identified in 7,461 patients; 1,841 ingested an unknown pesticide. In a subset of 808 patients, the history of ingestion was confirmed by laboratory analysis in 95% of patients. There was a large variation in case fatality between pesticides—from 0% to 42%. This marked variation in lethality was observed for compounds within the same chemical and/or WHO toxicity classification of pesticides and for those used for similar agricultural indications.
Conclusion
The human data provided toxicity rankings for some pesticides that contrasted strongly with the WHO toxicity classification based on rat toxicity. Basing regulation on human toxicity will make pesticide poisoning less hazardous, preventing hundreds of thousands of deaths globally without compromising agricultural needs. Ongoing monitoring of patterns of use and clinical toxicity for new pesticides is needed to identify highly toxic pesticides in a timely manner.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Suicide is a preventable global public health problem. About 1 million people die each year from suicide and many more harm themselves but survive. Although many people who commit suicide have a mental illness, stressful events (economic hardship or relationship difficulties, for example) can sometimes make life seem too painful to bear. Suicide attempts are frequently impulsive and use methods that are conveniently accessible. Strategies to reduce suicide rates include better treatment of mental illness and programs that help people at high risk of suicide deal with stress. Suicide rates can also be reduced by limiting access to common suicide methods. The single most important means of suicide worldwide is agricultural pesticide poisoning. Every year, between 250,000 and 370,000 people die from deliberate ingestion of pesticides (chemicals that kill animal pests or unwanted plants). Most of these suicides occur in rural areas of the developing world where high levels of pesticide use in agriculture combined with pesticide storage at home facilitate this particular method of suicide.
Why Was This Study Done?
To help reduce suicides through the ingestion of agricultural pesticides, the Food and Agriculture Organization of the United Nations recommends the withdrawal of the most toxic pesticides—World Health Organization (WHO) class I pesticides—from agricultural use. This strategy has proven successful in Sri Lanka where a ban on class I pesticides in 1995 and on the class II pesticide endosulfan in 1998 has reduced pesticide deaths by 50% over the past 20 years without decreasing agricultural output. Further reductions in suicides from pesticide ingestion could be achieved if regulatory restrictions on the sale and distribution of the most toxic class II pesticides were imposed. But such restrictions must balance agricultural needs against the impact of pesticides on public health. Unfortunately, the current WHO pesticide classification is based on toxicity in rats. Because rats handle pesticides differently from people, there is no guarantee that a pesticide with low toxicity in rodents is safe in people. Here, the researchers try to determine the relative human toxicity of agricultural pesticides in a prospective cohort study (a study in which people who share a characteristic—in this case, deliberate pesticide ingestion—are enrolled and followed to see how they fare).
What Did the Researchers Do and Find?
The researchers examined the case fatality (the proportion of patients dying after hospital admission) of different agricultural pesticides among patients who presented with pesticide self-poisoning at two Sri Lankan referral hospitals. Between April 2002 and November 2008, 9,302 people were admitted to the hospitals after swallowing a single pesticide. The researchers identified the pesticide ingested in 7,461 cases by asking the patient what he/she had taken or by identifying the container brought in by the patient or relatives. 10% of the patients died but there was a large variation in case fatality between pesticides. The herbicide paraquat was the most lethal pesticide, killing 42% of patients; several other pesticides killed no one. Compounds in the same chemical class and/or the same WHO toxicity class sometimes had very different toxicities. For example, dimethoate and malathione, both class II organophosphate insecticides, had case fatalities of 20.6% and 1.9%, respectively. Similarly, pesticides used for similar agricultural purposes sometimes had very different case fatalities.
What Do These Findings Mean?
These findings provide a toxicity ranking for pesticides that deviates markedly from the WHO toxicity classification based on rat toxicity. Although the findings are based on a study undertaken at just two Sri Lankan hospitals, they are likely to be generalizable to other hospitals and to other parts of rural Asia. However, because the study only included patients who were admitted to hospital after ingesting pesticides, the actual case fatalities for some pesticides may be somewhat different. Nevertheless, these findings have several important public health implications. For example, they suggest that the decision taken in January 2008 to withdraw paraquat, dimethoate, and fenthion from the Sri Lankan market should reduce deaths from pesticide poisoning in Sri Lanka by a further 33%–65% (equivalent to about 1,000 fewer suicides per year). More generally, they suggest that basing the regulation of pesticides on human toxicity has the potential to prevent hundreds and thousands of intentional and accidental deaths globally without compromising agricultural needs.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000357.
This study is further discussed in a PLoS Medicine Perspective by Matt Miller and Kavi Bhalla
The World Health Organization provides information on the global burden of suicide and on suicide prevention (in several languages) and on its classification of pesticides
The US Environmental Protection Agency provides information about all aspects of pesticides (in English and Spanish)
Toxtown, an interactive site from the US National Library of Science, provides information on environmental health concerns including exposure to pesticides (in English and Spanish)
The nonprofit organization Pesticide Action Network UK provides information about all aspects of pesticides
The US National Pesticide Information Center provides objective, science-based information about pesticides (in several languages)
The Food and Agriculture Organization of the United Nations leads international efforts to reduce hunger; as part of this effort, it has introduced pesticide policy reforms (in several languages)
MedlinePlus provides links to further resources about suicide and about pesticides (in English and Spanish)
doi:10.1371/journal.pmed.1000357
PMCID: PMC2964340  PMID: 21048990
9.  A Majority of the Cancer/Testis Antigens are Intrinsically Disordered Proteins 
Journal of cellular biochemistry  2011;112(11):3256-3267.
The Cancer/Testis Antigens (CTAs) are a group of heterogeneous proteins that are typically expressed in the testis but aberrantly expressed in several types of cancer. Although overexpression of CTAs is frequently associated with advanced disease and poorer prognosis, the significance of this correlation is unclear since the functions of the CTAs in the disease process remain poorly understood. Here, employing a bioinformatics approach, we show that a majority of CTAs are intrinsically disordered proteins (IDPs). IDPs are proteins that, under physiological conditions in vitro, lack rigid 3D structures either along their entire length or in localized regions. Despite the lack of structure, most IDPs can transition from disorder to order upon binding to biological targets and often promote highly promiscuous interactions. IDPs play important roles in transcriptional regulation and signaling via regulatory protein networks and are often associated with dosage sensitivity. Consistent with these observations, we find that several CTAs can bind DNA, and their forced expression appears to increase cell growth implying a potential dosage-sensitive function. Furthermore, the CTAs appear to occupy ‘hub’ positions in protein regulatory networks that typically adopt a ‘scale-free’ power law distribution. Taken together, our data provide a novel perspective on the CTAs implicating them in processing and transducing information in altered physiological states in a dosage-sensitive manner. Identifying the CTAs that occupy hub positions in protein regulatory networks would allow a better understanding of their functions as well as the development of novel therapeutics to treat cancer.
doi:10.1002/jcb.23252
PMCID: PMC3214731  PMID: 21748782
Cancer/Testis Antigens; Intrinsically Disordered Proteins; Dosage Sensitivity; Cancer
10.  Systematic exploration of synergistic drug pairs 
Two types of drug synergy, genetic and promiscuous, are explored in S. cerevisiae. The results suggest that promiscuous synergy predominates, and that propensity to synergize is an intrinsic drug property with the potential to accelerate the search for synergistic drug combinations.
Discovered 37 synergistic interactions among antifungal chemicalsPromiscuous synergy is the predominant form of drug synergyRate of synergy is an intrinsic property of drugs that can guide searches for drug synergy
Drug synergy allows a therapeutic effect to be achieved with lower doses of component drugs. Drug synergy can result when drugs target the products of genes that act in parallel pathways (‘specific synergy'). Such cases of drug synergy should tend to correspond to synergistic genetic interaction between the corresponding target genes. Alternatively, ‘promiscuous synergy' can arise when one drug non-specifically increases the effects of many other drugs, for example, by increased bioavailability. To assess the relative abundance of these drug synergy types, we examined 200 pairs of antifungal drugs in S. cerevisiae. We found 38 antifungal synergies, 37 of which were novel. While 14 cases of drug synergy corresponded to genetic interaction, 92% of the synergies we discovered involved only six frequently synergistic drugs. Although promiscuity of four drugs can be explained under the bioavailability model, the promiscuity of Tacrolimus and Pentamidine was completely unexpected. While many drug synergies correspond to genetic interactions, the majority of drug synergies appear to result from non-specific promiscuous synergy.
doi:10.1038/msb.2011.71
PMCID: PMC3261710  PMID: 22068327
chemical genetics; drug combinations; drug discovery; genetic interactions
11.  Sex Reversal in C57BL/6J XY Mice Caused by Increased Expression of Ovarian Genes and Insufficient Activation of the Testis Determining Pathway 
PLoS Genetics  2012;8(4):e1002569.
Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XYB6 mice impaired testis differentiation, but no ovarian tissue developed. If, however, a YAKR chromosome replaced the YB6 chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/− B6 XYAKR, Wt1+/− B6 XYAKR, B6 XYPOS, and B6 XYAKR fetuses. We propose that Wt1B6 and Sf1B6 are hypomorphic alleles of testis-determining pathway genes and that Wnt4B6 is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1B6 and/or Sf1B6 alleles that compromise testis differentiation and a Wnt4B6 allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a “weak” Sry allele, such as the one on the YPOS chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal.
Author Summary
It has been proposed that mice do not adequately model human disorders of sex development because testis determination is gene dosage-sensitive in humans, but initial studies suggested it is gene dosage-insensitive in mice. For example, XY humans with reduced functional WT1 or SF1 gene-dosage or increased WNT4 gene-dosage can be sex reversed, whereas the equivalent XY mice were not sex-reversed on the genetic backgrounds previously reported. However, because testis determination in C57BL/6J mice is very sensitive to disruption, we tested the hypothesis that these genes are dosage-sensitive in C57BL/6J XY mice. We found that C57BL/6J-YAKR mice with reduced Wt1 or Sf1 gene-dosage were sex-reversed, whereas decreased Wnt4 gene-dosage partially rescued testis development in four genetic systems where C57BL/6J XY mice develop ovarian tissue. Our results demonstrate that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, that genetic background affects this sensitivity (as we suspect is true in humans), and that the mouse models described here are good models for human dosage-sensitive sex reversal. The results from these and other experiments lead to the hypothesis that the Wt1 and/or Sf1 alleles found in C57BL/6J mice are relatively “weak” testis-determining genes and their Wnt4 allele is a hyperactive ovary-determining gene.
doi:10.1371/journal.pgen.1002569
PMCID: PMC3320579  PMID: 22496664
12.  Structural and Chemical Profiling of the Human Cytosolic Sulfotransferases  
PLoS Biology  2007;5(5):e97.
The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique “chemical fingerprints” for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural “priming” of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone.
Author Summary
We metabolize many hormones, drugs, and bioactive chemicals and toxins from the environment. One family of enzymes that participate in the metabolic process consists of the cytosolic sulfotransferases, or SULTs. SULTs have a variety of mechanisms of action—sometimes they inactivate the biological activity of the chemical (e.g., in the case of estrogen). At other times, the enzymes make the chemical more toxic (e.g., for certain carcinogens). Humans have 12 distinct SULT enzymes. Determining how each of these human enzymes recognizes and distinguishes between the thousands of chemicals we confront each day is essential for understanding hormone regulation, assessing environmental risk, and eventually developing better, more-effective drugs. We have studied the human SULT family of enzymes to profile which small molecules are recognized by each enzyme. We also visualized and compared the detailed structural features that determine which enzyme interacts with which molecule. By studying the entire family, we discovered new ways in which chemicals interact with each enzyme. Furthermore, we identified new inhibitors and inhibitory mechanisms. Finally, we discovered functions for many of the human enzymes that were previously uncharacterized.
Structural genomics and substrate screening provide "chemical fingerprints" and insights into substrate promiscuity for the human family of drug- and hormone-metabolizing cytosolic sulfotransferase enzymes.
doi:10.1371/journal.pbio.0050097
PMCID: PMC1847840  PMID: 17425406
13.  Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family 
PLoS Computational Biology  2013;9(9):e1003239.
Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined “fuzziness”, often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs.
Author Summary
Understanding how proteins recognize each other is central to deciphering the inner workings of living things and for biomedical research. It has long been known that the sequence of a protein, which is a string of different amino acids, can dictate how a protein molecule folds into a well-defined shape required for biological tasks. Many folded proteins recognize and bind with each other by a tight geometric fit similar to that between a lock and its key. Recently, however, it has become clear that some proteins function as a flexible string, in constant motion, without forming a stable shape. Understanding how such “disordered” proteins work is challenging. To gain insight, we studied a disordered protein region that causes a large family of human cancers. Employing an innovative combination of experimental and theoretical techniques, we describe a new mode of protein interaction based on multiple simple contacts between one type of amino acid (aromatic) in the disordered protein and another type (positively charged) on the partner protein. Because this mechanism also underlies the ability of the disordered protein to cause cancer, further investigation of this unprecedented mode of protein-protein interaction may open up new avenues for cancer therapy.
doi:10.1371/journal.pcbi.1003239
PMCID: PMC3784488  PMID: 24086122
14.  Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 Mimetics 
PLoS Medicine  2007;4(10):e316.
Background
The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.
Methods and Findings
We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.
Conclusions
Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.
Andreas Strasser and colleagues demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR−MEK−ERK signaling cascade is critical for BIM activation.
Editors' Summary
Background.
Normally, cell division (which produces new cells) and cell death are finely balanced to keep the human body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. One protein with a critical role in cell division that is often mutated in tumors is the epidermal growth factor receptor (EGFR). In normal cells, protein messengers bind to EGFR and activate its tyrosine kinase. This enzyme then adds phosphate groups to tyrosine (an amino acid) in proteins that form part of signaling cascades (for example, the MEK–ERK signaling cascade) that tell the cell to divide. In cancers that have mutations in EGFR, signaling is overactive so the cancer cells divide much more than they should. Some non-small cell lung cancers (NSCLC, the commonest type of lung cancer), for example, have activating mutations within the EGFR tyrosine kinase. Treatment with EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib induces the cells in these tumors to stop growing and die. This cell death causes tumor shrinkage (regression) and increases the life expectancy of patients with this type of NSCLC.
Why Was This Study Done?
Unfortunately, treatment with TKIs rarely cures NSCLC, so it would be useful to find a way to augment the effect that TKIs have on cancer cells. To do this, the molecular mechanisms that cause cancer-cell death and tumor regression in response to these drugs need to be fully understood. In this study, the researchers have used a combination of biochemical and genetic approaches to investigate how gefitinib kills NSCLC cells with mutated EGFR.
What Did the Researchers Do and Find?
The researchers first measured the sensitivity of NSCLC cell lines (tumor cells that grow indefinitely in dishes) to gefitinib-induced apoptosis. Gefitinib caused extensive apoptosis in two cell lines expressing mutant EGFR but not in one expressing normal EGFR. Next, they investigated the mechanism of gefitinib-induced apoptosis in the most sensitive cell line (H3255). Apoptosis is activated via two major pathways. Hallmarks of the “intrinsic” pathway include activation of a protein called BAX and cytochrome c release from subcellular compartments known as mitochondria. Gefitinib treatment induced both these events in H3255 cells. BAX (a proapoptotic member of the BCL-2 family of proteins) is activated when proapoptotic BH3-only BCL-2 proteins (for example, BIM; “BH3-only” describes the structure of these proteins) bind to antiapoptotic BCL2 proteins. Gefitinib treatment rapidly increased BIM activity in H3255 and HCC827 cells (but not in gefitinib-resistant cells) by increasing the production of BIM protein and the removal of phosphate groups from it, which increases BIM activity. Pharmacological blockade of the MEK–ERK signaling cascade, but not of other EGFR signaling cascades, also caused the accumulation of BIM. By contrast, blocking BIM expression using a technique called RNA interference reduced gefitinib-induced apoptosis. Finally, a combination of gefitinib and a BH3-mimicking compound called ABT-737 (which, like BIM, binds to antiapoptotic BCL-2 proteins) caused more apoptosis than gefitinib alone.
What Do These Findings Mean?
These findings (and those reported by Gong et al. and Costa et al.) indicate that activation of the proapoptotic BH3-only protein BIM is essential for gefitinib-induced killing of NSCLC cells that carry EGFR tyrosine kinase mutations. They also show that inhibition of the EGFR–MEK–ERK signaling cascade by gefitinib is essential for BIM activation. Because these findings come from studies on NSCLC cell lines, they need confirming in freshly isolated tumor cells and in tumors growing in people. However, the demonstration that a compound that mimics BH3 action enhances gefitinib-induced killing of NSCLC cells suggests that combinations of TKIs and drugs that affect the intrinsic pathway of apoptosis activation might provide a powerful strategy for treating cancers in which tyrosine kinase mutations drive tumor growth.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040316.
A perspective by Ingo Mellinghoff discusses this article and two related research articles
Wikipedia pages on epidermal growth factor receptor, apoptosis, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
CancerQuest provides information on all aspects of cancer from Emory University (in several languages)
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs
Information for patients from Cancerbackup on erlotinib and gefitinib
doi:10.1371/journal.pmed.0040316
PMCID: PMC2043013  PMID: 17973573
15.  Analysis of Gene Expression Using Gene Sets Discriminates Cancer Patients with and without Late Radiation Toxicity 
PLoS Medicine  2006;3(10):e422.
Background
Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%–10% of patients. Assuming that genetic susceptibility impacts this risk, we hypothesized that the cellular response of normal tissue to X-rays could discriminate patients with and without late radiation toxicity.
Methods and Findings
Prostate carcinoma patients without evidence of cancer 2 y after curative radiotherapy were recruited in the study. Blood samples of 21 patients with severe late complications from radiation and 17 patients without symptoms were collected. Stimulated peripheral lymphocytes were mock-irradiated or irradiated with 2-Gy X-rays. The 24-h radiation response was analyzed by gene expression profiling and used for classification. Classification was performed either on the expression of separate genes or, to augment the classification power, on gene sets consisting of genes grouped together based on function or cellular colocalization.
X-ray irradiation altered the expression of radio-responsive genes in both groups. This response was variable across individuals, and the expression of the most significant radio-responsive genes was unlinked to radiation toxicity. The classifier based on the radiation response of separate genes correctly classified 63% of the patients. The classifier based on affected gene sets improved correct classification to 86%, although on the individual level only 21/38 (55%) patients were classified with high certainty. The majority of the discriminative genes and gene sets belonged to the ubiquitin, apoptosis, and stress signaling networks. The apoptotic response appeared more pronounced in patients that did not develop toxicity. In an independent set of 12 patients, the toxicity status of eight was predicted correctly by the gene set classifier.
Conclusions
Gene expression profiling succeeded to some extent in discriminating groups of patients with and without severe late radiotherapy toxicity. Moreover, the discriminative power was enhanced by assessment of functionally or structurally related gene sets. While prediction of individual response requires improvement, this study is a step forward in predicting susceptibility to late radiation toxicity.
Expression profiling can discriminate between groups of patients with and without severe late radiotherapy toxicity but not (yet) predict individual responses.
Editors' Summary
Background.
More than half the people who develop cancer receive radiotherapy as part of their treatment. That is, tumor cells are destroyed by exposing them to a source of ionizing radiation such as X-rays. Ionizing radiation damages the genetic material of cancer cells so that they can no longer divide. Unfortunately, it also damages nearby normal cells, although they are less sensitive to radiation than the cancer cells. Radiotherapists minimize how much radiation hits normal tissues by carefully aiming the X-rays at the tumor. Even so, patients often develop side effects such as sore skin or digestive problems during or soon after radiotherapy; the exact nature of the side effects depends on the part of the body exposed to the X-rays. In addition, a few patients develop severe late radiation toxicity, months or years after their treatment. Like early toxicity, late toxicity occurs in the normal tissues near the tumor site. For example, in prostate cancer—a tumor that forms in a gland in the male reproductive system that lies between the bladder and the end of the gut (the rectum)—late radiation toxicity affects rectal, bladder, and sexual function in 5%–10% of patients.
Why Was This Study Done?
It is not known why some patients develop late radiation toxicity, and it is impossible to predict before treatment which patients will have long-term health problems after radiotherapy. It would be useful to know this, because radiation levels might be reduced in those patients, while larger doses of radiation could be given to patients at low risk of late complications to ensure a complete eradication of their cancer. One theory is that some patients are genetically predisposed to develop severe late radiation toxicity. In other words, their genetic make-up makes it more likely that their tissues develop long-term complications after radiation damage. In this study, the researchers looked for markers of a genetic predisposition for late radiation toxicity by comparing radiation-induced changes in the pattern of cellular proteins in patients who had late radiation toxicity after radiotherapy with the changes seen in patients who did not develop such complications.
What Did the Researchers Do and Find?
The researchers recruited 38 patients who had been treated successfully with radiotherapy for prostate cancer two years previously. Of these, 21 had developed severe late radiation toxicity. They isolated lymphocytes (a type of immune system cell) from the patients' blood, stimulated the lymphocytes to divide, exposed them to X-rays, and analyzed the pattern of genes active in these cells—their gene expression profile—before and after irradiation. The researchers found that irradiation induced the expression of numerous genes in the lymphocytes, including many well-known radiation-responsive genes. They then used an analytical process called “random cross-validation” to look for a gene expression profile (or molecular signature) that was associated with late radiation toxicity. They report that a signature based on the radiation response of 50 individual genes correctly classified 63% of the patient population in terms of whether the patient had developed late radiation toxicity. A signature based on the radiation response of gene sets containing genes linked by function or cellular localization correctly classified 86% of the patient population.
What Do These Findings Mean?
Gene expression profiling identified groups of patients who had had severe late radiation toxicity pretty well, particularly when sets of related genes were used to classify the patients. The approach was not so good, however, at identifying individual patients who had had problems, being correct and certain only half the time. Additional studies are needed, therefore, before this promising approach can be used clinically to predict patient responses to radiotherapy. Overall, the study supports the idea that some patients are genetically predisposed to develop late radiation toxicity, and it also provides clues about which cellular pathways help to determine late radiation toxicity. Most of the genes and gene sets that discriminated between the patients with and without late radiation toxicity are involved in protein metabolism, apoptosis (a special sort of cell death), and stress signaling networks (pathways that protect cells from damage). This information, if confirmed, might help researchers to develop therapeutic interventions to minimize late radiation toxicity in vulnerable individuals.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030422.
US National Cancer Institute patient information on radiotherapy and on prostate cancer
American Cancer Society information on radiation therapy
Cancer Research UK patient information on radiotherapy
Wikipedia pages on radiotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030422
PMCID: PMC1626552  PMID: 17076557
16.  Intrinsic disorder and protein multibinding in domain, terminal, and linker regions† 
Molecular bioSystems  2010;6(10):1821-1828.
Intrinsic disorder is believed to contribute to the ability of some proteins to interact with multiple partners which is important for protein functional promiscuity and regulation of the cross-talk between pathways. To better understand the mechanisms of molecular recognition through disordered regions, here, we systematically investigate the coupling between disorder and binding within domain families in a structure interaction network and in terminal and inter-domain linker regions. We showed that the canonical domain–domain interaction model should take into account contributions of N- and C-termini and inter-domain linkers, which may form all or part of the binding interfaces. For the majority of proteins, binding interfaces on domain and terminal regions were predicted to be less disordered than non-interface regions. Analysis of all domain families revealed several exceptions, such as kinases, DNA/RNA binding proteins, certain enzymes, and regulatory proteins, which are candidates for disorder-to-order transitions that can occur upon binding. Domain interfaces that bind single or multiple partners do not exhibit significant difference in disorder content if normalized by the number of interactions. In general, protein families with more diverse interactions exhibit less average disorder over all members of the family. Our results shed light on recent controversies regarding the relationship between disorder and binding of multiple partners at common interfaces. In particular, they support the hypothesis that protein domains with many interacting partners should have a pleiotropic effect on functional pathways and consequently might be more constrained in evolution.
doi:10.1039/c005144f
PMCID: PMC2955455  PMID: 20544079
17.  A Preformed Binding Interface in the Unbound Ensemble of an Intrinsically Disordered Protein: Evidence from Molecular Simulations 
PLoS Computational Biology  2012;8(7):e1002605.
Intrinsically disordered proteins play an important role in cellular signalling, mediated by their interactions with other biomolecules. A key question concerns the nature of their binding mechanism, and whether the bound structure is induced only by proximity to the binding partner. This is difficult to answer through experiment alone because of the very heterogeneous nature of the unbound ensemble, and the probable rapid interconversion of the various unbound structures. Here we report the most extensive set of simulations on NCBD to date: we use large-scale replica exchange molecular dynamics to explore the unbound state. An important feature of the study is the use of an atomistic force field that has been parametrised against experimental data for weakly structured peptides, together with an accurate explicit water model. Neither the force field nor the starting conformations are biased towards a particular structure. The regions of NCBD that have high helical propensity in the simulations correspond closely to helices in the ‘core’ unbound conformation determined by NMR, although no single member of the simulated unbound ensemble closely resembles the core conformation, or either of the two known bound conformations. We have validated the results against NMR spectroscopy and SAXS measurements, obtaining reasonable agreement. The two helices which most stabilise the binding of NCBD with ACTR are formed readily; the third helix, which is less important for binding but is involved in most of the intraprotein contacts of NCBD in the bound conformation, is formed more rarely, and tends not to coexist with the other helices. These results support a mechanism by which NCBD gains the advantages of disorder, while forming binding-competent structures in the unbound state. We obtain support for this mechanism from coarse-grained simulations of NCBD with, and without, its binding partner.
Author Summary
While many proteins have a specific ‘native’ conformation, so-called intrinsically disordered proteins (IDPs) adopt many different conformations in rapid succession—a characteristic that may be advantageous for rapid binding and promiscuous association. However, this characteristic also makes it very hard to make experimental measurements over times that are short enough to see changes of conformation. In this work, we use the results of a large-scale molecular simulation to explore conformations of NCBD, which is an IDP that adopts specific conformations when it binds either of two other proteins (ACTR and IRF-3). Our results point to the following hypothesis: to help NCBD bind ACTR, those parts of NCBD that make contact with it in the bound conformation are biased towards that conformation, even in the absence of ACTR. Other parts of NCBD tend to avoid the ACTR-bound conformation, to help ensure that NCBD is disordered when unbound.
doi:10.1371/journal.pcbi.1002605
PMCID: PMC3400577  PMID: 22829760
18.  A Plant-Derived Morphinan as a Novel Lead Compound Active against Malaria Liver Stages  
PLoS Medicine  2006;3(12):e513.
Background
The global spread of multidrug–resistant malaria parasites has led to an urgent need for new chemotherapeutic agents. Drug discovery is primarily directed to the asexual blood stages, and few drugs that are effective against the obligatory liver stages, from which the pathogenic blood infection is initiated, have become available since primaquine was deployed in the 1950s.
Methods and Findings
Using bioassay-guided fractionation based on the parasite's hepatic stage, we have isolated a novel morphinan alkaloid, tazopsine, from a plant traditionally used against malaria in Madagascar. This compound and readily obtained semisynthetic derivatives were tested for inhibitory activity against liver stage development in vitro (P. falciparum and P. yoelii) and in vivo (P. yoelii). Tazopsine fully inhibited the development of P. yoelii (50% inhibitory concentration [IC50] 3.1 μM, therapeutic index [TI] 14) and P. falciparum (IC50 4.2 μM, TI 7) hepatic parasites in cultured primary hepatocytes, with inhibition being most pronounced during the early developmental stages. One derivative, N-cyclopentyl-tazopsine (NCP-tazopsine), with similar inhibitory activity was selected for its lower toxicity (IC50 3.3 μM, TI 46, and IC50 42.4 μM, TI 60, on P. yoelii and P. falciparum hepatic stages in vitro, respectively). Oral administration of NCP-tazopsine completely protected mice from a sporozoite challenge. Unlike the parent molecule, the derivative was uniquely active against Plasmodium hepatic stages.
Conclusions
A readily obtained semisynthetic derivative of a plant-derived compound, tazopsine, has been shown to be specifically active against the liver stage, but inactive against the blood forms of the malaria parasite. This unique specificity in an antimalarial drug severely restricts the pressure for the selection of drug resistance to a parasite stage limited both in numbers and duration, thus allowing researchers to envisage the incorporation of a true causal prophylactic in malaria control programs.
A derivative of a morphinan alkaloid, tazopsine, from a plant used against malaria in Madagascar, is active against the hepatic stages ofPlasmodium species.
Editors' Summary
Background.
The parasite that causes malaria has quickly developed resistance to many of the drugs that are commonly used to treat this disease. As a result, new drugs and drug combinations are needed. In some parts of the world where antimalarial drugs are failing due to resistance, or are not available to everyone, people often turn to traditional herbal remedies instead. These traditional plant remedies can be a useful starting point for development of new drugs, but the process of developing effective new drugs from plant remedies is long and complicated. An important initial step is to isolate and identify the active compounds from plants and then see how well these compounds perform against malaria parasites in laboratory tests. If the tests are successful, such compounds could then progress to experiments in animals and possibly eventually human trials. One plant used widely in Madagascar for treatment of malaria is Strychnopsis thouarsii; the traditional remedy consists of the plant stem bark boiled in water.
Why Was This Study Done?
The group of researchers doing this study wanted to discover candidates for new malaria drugs. They therefore wanted to find out which molecular compounds in the stem bark of S. thouarsii contained antimalarial activity, and what particular stage of the malaria parasite's life cycle these compounds had an effect on. The researchers suspected that the agents in this plant bark had some activity against the “liver stage” of malaria infection in humans. This is the first stage of infection, after a person has been bitten by a malaria-infected mosquito, and before blood cells are invaded by malaria parasites (which then causes the disease symptoms). Very few drugs currently in existence have an effect on the “liver stage” of infection, but activity at this stage would be tremendously useful because it could mean a drug is better for prevention of malaria than others in existence.
What Did the Researchers Do and Find?
First, the researchers wanted to take the traditional herbal remedy—of S. thouarsii bark boiled in water—and find out precisely which molecule in that remedy was responsible for the antimalarial activity. They therefore used a method called chromatography to progressively separate the herbal extract into its distinct components. At each stage of separation, the extract was checked for activity against malaria using a laboratory test. Inactive extracts were disregarded, and the active component then taken on to a further separation round. After many rounds of separation and testing, the researchers got down to a single, apparently new, molecule that was active against malaria in the laboratory test, and this molecule was named tazopsine (in the Malagasy language the word Tazo refers to malaria). In order to find out how effective the molecule was at killing malaria parasites, the researchers took human or mouse liver cells cultured in the laboratory, infected them with malaria parasites (either the malaria parasite that normally infects humans, or a related species that infects mice), and then added tazopsine at different concentrations. The compound completely killed the malaria parasites even at very low concentrations, and had activity against malaria infecting either liver cells or red blood cells. Tazopsine was then given to mice injected with a species of the malaria parasite. The compound protected most mice against malaria infection when it was used at a dosage level lower than the toxic dose. The researchers then tried making a series of different variants of tazopsine in the hope that some variants would be less toxic, but equally active as, the original compound. They found one variant, named NCP-tazopsine, that was much less toxic but just as active as tazopsine, but only against the malaria infecting liver cells.
What Do These Findings Mean?
In these experiments a new molecule, tazopsine, was discovered from a Malagasy plant, and it was found to be active against liver-stage malaria parasites, in laboratory experiments and in mice. This molecule or variants of it could in future become candidate antimalarial drugs in humans. However, much work would need to be done before testing could get to that stage. Different variants of molecules related to tazopsine would need to be tested to find one that has low toxicity, and these variants would need to be fully evaluated in animals to see how they are handled in the body before any trials could begin in humans.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030513
The World Health Organization publishes a minisite containing links to information about all aspects of malaria worldwide, including treatment, prevention, and current programmes for malaria control
Medicines for Malaria Venture is a collaboration between public and private organizations (including the pharmaceutical industry) that aims to fund and manage the development of new drugs for treatment and prevention of malaria
Wikipedia entries for drug discovery and drug development (note: Wikipedia is an internet encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030513
PMCID: PMC1716192  PMID: 17194195
19.  Synthetic incoherent feedforward circuits show adaptation to the amount of their genetic template 
Variable gene dosage is a major source of fluctuations in gene expression in both endogenous and synthetic circuits. Synthetic incoherent feedforward regulatory motifs using RNA interference are shown to robustly adapt to changes in DNA template amounts in mammalian cells.
Variable gene dosage is a major source of fluctuations in gene product levels in both endogenous and synthetic circuits.To mitigate gene expression variability, we designed, simulated, constructed, and tested regulatory circuits, implementing an incoherent feedforward motif.A number of control mechanisms including transcription and post-transcriptional regulation were tested in mammalian cells.Feedforward regulation displayed better adaptation than negative feedback, and circuits based on RNA interference were the most robust to variation in DNA template amounts.
Natural and synthetic biological networks must function reliably in the face of fluctuating stoichiometry of their molecular components. These fluctuations are caused in part by changes in relative expression efficiency and the DNA template amount of the network-coding genes. Indeed, changes in gene dosage are clearly a major source of variation in cells, and yet those changes are very common in both normal processes (sex determination, ploidy change) and disease (gene amplification in cancer). In synthetic networks, the problem is exacerbated due to commonly used transient delivery methods that result in very large cell-to-cell variability in gene dosage. The basic question on gene dosage compensation in nature (Veitia et al, 2008; Acar et al, 2010) and a practical challenge of overcoming sensitivity to DNA copy number in synthetic circuits prompted us to investigate mechanisms to reduce this variability using sophisticated internal regulatory mechanisms. Indeed, the baseline expression unit in many synthetic circuits is an open-loop promoter-ORF combination. We hypothesized that some sort of internal regulation will result in ‘expression units' whose gene product (i.e. protein) output will depend only mildly on the intracellular concentration of its DNA template. In other words, we searched for architecture that would lead to ‘adaptation' of the gene product to the amount of DNA template.
By examining large body of published work, we found frequent reference to a so-called ‘incoherent feedforward' network motif (Mangan and Alon, 2003). The canonical three-node incoherent loop contains input, auxiliary regulator, and output nodes. The output is controlled directly by the input and the auxiliary regulator. The latter is also controlled by the input, introducing an additional indirect effect of the input on the output. In incoherent loops, the input controls the auxiliary regulator node in such a way that input's overall indirect action on the output via this node counteracts its direct effect. In a motif named ‘type I incoherent feedforward loop' (I1-FFL), the input's direct effect is activating, as is its control of the auxiliary node, while the auxiliary node controls the output through repression. One of the most studied properties of such motifs is their transient response to persistent stimulus, that is visually characterized as a ‘bump' or ‘pulse' (hence the name ‘pulse generator') that then goes back to the original steady state of the system (Basu et al, 2004). We hypothesized that changing DNA amount could serve as an input to an incoherent circuit if the auxiliary regulator and the output nodes are coexpressed from this DNA; in other words DNA can be viewed as an ‘activator' of both the regulator and the output. We conjectured that this might lead to adaptation to changes in DNA template.
We designed and simulated in silico a number of network architectures that all exhibit incoherent feedforward connectivity. We also compared them with the well-studied feedback loop circuit that in theory weakens but does not eliminate gene product dependency on the DNA template amount. The schematics of the circuits are shown in Figure 1.
Experimental measurement of input–output response of these circuits, or their transfer function, indeed uncovered adaptation of the output to DNA template abundance. Such adaptation has not been observed with feedback loop, as expected. Among various architectures, the post-transcriptional circuits showed faster adaptation, higher absolute expression levels and lower ‘noise' (Figure 4).
We also simulated and measured stochastic variability in the circuits by collecting all the cells with similar input values and statistically analyzing output values in those cells. We found that substantial noise component could not be accounted for by known noise sources, and concluded that the very step of negative regulation, both by a repressor LacI and by a microRNA, significantly increases cell-to-cell variability. This needs to be addressed in further studies. We also found that the negative feedback loop did not result in reduced noise as we expected, yet it did not result in noise increase as in the incoherent motif. This means that there may be effective noise reduction but it is not sufficient to produce narrow distributions of outputs for a given input.
We conclude that expression units that incorporate incoherent feedforward control of the gene product provide adaptation to the amount of DNA template and can be superior to simple combinations of constitutive promoters with ORFs. We also emphasize the relevance of our findings to the long-standing question of gene dosage compensation in cells, and note that similar incoherent architectures with microRNA negative regulators have been found in cells, suggesting that their physiological role is to curb variability in gene dosage and/or promoter strength.
Natural and synthetic biological networks must function reliably in the face of fluctuating stoichiometry of their molecular components. These fluctuations are caused in part by changes in relative expression efficiency and the DNA template amount of the network-coding genes. Gene product levels could potentially be decoupled from these changes via built-in adaptation mechanisms, thereby boosting network reliability. Here, we show that a mechanism based on an incoherent feedforward motif enables adaptive gene expression in mammalian cells. We modeled, synthesized, and tested transcriptional and post-transcriptional incoherent loops and found that in all cases the gene product adapts to changes in DNA template abundance. We also observed that the post-transcriptional form results in superior adaptation behavior, higher absolute expression levels, and lower intrinsic fluctuations. Our results support a previously hypothesized endogenous role in gene dosage compensation for such motifs and suggest that their incorporation in synthetic networks will improve their robustness and reliability.
doi:10.1038/msb.2011.49
PMCID: PMC3202791  PMID: 21811230
feedforward motifs; gene dosage and noise; mammalian cells; microRNAs; negative autoregulation
20.  Modulation of allostery by protein intrinsic disorder 
Nature  2013;498(7454):390-394.
Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical1 and empirical2 observations indicate that allostery is also manifest in intrinsically disordered proteins (IDPs), which account for a significant proportion of the proteome3,4. Many IDPs are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub IDP5. E1A can induce drastic epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators like the general transcriptional coactivator CREB binding protein (CBP), its paralog p300, and the retinoblastoma protein (pRb)6,7. Little is known about the allosteric effects at play in E1A-CBP-pRb interactions, or more generally in hub IDP interaction networks. Here, we utilized single-molecule Förster/fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved essential for these studies, which are challenging due to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A-CBP-pRb interactions display either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary vs. binary E1A complexes, and may permit a context-specific tuning of associated downstream signaling outputs. Such a modulation of allosteric interactions is likely a common mechanism in molecular hub IDP function.
doi:10.1038/nature12294
PMCID: PMC3718496  PMID: 23783631
adenovirus E1A; p300/CBP; retinoblastoma protein; intrinsically disordered protein; allostery; single-molecule fluorescence
21.  Just how versatile are domains? 
Background
Creating new protein domain arrangements is a frequent mechanism of evolutionary innovation. While some domains always form the same combinations, others form many different arrangements. This ability, which is often referred to as versatility or promiscuity of domains, its a random evolutionary model in which a domain's promiscuity is based on its relative frequency of domains.
Results
We show that there is a clear relationship across genomes between the promiscuity of a given domain and its frequency. However, the strength of this relationship differs for different domains. We thus redefine domain promiscuity by defining a new index, DV I ("domain versatility index"), which eliminates the effect of domain frequency. We explore links between a domain's versatility, when unlinked from abundance, and its biological properties.
Conclusion
Our results indicate that domains occurring as single domain proteins and domains appearing frequently at protein termini have a higher DV I. This is consistent with previous observations that the evolution of domain re-arrangements is primarily driven by fusion of pre-existing arrangements and single domains as well as loss of domains at protein termini. Furthermore, we studied the link between domain age, defined as the first appearance of a domain in the species tree, and the DV I. Contrary to previous studies based on domain promiscuity, it seems as if the DV I is age independent. Finally, we find that contrary to previously reported findings, versatility is lower in Eukaryotes. In summary, our measure of domain versatility indicates that a random attachment process is sufficient to explain the observed distribution of domain arrangements and that several views on domain promiscuity need to be revised.
doi:10.1186/1471-2148-8-285
PMCID: PMC2588589  PMID: 18854028
22.  Common Features at the Start of the Neurodegeneration Cascade 
PLoS Biology  2012;10(5):e1001335.
A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis.
Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these “neurotoxic proteins” triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases.
Author Summary
Neurodegenerative diseases like Alzheimer's or Parkinson's are currently incurable. They are caused by different proteins that, under certain circumstances, aggregate and become toxic as we grow older, but the molecular events underlying this process remain unclear. The lack of a well-defined structure, and the tendency of these “neurotoxic proteins” to aggregate make them difficult to study using conventional techniques. Here, we use an established single-molecule manipulation technique combined with a new protein-engineering strategy to show that all these proteins can adopt a rich collection of structures (conformers) that includes a high proportion of mechanostable conformers, which are associated with toxicity and disease. We also find that a known drug can block the formation of these mechanostable structures in different neurotoxic proteins. We suggest that the most mechanostable conformers, or their precursors, may trigger the pathogenic cascade that results in toxicity. We thus propose that these mechanostable structures are ideal targets for early diagnosis, prevention, and treatment of these fatal diseases.
doi:10.1371/journal.pbio.1001335
PMCID: PMC3362641  PMID: 22666178
23.  Intrinsic Disorder in the Human Spliceosomal Proteome 
PLoS Computational Biology  2012;8(8):e1002641.
The spliceosome is a molecular machine that performs the excision of introns from eukaryotic pre-mRNAs. This macromolecular complex comprises in human cells five RNAs and over one hundred proteins. In recent years, many spliceosomal proteins have been found to exhibit intrinsic disorder, that is to lack stable native three-dimensional structure in solution. Building on the previous body of proteomic, structural and functional data, we have carried out a systematic bioinformatics analysis of intrinsic disorder in the proteome of the human spliceosome. We discovered that almost a half of the combined sequence of proteins abundant in the spliceosome is predicted to be intrinsically disordered, at least when the individual proteins are considered in isolation. The distribution of intrinsic order and disorder throughout the spliceosome is uneven, and is related to the various functions performed by the intrinsic disorder of the spliceosomal proteins in the complex. In particular, proteins involved in the secondary functions of the spliceosome, such as mRNA recognition, intron/exon definition and spliceosomal assembly and dynamics, are more disordered than proteins directly involved in assisting splicing catalysis. Conserved disordered regions in spliceosomal proteins are evolutionarily younger and less widespread than ordered domains of essential spliceosomal proteins at the core of the spliceosome, suggesting that disordered regions were added to a preexistent ordered functional core. Finally, the spliceosomal proteome contains a much higher amount of intrinsic disorder predicted to lack secondary structure than the proteome of the ribosome, another large RNP machine. This result agrees with the currently recognized different functions of proteins in these two complexes.
Author Summary
In eukaryotic cells, introns are spliced out of proteincoding mRNAs by a highly dynamic and extraordinarily plastic molecular machine called the spliceosome. In recent years, multiple regions of intrinsic structural disorder were found in spliceosomal proteins. Intrinsically disordered regions lack stable native three-dimensional structure in solutions, which makes them structurally flexible and/or able to switch between different conformations. Hence, intrinsically disordered regions are the ideal candidate responsible for the spliceosome's plasticity. Intrinsically disordered regions are also frequently the sites of post-translational modifications, which were also proven to be important in spliceosome dynamics. In this article, we describe the results of a structural bioinformatics analysis focused on intrinsic disorder in the spliceosomal proteome. We systematically analyzed all known human spliceosomal proteins with regards to the presence and type of intrinsic disorder. Almost a half of the combined sequence of these spliceosomal proteins is predicted to be intrinsically disordered, and the type of intrinsic disorder in a protein varies with its function and its location in the spliceosome. The parts of the spliceosome that act earlier in the process are more disordered, which corresponds to their role in establishing a network of interactions, while the parts that act later are more ordered.
doi:10.1371/journal.pcbi.1002641
PMCID: PMC3415423  PMID: 22912569
24.  Profiling 976 ToxCast Chemicals across 331 Enzymatic and Receptor Signaling Assays 
Chemical Research in Toxicology  2013;26(6):878-895.
Understanding potential health risks is a significant challenge due to the large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study analyzes 976 chemicals (including failed pharmaceuticals, alternative plasticizers, food additives, and pesticides) in Phases I and II of the U.S. EPA’s ToxCast project across 331 cell-free enzymatic and ligand-binding high-throughput screening (HTS) assays. Half-maximal activity concentrations (AC50) were identified for 729 chemicals in 256 assays (7,135 chemical–assay pairs). Some of the most commonly affected assays were CYPs (CYP2C9 and CYP2C19), transporters (mitochondrial TSPO, norepinephrine, and dopaminergic), and GPCRs (aminergic). Heavy metals, surfactants, and dithiocarbamate fungicides showed promiscuous but distinctly different patterns of activity, whereas many of the pharmaceutical compounds showed promiscuous activity across GPCRs. Literature analysis confirmed >50% of the activities for the most potent chemical–assay pairs (54) but also revealed 10 missed interactions. Twenty-two chemicals with known estrogenic activity were correctly identified for the majority (77%), missing only the weaker interactions. In many cases, novel findings for previously unreported chemical–target combinations clustered with known chemical–target interactions. Results from this large inventory of chemical–biological interactions can inform read-across methods as well as link potential targets to molecular initiating events in adverse outcome pathways for diverse toxicities.
doi:10.1021/tx400021f
PMCID: PMC3685188  PMID: 23611293
25.  Protein Under-Wrapping Causes Dosage Sensitivity and Decreases Gene Duplicability 
PLoS Genetics  2008;4(1):e11.
A fundamental issue in molecular evolution is how to identify the evolutionary forces that determine the fate of duplicated genes. The dosage balance hypothesis has been invoked to explain gene duplication patterns at the genomic level under the premise that a dosage imbalance among protein-complex subunits or interacting partners is often deleterious. Here we examine this hypothesis by investigating the molecular basis of dosage sensitivity. We focus on the extent of protein wrapping, which indicates how strongly the structural integrity of a protein relies on its interactive context. From this perspective, we predict that the duplicates of a highly under-wrapped protein or protein subunit should (1) be more sensitive to dosage imbalance and be less likely to be retained and (2) be more likely to survive from a whole-genome duplication (WGD) than from a non-WGD because a WGD causes little or no dosage imbalance. Our under-wrapping analysis of more than 12,000 protein structures strongly supports these predictions and further reveals that the effect of dosage sensitivity on gene duplicability decreases with increasing organismal complexity.
Author Summary
A gene duplication provides an extra gene copy that can be free to accumulate mutations and gain a new function. Therefore, gene duplication plays a very important role in evolution. However, the presence of an additional gene copy can sometimes be deleterious because it can lead to an excessive dosage relative to those of its interacting partners. This dosage imbalance effect in turn influences the fate of duplicated genes in evolution. Our study gives the first description to our knowledge of the molecular/structural basis for the dosage imbalance effect. We study the relationships between gene family size and extent of protein under-wrapping, a molecular quantifier of the reliance of the protein on binding partnerships to maintain structural integrity, indicative of the extent of structure protection from disruptive hydration. Using more than 12,000 protein three-dimensional structures from six organisms that range from bacteria to human, we show an inverse relationship between extent of protein under-wrapping and family size. That is, a duplication is unlikely to be tolerated if the protein is highly under-wrapped (i.e., its structure requires substantial stabilizing interactions with other proteins). We also show that the effect of dosage imbalance is more apparent in unicellular organisms but is buffered to some extent in higher eukaryotes.
doi:10.1371/journal.pgen.0040011
PMCID: PMC2211539  PMID: 18208334

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