Inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network

Evolution or engineering of novel metabolic pathways can endow microbes with new abilities to degrade anthropogenic pollutants or synthesize valuable chemicals. Most studies of the evolution of new pathways have focused on the origins and quality of function of the enzymes involved. However, there is an additional layer of complexity that has received less attention. Introduction of a novel pathway into an existing metabolic network can result in inhibitory cross-talk due to adventitious interactions between metabolites and macromolecules that have not previously encountered one another. Here, we report a thorough examination of inhibitory cross-talk between a novel metabolic pathway for synthesis of pyridoxal 5′-phosphate and the existing metabolic network of Escherichia coli. We demonstrate multiple problematic interactions, including (i) interference by metabolites in the novel pathway with metabolic processes in the existing network, (ii) interference by metabolites in the existing network with the function of the novel pathway, and (iii) diversion of metabolites from the novel pathway by promiscuous activities of enzymes in the existing metabolic network. Identification of the mechanisms of inhibitory cross-talk can reveal the types of adaptations that must occur to enhance the performance of a novel metabolic pathway as well as the fitness of the microbial host. These findings have important implications for evolutionary studies of the emergence of novel pathways in nature as well as genetic engineering of microbes for “green” manufacturing processes.     

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen

Entomopathogenic fungi and their insect hosts represent a model system for examining invertebrate-pathogen coevolutionary selection processes. Here we report the characterization of competing components of an arms race consisting of insect protective antimicrobial compounds and evolving fungal mechanisms of detoxification. The insect pathogenic fungus Beauveria bassiana has a remarkably wide host range; however, some insects are resistant to fungal infection. Among resistant insects is the tenebrionid beetle Tribolium castaneum that produces benzoquinone-containing defensive secretions. Reduced fungal germination and growth was seen in media containing T. castaneum dichloromethane extracts or synthetic benzoquinone. In response to benzoquinone exposure, the fungus expresses a 1,4-benzoquinone oxidoreductase, BbbqrA, induced >40-fold. Gene knockout mutants (ΔBbbqrA) showed increased growth inhibition, whereas B. bassiana overexpressing BbbqrA (Bb::BbbqrA^O) displayed increased resistance to benzoquinone compared with wild type. Increased benzoquinone reductase activity was detected in wild-type cells exposed to benzoquinone and in the overexpression strain. Heterologous expression and purification of BbBqrA in Escherichia coli confirmed NAD(P)H-dependent benzoquinone reductase activity. The ΔBbbqrA strain showed decreased virulence toward T. castaneum, whereas overexpression of BbbqrA increased mortality versus T. castaneum. No change in virulence was seen for the ΔBbbqrA or Bb::BbbqrA^O strains when tested against the greater wax moth Galleria mellonella or the beetle Sitophilus oryzae, neither of which produce significant amounts of cuticular quinones. The observation that artificial overexpression of BbbqrA results in increased virulence only toward quinone-secreting insects implies the lack of strong selection or current failure of B. bassiana to counteradapt to this particular host defense throughout evolution.Coevolution between hosts and their parasites is considered to be a major driving force of natural selection, and significant literature exists on theoretical and ecological outcomes derived from such interactions. The Red Queen hypothesis, eponymously named after the character in Lewis Carroll’s book Through the Looking Glass, who says to Alice, “…it takes all the running you can do, to keep in the same place…” serves as a concise metaphor for the selection pressures on hosts to constantly change defense mechanisms against pathogens and the counterselection on the pathogens for continuously developing means for overcoming such evolving defenses, resulting in the appearance of both organisms “running in place” (1). Variations of the Red Queen as well as alternative theories, most unified around the centrality of biotic interactions as a driving force, have been proposed as explanations for (i) taxon extinction [Van Valen’s original Red Queen’s hypothesis (2)]; (ii) the evolution and maintenance of sex, genetic recombination, and immune systems (3–5); (iii) as a framework for understanding invasions of exotic species (6); and (iv) as mechanism(s) selecting for host–pathogen coevolution [i.e., broad versus specific host ranges and/or host tolerance (7–9)].For fungal pathogens, the best-known examples of coevolutionary interactions are the pathogen effector and corresponding plant host target genes that have defined a gene-for-gene disease susceptibility model in which the outcome of infection is based on avirulence (Avr) genes in the pathogen and dominant resistance (R) genes in the host (10, 11). The fungal protein can trigger immunity due to R protein effector recognition, thus limiting or preventing disease. In these systems, certain Avr mutations or lack of the appropriate R gene product leads to infection. Avr systems, however, form only part of the landscape of interactions between plant fungal pathogens and their hosts. Invading microbes must overcome exterior defenses, e.g., the plant epicuticle and cell wall, and interior defenses, e.g., immune-related defenses that are both preformed and induced (11–13). Within a broader context, resistance to host antimicrobial compounds, host-induced oxidative and/or other stress, and host immune evasion can also be considered as points of interactions that can result in an evolutionary arms race. Despite the ubiquity of fungal–arthropod interactions, there are few examples in which the underlying molecular and biochemical mechanism(s) that define the competing aspects of a coevolutionary arms race have been described. Intriguingly, although effector-like sequences have been found in the genome of entomopathogenic fungi, gene-for-gene avirulence mechanisms have yet to be reported for insect pathogenic fungi (14).Ecologically important as regulators of insect populations, and distributed throughout almost all ecosystems, entomopathogenic fungi are also of significant interest as potential biological control agents against different insect pests, in particular, as more environmentally friendly alternatives to chemical pesticides (15). Several isolates of the Ascomycetes Beauveria bassiana and Metarhizium anisopliae have been successfully used worldwide to control insects of both agricultural and medical importance (16–19). B. bassiana is known as a broad host range arthropod pathogen capable of infecting a wide range of target hosts. Infection occurs via attachment and penetration of the host cuticle, which represents the first and likely most important line of defense against microbial pathogens (20, 21). Mortality typically occurs 3–10 d postinfection, after which the fungus sporulates on the host cadaver. However, for B. bassiana, completion of its life cycle does not require parasitism of a host because the fungus is a facultative pathogen that can grow as a saprophyte as well as form intimate associations with plants (22, 23).Despite its broad host range, an enduring mystery has been the observation that select insect species are recalcitrant to infection by B. bassiana, even though other closely related species are susceptible. Tenebrionid beetles including Tribolium castaneum (the red flour beetle) and Ulomoides dermestoides are two such resistant insect species. The former is of particular importance as a worldwide pest of stored products, resulting in significant agricultural losses per year that often disproportionately affect developing nations (24, 25). The latter, curiously enough, is consumed as an alternative medicine for the treatment of a range of illnesses including diabetes and cancer (26). The genome of T. castaneum is available, and it has become an important model system for insect development (27, 28). Both beetle species are known to produce quinone-containing cuticular secretions hypothesized to act as antimicrobial defense compounds (29, 30). These tenebrionids use prothoracic and abdominal glands to produce quinone-containing defensive secretions originally identified as displaying repellent and/or irritant against predators. Secreted quinone derivatives are also involved in cuticle tanning and sclerotization, and these compounds have been shown to possess potent antimicrobial activities (31–33). The chemical structures of the major components of these beetle secretions have been shown to consist of methyl-1,4-benzoquinone and ethyl-1,4-benzoquinone, together with the carrier alkene 1-pentadecene (34, 35).Biodegradation of quinone derivatives by fungi is a well-known process in the lignin-degrading fungi, where quinones are the product of the oxidation of lignin-related aromatic compounds by ligninases (36). The pathway involves the induction of NAD(P)H:quinone reductases, which catalyze the conversion of cytotoxic benzoquinone to (nontoxic) hydroquinone. Several quinone reductases have been purified and characterized and their genes cloned in both white- and brown-rot fungi (basidiomycetes) as part of lignin degradation pathways (37–40). However, entomopathogenic fungi are incapable of lignin degradation, and no information is available regarding the presence and/or function of these enzymes in this group of fungi. However, entomopathogenic fungi are able to assimilate various hydrocarbon and lipid components of the insect cuticle, responding to surface cues on their hosts to initiate programs for infection (41–43).In this work we demonstrate the antifungal properties of tenebrionid cuticular secretions and characterize a counteracting fungal NAD(P)H:1,4-benzoquinone oxidoreductase (BbBqrA) in B. bassiana. Gene expression and enzyme activity assays revealed induction of BbbqrA expression and increased BbBqrA activity in the presence of exogenous benzoquinone. Heterologous expression and characterization of the BbbqrA enzyme confirmed NADP(H)-dependent benzoquinone reductase activity. Fungi containing a targeted disruption of BbbqrA showed decreased ability to infect T. castaneum, but no changes in virulence when tested against nonquinone-producing beetles (Sitophilus oryzae, rice weevil) or the Lepidopertan host, the greater wax moth Galleria mellonella. Conversely, a B. bassiana fungal strain engineered to overexpress BbbqrA displayed increased virulence against T. castaneum with, again, no changes in virulence when tested against G. mellonella or S. oryzae. These results link BbbqrA to degradation of tenebrionid quinone-containing defensive secretions, identifying a host-specific virulence factor that is part of a coevolutionary arms race between a pathogen and its host. These data reveal a biochemical mechanism that can act as one factor to limit host range.     

Dynamic functional evolution of an odorant receptor for sex-steroid-derived odors in primates

Odorant receptors are among the fastest evolving genes in animals. However, little is known about the functional changes of individual odorant receptors during evolution. We have recently demonstrated a link between the in vitro function of a human odorant receptor, OR7D4, and in vivo olfactory perception of 2 steroidal ligands—androstenone and androstadienone—chemicals that are shown to affect physiological responses in humans. In this study, we analyzed the in vitro function of OR7D4 in primate evolution. Orthologs of OR7D4 were cloned from different primate species. Ancestral reconstruction allowed us to reconstitute additional putative OR7D4 orthologs in hypothetical ancestral species. Functional analysis of these orthologs showed an extremely diverse range of OR7D4 responses to the ligands in various primate species. Functional analysis of the nonsynonymous changes in the Old World Monkey and Great Ape lineages revealed a number of sites causing increases or decreases in sensitivity. We found that the majority of the functionally important residues in OR7D4 were not predicted by the maximum likelihood analysis detecting positive Darwinian selection.     

Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling

Enpp1 is a membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing extracellular nucleotide triphosphates to produce pyrophosphate. Enpp1 dysfunction causes human diseases characterized by ectopic calcification. Enpp1 also inhibits insulin signaling, and an Enpp1 polymorphism is associated with insulin resistance. However, the precise mechanism by which Enpp1 functions in these cellular processes remains elusive. Here, we report the crystal structures of the extracellular region of mouse Enpp1 in complex with four different nucleotide monophosphates, at resolutions of 2.7–3.2 Å. The nucleotides are accommodated in a pocket formed by an insertion loop in the catalytic domain, explaining the preference of Enpp1 for an ATP substrate. Structural mapping of disease-associated mutations indicated the functional importance of the interdomain interactions. A structural comparison of Enpp1 with Enpp2, a lysophospholipase D, revealed marked differences in the domain arrangements and active-site architectures. Notably, the Enpp1 mutant lacking the insertion loop lost the nucleotide-hydrolyzing activity but instead gained the lysophospholipid-hydrolyzing activity of Enpp2. Our findings provide structural insights into how the Enpp family proteins evolved to exert their diverse cellular functions.     

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas

Carbonic anhydrase (CA) is one of nature’s fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme’s sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO₂ absorption 25-fold compared with the noncatalyzed reaction.Releasing over 9 billion metric tons of CO₂ worldwide each year, coal-fired power plants are the leading anthropogenic source of CO₂ emission. A potential solution to reduce the release of CO₂ into the atmosphere is the implementation of carbon capture and sequestration (CCS) technology on coal- and natural-gas–fired power plants. Currently, one of the most viable postcombustion CCS technology options uses an amine solvent to remove CO₂ from the flue gas (1). Unfortunately, the large amount of energy required to release the CO₂ and regenerate the solvent would significantly increase the cost of electricity generated (2). The ideal solvent capture process uses a solvent such as an aqueous amine with fast CO₂ absorption kinetics and a low heat of desorption to minimize the solvent regeneration energy. However, a high rate of CO₂ absorption kinetics for an amine solvent correlates with a higher temperature of desorption, whereas solvents with low heat of desorption tend to have much slower adsorption kinetics (3, 4).With reaction rates approaching the limits of diffusion, carbonic anhydrase (CA) is one of the fastest enzymes known. The active site of CAs can turnover CO₂ and water to bicarbonate and a proton up to a million times per second, and are used by almost every living organism to maintain pH balance and transport carbon dioxide. It has been shown that CAs can be used to accelerate the capture of CO₂ (5) by serving as a catalyst in alkaline capture solvents with slow absorption kinetics (6, 7). The potential improvements of CAs on CCS process economics stems from several considerations. The faster absorption and desorption kinetics allows for smaller processing equipment with reduced capital and operating costs (6). The improved kinetics also allows the use of lower temperature capture and solvent regeneration process conditions, and decreases energy losses in the capture process. Another benefit is the improved range of solvent candidates with attractive thermochemical stability (e.g., low vapor pressure, low formation of heat stable salts, etc.), but otherwise unacceptable uncatalyzed absorption kinetics. In addition, the use of a CA as an accelerant has the potential of lowering solvent replacement costs. However, implementing natural CAs in large-scale carbon capture processes has been limited by inactivation of the enzyme under the harsh alkaline conditions of an amine solvent and the elevated temperature required for CO₂ desorption. To date, it has been assumed that a protein catalyst could not tolerate exposure to the high temperatures and alkaline environment of amine solvent capture and desorption process (Fig. 1).Open in a separate windowFig. 1.Flue gas from a coal-fired power plant is piped into an absorber column (blue) where CO₂ chemisorbs into an amine solvent, catalyzed by CA, and is hydrated to a proton and a bicarbonate ion. The CO₂-depleted flue gas is released into the atmosphere and the HCO₃⁻-loaded amine solvent and CA is transferred to a second column where CO₂ is stripped at elevated temperatures (>87 °C), resulting in solvent regeneration. The pure CO₂ stream can be compressed and stored in depositories or used in industrial processes. The regenerated solvent is returned to the absorber column to repeat the process.Directed evolution is an efficient protein engineering strategy for the generation of very large changes in active site or surface residues to give enzyme variants capable of catalyzing new chemistries or ones which act on substrates far afield from those found in nature. Such novel enzymes have enabled efficient biocatalytic pharmaceutical manufacturing processes (8–12). In this article we describe the use of directed evolution in combination with the protein sequence activity relationships (ProSAR) algorithm (13) to create new variants of CA that function under some of the harshest industrial conditions yet applied to enzymes. We created a highly stable form of the CA from Desulfovibrio vulgaris (DvCA) that accelerates CO₂ absorption at temperatures above 100 °C in alkaline solvents required for efficient carbon capture.     

Evidence for regulation of a thyrotropin-releasing hormone degradation pathway in GH3 cells

GH3 cells, cloned from a rat anterior pituitary tumor, synthesize and secrete PRL in response to TRH. One of the pathways of TRH degradation is removal of the N-terminal pyroglutamyl residue catalyzed by pyroglutamyl peptide hydrolase (PPH; EC 3.4.11.8). We recently described the synthesis and properties of 5-oxoprolinal, a specific and potent (Ki = 26 nM) inhibitor of PPH. The effect of long term exposure of GH3 cells to 5-oxoprolinal on PPH activity was studied by incubating cells with inhibitor for 3 days, harvesting, washing to remove inhibitor, and assaying for PPH. Unexpectedly, we found a marked (300%) increase in PPH activity. This effect was dependent on the concentration of 5-oxoprolinal (EC50 = 10(-7) M) and was time dependent, with a rapid increase in enzyme activity occurring during the first 24 h. Cycloheximide did not block the increase. The results suggest that the activity of PPH in GH3 cells is subject to complex regulatory mechanisms.     

Definition of a third VLR gene in hagfish

Jawless vertebrates (cyclostomes) have an alternative adaptive immune system in which lymphocytes somatically diversify their variable lymphocyte receptors (VLR) through recombinatorial use of leucine-rich repeat cassettes during VLR gene assembly. Three types of these anticipatory receptors in lampreys (VLRA, VLRB, and VLRC) are expressed by separate lymphocyte lineages. However, only two VLR genes (VLRA and VLRB) have been found in hagfish. Here we have identified a third hagfish VLR, which undergoes somatic assembly to generate sufficient diversity to encode a large repertoire of anticipatory receptors. Sequence analysis, structural comparison, and phylogenetic analysis indicate that the unique hagfish VLR is the counterpart of lamprey VLRA and the previously identified hagfish “VLRA” is the lamprey VLRC counterpart. The demonstration of three orthologous VLR genes in both lampreys and hagfish suggests that this anticipatory receptor system evolved in a common ancestor of the two cyclostome lineages around 480 Mya.Phylogenetic studies of immunity indicate the emergence of two types of recombinatorial adaptive immune systems (AISs) in vertebrates (1, 2). All of the extant jawed vertebrates generate a vast repertoire of Ig-domain–based T- and B-cell antigen receptors primarily by the recombinatorial assembly of Ig V-(D)-J gene segments and somatic hypermutation (3). The extant jawless vertebrates, lampreys and hagfish, instead have an alternative AIS that is based on variable lymphocyte receptors (VLRs), the diversity of which is generated through recombinatorial use of leucine-rich repeat (LRR) cassettes (4–6). The germ-line VLR genes are incomplete in that they only contain coding sequences for the leader sequence, incomplete amino- and carboxyl-terminal LRR subunits (LRRNT and LRRCT) and the stalk region (4, 7, 8). However, each germ-line VLR gene is flanked by hundreds of different LRR-encoding sequences, which can be used as templates to add LRR sequences during the assembly of a mature VLR gene (4, 8–11). This gene conversion-like process is postulated to involve the activation-induced cytidine deaminase (AID) orthologs, cytidine deaminases 1 and 2 (CDA1 and CDA2) (8, 12). The combinatorial VLR assembly can generate a vast repertoire of anticipatory receptors comparable in diversity to the repertoire of Ig-domain–based antigen receptors in jawed vertebrates (8, 9).Three VLR genes have been identified in lampreys (VLRA, VLRB, and VLRC), but only two (VLRA and VLRB) have been identified so far in hagfish (4, 7, 8, 13). Lamprey VLRB-expressing cells can respond to immunization by undergoing lymphoblastoid transformation, clonal expansion, and secretion of their antigen-specific VLRB antibodies (9, 14). VLRA- and VLRC-bearing cells also proliferate in response to antigen stimulation, but do not differentiate into antibody secreting cells; instead they maintain cell surface expression of their receptors, while increasing the expression of proinflammatory cytokines, macrophage migration inhibitory factor (MIF), and interleukin-17 (IL-17) (12). CDA1-expressing progenitors assemble their VLRA and VLRC genes to become VLRA⁺ and VLRC⁺ lymphocytes in a thymus-equivalent region of the gills termed the thymoid (15, 16). Conversely, VLRB assembly coincides with CDA2 expression during VLRB⁺ lymphocyte development in hematopoietic tissues (15). The T- and B-like characteristics of the lamprey lymphocytes imply that jawless vertebrates have humoral and cellular arms of adaptive immunity comparable to those of jawed vertebrates.In the present study, we sought to determine whether or not hagfish have a third VLR gene. In comparing the sequences of the two known hagfish VLRs with those of the three lamprey VLRs, we noticed that the highly variable inserts in the LRRCT module of the currently designated hagfish “VLRA” are more similar to those of lamprey VLRC than to those of lamprey VLRA (13, 17, 18). This led us to hypothesize that a third hagfish VLR, if present, would be the true counterpart of lamprey VLRA. Through a similarity search against the hagfish database, we identified a fragment of a potential third VLR gene, which was in turn used to clone and sequence a previously uncharacterized VLR gene. Here, we report the characterization of this third VLR type in pacific hagfish (Eptatretus stoutii) and its phylogenetic and structural relationship with previously identified VLRs. Our findings suggest a modified nomenclature for the hagfish VLRs.     

人工进化丙型肝炎病毒C和E1区噬菌体展示文库的构建及初步筛选

目的构建人工进化丙型肝炎病毒(HCV)C和E1区基因噬菌体展示文库,并进行初步筛选。方法应用DNA改组技术进行不同基因型的HCV C和E1区基因的人工进化,克隆于噬菌体载体,以辅助噬菌体M13K07援救后,构建噬菌体展示文库,应用HCV C和E1区单克隆抗体进行初步筛选。随机选取筛选后的20个噬菌体克隆,用高滴度HCV阳性血清通过双抗体夹心酶联免疫吸附法进行抗原抗体结合反应,以正常人血清作为对照。结果人工进化HCV C和E1区噬菌体展示文库库容达1．64×106,重组率为0．86。以C和E1区单克隆抗体淘筛4轮,噬菌体展示文库得到特异性富集。筛选获得12个阳性克隆。结论所构建的噬菌体展示文库的库容和多样性符合筛选的要求。筛选获得的阳性克隆具有较好的抗原抗体结合反应活性,表现出较好的亲和力。     

EBS7 is a plant-specific component of a highly conserved endoplasmic reticulum-associated degradation system in Arabidopsis

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an essential part of an ER-localized protein quality-control system for eliminating terminally misfolded proteins. Recent studies have demonstrated that the ERAD machinery is conserved among yeast, animals, and plants; however, it remains unknown if the plant ERAD system involves plant-specific components. Here we report that the Arabidopsis ethyl methanesulfonate-mutagenized brassinosteroid-insensitive 1 suppressor 7 (EBS7) gene encodes an ER membrane-localized ERAD component that is highly conserved in land plants. Loss-of-function ebs7 mutations prevent ERAD of brassinosteroid insensitive 1-9 (bri1-9) and bri1-5, two ER-retained mutant variants of the cell-surface receptor for brassinosteroids (BRs). As a result, the two mutant receptors accumulate in the ER and consequently leak to the plasma membrane, resulting in the restoration of BR sensitivity and phenotypic suppression of the bri1-9 and bri1-5 mutants. EBS7 accumulates under ER stress, and its mutations lead to hypersensitivity to ER and salt stresses. EBS7 interacts with the ER membrane-anchored ubiquitin ligase Arabidopsis thaliana HMG-CoA reductase degradation 1a (AtHrd1a), one of the central components of the Arabidopsis ERAD machinery, and an ebs7 mutation destabilizes AtHrd1a to reduce polyubiquitination of bri1-9. Taken together, our results uncover a plant-specific component of a plant ERAD pathway and also suggest its likely biochemical function.Endoplasmic reticulum (ER)-associated degradation (ERAD) is an integral part of an ER-mediated protein quality-control system in eukaryotes, which permits export of only correctly folded proteins but retains misfolded proteins in the ER for repair via additional folding attempts or removal through ERAD. Genetic and biochemical studies in yeast and mammalian cells have revealed that the core ERAD machinery is highly conserved between yeast and mammals and that ERAD involves four tightly coupled steps: substrate selection, retrotranslocation through the ER membrane, ubiquitination, and proteasome-mediated degradation (1, 2).Because the great majority of secretory/membrane proteins are glycosylated in the ER, diversion of most ERAD substrates from their futile folding cycles into ERAD is initiated through progressive mannose trimming of their asparagine-linked glycans (N-glycans) by ER/Golgi-localized class I mannosidases, including homologous to α-mannosidase 1 (Htm1) and its mammalian homologs ER degradation-enhancing α-mannosidase-like proteins (EDEMs) (3). The processed glycoproteins are captured by two ER resident proteins, yeast amplified in osteosarcoma 9 (OS9 in mammals) homolog (Yos9) and HMG-CoA reductase degradation 3 (Hrd3) [suppressor/enhancer of Lin-12–like (SEL1L) in mammals], which recognize the mannose-trimmed N-glycans and surface-exposed hydrophobic amino acid residues, respectively (4, 5). The selected ERAD clients are delivered to an ER membrane-anchored ubiquitin ligase (E3), which is the core component of the ERAD machinery (6), for polyubiquitination. Yeast has two known ERAD E3 ligases, Hrd1 and degradation of alpha 10 (Doa10), both containing a catalytically active RING finger domain, whereas mammals have a large collection of ER membrane-anchored E3 ligases, including Hrd1 and gp78 (7). The yeast Hrd1/Doa10-containing ERAD complexes target different substrates, with the former ubiquitinating substrates with misfolded transmembrane or luminal domains and the latter acting on clients with cytosolic structural lesions (8).Because of the cytosolic location of the E3′s catalytic domain and proteasome, all ERAD substrates must retrotranslocate through the ER membrane. It is well known that the retrotranslocation step is tightly coupled with substrate ubiquitination and is powered by an AAA-type ATPase, cell division cycle 48 (Cdc48) in yeast and p97 in mammals. However, the true identity of the retrotranslocon remains controversial. Earlier studies implicated the secretory 61 (Sec61) translocon, degradation in the endoplasmic reticulum 1 (Der1) [Der1-like proteins (Derlins) in mammals], and Hrd1 in retrotranslocating ERAD substrates (9). After retrotranslocation, ubiquitinated ERAD clients are delivered to the cytosolic proteasome with the help of Cdc48/p97 and their associated factors for proteolysis (10). In addition to the above-mentioned proteins, the yeast/mammalian ERAD systems contain several other components, including several ubiquitin-conjugating enzymes (E2), a membrane-anchored E2-recruiting factor, Cue1 that has no mammalian homolog, a scaffold protein U1-Snp1–associating 1 (Usa1) [homocysteine-induced ER protein (HERP) in mammals] of the E3 ligases, and a membrane-anchored Cdc48-recruiting factor, Ubx2 (Ubxd8 in mammals) (6).For many years ERAD has been known to operate in plants (11), but the research on the plant ERAD pathway lagged far behind similar studies in yeast and mammalian systems. Recent molecular and genetic studies in the reference plant Arabidopsis, especially two Arabidopsis dwarf mutants, brassinosteroid-insensitive 1-5 (bri1-5) and bri1-9, carrying ER-retained mutant variants of the brassinosteroid receptor (BR) BRASSINOSTEROID-INSENSITIVE 1 (BRI1) (12–14), revealed that the ERAD system also is conserved in plants (reviewed in refs. 15 and 16). For example, the ERAD N-glycan signal to mark misfolded glycoproteins in Arabidopsis was found to be the same as that in yeast/mammalian cells (17, 18). Both forward and reverse genetic studies have shown that Arabidopsis homologs of the yeast/mammalian ERAD components, including Yos9/OS9 (19, 20), Hrd3/Sel1L (21, 22), Hrd1 (21), EDEMs (23), and a membrane-anchored E2 (24), are involved in degrading misfolded glycoproteins. However, it remains unknown if the plant ERAD requires one or more plant-specific components to degrade terminally misfolded proteins efficiently. In this study, we took a forward genetic approach to identify a novel Arabidopsis ERAD mutant, ethyl methanesulfonate-mutagenized bri1 suppressor 7 (ebs7), and subsequently cloned the corresponding EBS7 gene. We discovered that EBS7 encodes an ER-localized membrane protein that is highly conserved in land plants but lacks a homolog in yeast or mammals. Our biochemical studies strongly suggested that EBS7 plays a key role in an Arabidopsis ERAD process by regulating the protein stability of the Arabidopsis thaliana HRD1a (AtHrd1a).     

Plasmodium falciparum: higher incidence of molecular resistance markers for sulphadoxine than for pyrimethamine in Kasangati, Uganda

In November of 2000, Uganda changed its anti-malarial policy to replace chloroquine (CQ) with a combination of CQ and sulphadoxine-pyrimethamine (SP) as the first line agents. Information was limited on the efficacy of either drug. The present study was designed to provide baseline information on the efficacy of SP and the prevalence of molecular markers that are associated with SP resistance. Blood samples were collected on filter paper from 169 consenting patients who were diagnosed with malaria. Patients were treated with SP and followed for 14 days using the WHO clinical guidelines. The samples were analysed for molecular resistance markers and correlation of the molecular markers with clinical findings was assessed. SP monotherapy was efficacious for 140 of 163 (85.9%) treated patients. We found a high level of mutations in alleles which have previously been reported to be associated with SP resistance, but there was no correlation between clinical outcomes and molecular markers. With the exception of codon S108 in dhfr (dhfr S108N was at 94.9%), frequencies of dihydropteroate synthase (dhps) mutant and mixed alleles combined (A437G 89% and K540E 83.9%) were higher than those of dihydrofolate reductase (dhfr) (N51I 58.4%, C59R 31.3%).     

Artificial gene amplification reveals an abundance of promiscuous resistance determinants in Escherichia coli

Duplicated genes provide an important raw material for adaptive evolution. However, the relationship between gene duplication and the emergence of new biochemical functions is complicated, and it has been difficult to quantify the likelihood of evolving novelty in any systematic manner. Here, we describe a comprehensive search for artificially amplified genes that are able to impart new phenotypes on Escherichia coli, provided their expression is up-regulated. We used a high-throughput, library-on-library strategy to screen for resistance to antibiotics and toxins. Cells containing a complete E. coli ORF library were exposed to 237 toxin-containing environments. From 86 of these environments, we identified a total of 115 cases where overexpressed ORFs imparted improved growth. Of the overexpressed ORFs that we tested, most conferred small but reproducible increases in minimum inhibitory concentration (≤16-fold) for their corresponding antibiotics. In many cases, proteins were acting promiscuously to impart resistance. In the absence of toxins, most strains bore no fitness cost associated with ORF overexpression. Our results show that even the genome of a nonpathogenic bacterium harbors a substantial reservoir of resistance genes, which can be readily accessed through overexpression mutations. During the growth of a population under selection, these mutations are most likely to be gene amplifications. Therefore, our work provides validation and biochemical insight into the innovation, amplification, and divergence model of gene evolution under continuous selection [Bergthorsson U, Andersson DI, Roth JR (2007) Proc Natl Acad Sci USA 104:17004-17009], and also illustrates the high frequency at which novel traits can evolve in bacterial populations.     

Evolution of the ssrA degradation tag in Mycoplasma: specificity switch to a different protease

Stalled ribosomes in bacteria are rescued by the tmRNA system. In this process, the nascent polypeptide is modified by the addition of a short C-terminal sequence called the ssrA tag, which is encoded by tmRNA and allows normal termination and release of ribosomal subunits. In most bacteria, ssrA-tagged proteins are degraded by the AAA+ protease, ClpXP. However, in bacterial species of the genus Mycoplasma, genes for ClpXP and many other proteins were lost through reductive evolution. Interestingly, Mycoplasma ssrA tag sequences are very different from the tags in other bacteria. We report that ssrA-tagged proteins in Mesoplasma florum, a Mycoplasma species, are efficiently recognized and degraded by the AAA+ Lon protease. Thus, retaining degradation of ssrA-tagged translation products was apparently important enough during speciation of Mycoplasma to drive adaptation of the ssrA tag to a different protease. These results emphasize the importance of coupling proteolysis with tmRNA-mediated tagging and ribosome rescue.     

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.     

Generation of a highly inducible Gal4-->Fluc universal reporter mouse for in vivo bioluminescence imaging

Full understanding of the functional complexity of the protein interactome requires mapping of biomolecular complexes within the cellular environment over biologically relevant time scales. New approaches to imaging interacting protein partners in vivo will allow the study of functional proteomics of human biology and disease within the context of living animals. Herein, we describe a universal transgenic reporter mouse strain that expresses firefly luciferase (Fluc) under the regulatory control of a concatenated Gal4 promoter (Tg^G4F(+/−)). Using an adenovirus to deliver a fused binding-domain-activator chimera (Gal4BD-VP16), induction of bioluminescence in Tg^G4F(+/−) tissues of up to 4 orders of magnitude was observed in fibroblasts, liver, respiratory epithelia, muscle, and brain. The Tg^G4F(+/−) reporter strain allowed noninvasive detection of viral infectivity, duration of the infection as well as viral clearance in various tissues in vivo. To demonstrate protein–protein interactions in live mice, the well characterized interaction between tumor suppressor p53 (fused to Gal4BD) and large T antigen (TAg) (fused to VP16) was visualized in vivo by using a two-hybrid strategy. Hepatocytes of Tg^G4F(+/−) mice transfected with p53/TAg demonstrated 48-fold greater induction of Fluc expression in vivo than noninteracting pairs. Furthermore, to demonstrate the feasibility of monitoring experimental therapy with siRNA in vivo, targeted knockdown of p53 resulted in markedly reduced light output, whereas use of a control siRNA had no effect on protein interaction-dependent induction of Fluc. Thus, this highly inducible Gal4→Fluc conditional reporter strain should facilitate imaging studies of protein interactions, signaling cascades, viral dissemination, and therapy within the physiological context of the whole animal.