The Role of Biomacromolecular Crowding,Ionic Strength,and Physicochemical Gradients in the Complexities of Life's Emergence

Summary: We have developed a general scenario of prebiotic physicochemical evolution during the Earth''s Hadean eon and reviewed the relevant literature. We suggest that prebiotic chemical evolution started in microspaces with membranous walls, where external temperature and osmotic gradients were coupled to free-energy gradients of potential chemical reactions. The key feature of this scenario is the onset of an emergent evolutionary transition within the microspaces that is described by the model of complex vectorial chemistry. This transition occurs at average macromolecular crowding of 20 to 30% of the cell volume, when the ranges of action of stabilizing colloidal forces (screened electrostatic forces, hydration, and excluded volume forces) become commensurate. Under these conditions, the macromolecules divide the interior of microspaces into dynamically crowded macromolecular regions and topologically complementary electrolyte pools. Small ions and ionic metabolites are transported vectorially between the electrolyte pools and through the (semiconducting) electrolyte pathways of the crowded macromolecular regions from their high electrochemical potential (where they are biochemically produced) to their lower electrochemical potential (where they are consumed). We suggest a sequence of tentative transitions between major evolutionary periods during the Hadean eon as follows: (i) the early water world, (ii) the appearance of land masses, (iii) the pre-RNA world, (iv) the onset of complex vectorial chemistry, and (v) the RNA world and evolution toward Darwinian thresholds. We stress the importance of high ionic strength of the Hadean ocean (short Debye''s lengths) and screened electrostatic interactions that enabled the onset of the vectorial structure of the cytoplasm and the possibility of life''s emergence.

Architecture is what ultimately distinguishes a living cell from a soup of the chemicals of which it is composed.—Franklin Harold (40)

     

Wait times in the emergency department for patients with mental illness

Background:

It has been suggested that patients with mental illness wait longer for care than other patients in the emergency department. We determined wait times for patients with and without mental health diagnoses during crowded and noncrowded periods in the emergency department.

Methods:

We conducted a population-based retrospective cohort analysis of adults seen in 155 emergency departments in Ontario between April 2007 and March 2009. We compared wait times and triage scores for patients with mental illness to those for all other patients who presented to the emergency department during the study period.

Results:

The patients with mental illness (n = 51 381) received higher priority triage scores than other patients, regardless of crowding. The time to assessment by a physician was longer overall for patients with mental illness than for other patients (median 82, interquartile range [IQR] 41–147 min v. median 75 [IQR 36–140] min; p < 0.001). The median time from the decision to admit the patient to hospital to ward transfer was markedly shorter for patients with mental illness than for other patients (median 74 [IQR 15–215] min v. median 152 [IQR 45–605] min; p < 0.001). After adjustment for other variables, patients with mental illness waited 10 minutes longer to see a physician compared with other patients during noncrowded periods (95% confidence interval [CI] 8 to 11), but they waited significantly less time than other patients as crowding increased (mild crowding: −14 [95% CI −12 to −15] min; moderate crowding: −38 [95% CI −35 to −42] min; severe crowding: −48 [95% CI −39 to −56] min; p < 0.001).

Interpretation:

Patients with mental illness were triaged appropriately in Ontario’s emergency departments. These patients waited less time than other patients to see a physician under crowded conditions and only slightly longer under noncrowded conditions.In a 2008 report, the Schizophrenia Society of Ontario recommended adding a psychiatric wait times component to the Ontario government’s Emergency Room Wait Times Strategy.¹ They suggested that patients who present to the emergency department in psychiatric distress wait longer for care than other patients and that they are given a low priority triage score² (all patients are assigned a triage score when they first arrive at the emergency department, which may determine when and where they are seen by a physician).³ The Kirby Report, a senate report on mental illness and addiction in Canada, also decried differential emergency care for patients with mental illness.⁴A recent study found that patients with acute myocardial infarction are given lower priority care in the emergency department if they have a charted history of depression.⁵ However, whether patients who present to the emergency department for mental illness receive slower care than other patients is not known. In this study, we compared the emergency department wait times and triage scores for patients with affective and psychotic disorders to those for other patients, both in noncrowded conditions and during periods of crowding. Because we believe that triage nurses apply triage principles consistently to all emergency patients while physicians may be less likely to adhere to the guidelines, we hypothesized that there would be no “down-triage” (assigning a lower priority triage score) of these patients, but that patients with mental illness would have longer delays to see a physician, relative to other patients.     

Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.Protein-protein interactions are essential to virtually every cellular process, and their understanding is of great interest in basic science as well as in the development of effective therapeutics. Existing techniques to detect and screen pairs of interacting proteins in vivo include the yeast two-hybrid system (1) and protein-fragment complementation assay (PCA)² (2–6), where the association of two interacting proteins either turns on a target gene that is necessary for cell survival or leads to the reconstitution of enzymes or green fluorescence protein (GFP) or its variants. The application of protein-protein interactions that are probed with yeast two-hybrid and PCA has been focused mainly on the interactions occurring in the nucleus or cytosol. To study interactions among secretory proteins and membrane-associated proteins, a variant of yeast two-hybrid has been developed for detecting protein-protein interactions occurring in the secretory pathway (7, 8). However, most existing methods are designed to map connectivity information for pairwise interactions and are not suitable for measuring the affinity between two interacting proteins, comparing interaction strength of different pairs, or ranking multiple binders to the interaction “hub” according to their binding affinity.Quantitative estimation of protein-protein interactions in vivo will require the amount of the complex to be directly measured or the level of reconstituted reporters to be directly proportional to the strength of the interactions. To achieve quantitative analysis of protein interactions in eukaryotic expression system, we have designed a yeast surface two-hybrid (YS2H) system that can express a pair of proteins, one protein as a fusion to a yeast cell wall protein, agglutinin, and the other in a secretory form. When two proteins interact in this system, they associate in the secretory pathway, and the prey that would otherwise be released into the media is captured on the surface by the bait. We have devised two different schemes to quantitatively estimate the affinity of two interacting molecules: flow cytometric detection of antibody binding to the epitope tags fused to the prey and the bait, and the GFP readout from the complementation of split GFP fragments fused to the prey and the bait. They are induced under a bi-directional promoter to promote a synchronized and comparable level of expression.Herein we demonstrate the quantitative nature of YS2H in predicting the affinity between two interacting proteins, particularly in the range of 100 pm to 10 μm. This feature allowed us to examine specific interactions between antigen and antibody, to identify hot spots of allosteric activation in integrins, and to isolate camelid heavy chain-only antibodies against botulinum neurotoxin as components of therapeutic agents to treat botulism (9). With the incorporation of PCA technique into the YS2H, our system may be developed into an in vivo tool to measure the kinetics of protein-protein interactions. Potential applications of YS2H include affinity maturation of antibodies, differentiation of weak to high affinity binders to the hub protein in interaction networks, and confirmation of hypothetical interacting pairs of proteins in a high throughput manner.     

Crowded Cell-like Environment Accelerates the Nucleation Step of Amyloidogenic Protein Misfolding

To understand the role of a crowded physiological environment in the pathogenesis of neurodegenerative diseases, we report the following. 1) The formation of fibrous aggregates of the human Tau fragment Tau-(244–441), when hyperphosphorylated by glycogen synthase kinase-3β, is dramatically facilitated by the addition of crowding agents. 2) Fibril formation of nonphosphorylated Tau-(244–441) is only promoted moderately by macromolecular crowding. 3) Macromolecular crowding dramatically accelerates amyloid formation by human prion protein. A sigmoidal equation has been used to fit these kinetic data, including published data of human α-synuclein, yielding lag times and apparent rate constants for the growth of fibrils for these amyloidogenic proteins. These biochemical data indicate that crowded cell-like environments significantly accelerate the nucleation step of fibril formation of human Tau fragment/human prion protein/human α-synuclein (a significant decrease in the lag time). These results can in principle be predicted based on some known data concerning protein concentration effects on fibril formation both in vitro and in vivo. Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of neurodegenerative diseases by accelerating amyloidogenic protein misfolding and inducing human prion fibril fragmentation, which is considered to be an essential step in prion replication.Amyloid fibrils associated with neurodegenerative diseases such as Alzheimer disease, Parkinson disease, Huntington disease, and transmissible spongiform encephalopathy (TSE)³ (1–5) can be considered biologically relevant failures of the cellular protein quality control mechanisms (6) consisting of molecular chaperones and proteases (7). Up to now, about 20 different proteins with unrelated sequences and tertiary structures are known to form fibrous aggregates associated with various neurodegenerative diseases. These amyloidogenic proteins include both natively unfolded proteins, such as human Tau protein (3) and human α-synuclein (8), and folded globular proteins such as human prion protein (4). There are two faces of protein misfolding in neurodegeneration as follows: a gain of toxic function and a loss of physiological function, which can even occur in combination (9).Human Tau protein, a marker for Alzheimer disease, forms filaments in the brains of patients with Alzheimer disease (3, 10, 11). It has been found that hyperphosphorylation of Tau reduces the binding affinity between Tau and tubulin and contributes to the self-association of Tau and the formation of Tau paired helical filaments (3, 11–13). It has been proposed that glycogen synthase kinase-3β (GSK-3β) hyperphosphorylation of Tau plays an important role in Alzheimer disease (14, 15), and GSK-3β induces an Alzheimer disease-like hyperphosphorylation of Tau when overexpressed in cultured human neurons (16).A large body of data strongly suggests Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, and other TSEs are caused by prions (4). Prions are infectious proteins that can transmit biological information by propagating protein misfolding and aggregation (17). The infectious agent is believed to consist entirely of the prion protein (PrP) and is devoid of nucleic acid (4, 17). Prion biogenesis is associated with the normal protease-sensitive form of the protein (cellular PrP molecule, PrP^C) undergoing structural change into an abnormal, protease-resistant, disease-causing isoform of prion protein (PrP^Sc) (4, 17). Although the mechanism by which PrP^C is converted to PrP^Sc in TSE-infected cells and in vivo is not clear, data from cell-free reactions suggest this process is akin to autocatalytic polymerization (18).Misfolding of Tau and prion proteins has been traditionally and widely studied in dilute solutions (10, 19–21). However, the physiological environment is poorly modeled by such dilute solutions, and biochemical reactions in vivo differ greatly from those in dilute solutions (22). The proteins associated with neurodegenerative diseases form fibrils in a physiological environment crowded with other background macromolecules (22–26), such as proteins, glycosaminoglycans, and proteoglycans (23). Crowding is not confined to cellular interiors but also occurs in the extracellular matrix of tissues (24) and takes place at membrane surfaces (27). For example, blood plasma contains ∼80 g/liter protein, a concentration sufficient to cause significant crowding effects (24). Polysaccharides also contribute to crowding, especially in the extracellular matrix of tissues such as collagen (23, 26). The conversion of PrP from a normal soluble conformation PrP^C to its pathogenic conformation PrP^Sc is believed to occur on the cell surface, in the endocytic vesicles, or in the crowded extracellular matrix (18). Thus, macromolecular crowding on the cell surface and in the extracellular matrix may play an important role in the conformational transition and amyloid formation of PrP in vivo, which have not been fully characterized yet. In vitro, such a crowded environment can be achieved experimentally by adding high concentrations of single or mixed nonspecific crowding agents to the system (23–31). Recently, it has been demonstrated that macromolecular crowding significantly enhances the rate of amyloid formation of α-synuclein (32, 33), amyloid-β peptides (27), and human apolipoprotein C-II (34). However, the role of the crowded physiological environment in the pathogenesis of neurodegenerative diseases is poorly understood so far.To address the contributions of crowded physiological environments on the pathogenesis of neurodegenerative diseases, we report here that macromolecular crowding dramatically accelerates fibril formation by human Tau fragment and by human prion protein under physiological conditions. Our results indicate that macromolecular crowding significantly accelerates the nucleation step of fibril formation of human Tau fragment/human prion protein/human α-synuclein by fitting the data to a sigmoidal equation (35, 36). Furthermore, macromolecular crowding causes human prion protein to form short fibrils and nonfibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions.     

The contrasting effect of macromolecular crowding on amyloid fibril formation

Background

Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.

Methodology/Principal Findings

As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.

Conclusions/Significance

We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.     

Exploring the world of phospholipids and their interactions with proteins: the work of William Dowhan

The Gene Encoding the Phosphatidylinositol Transfer Protein Is Essential for Cell Growth (Aitken, J. F., van Heusden, G. P., Temkin, M., and Dowhan, W. (1990) J. Biol. Chem. 265, 4711–4717)A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein (Bogdanov, M., Sun, J., Kaback, H. R., and Dowhan, W. (1996) J. Biol. Chem. 271, 11615–11618)William Dowhan''s curiosity about the connections between phospholipids and proteins associated with them goes back as far as his days as a graduate student with Esmond Snell at the University of California, Berkeley. In these two JBC Classics, his group''s ability to manipulate biochemical and molecular genetics tools to answer fundamental questions about lipid biology shines through. “William Dowhan and his research group have made many contributions to the biochemistry of phospholipid metabolism and membrane biogenesis,” says Robert Simoni at Stanford University.Open in a separate windowBill Dowhan (right) is shown here with the late Chris Raetz (left), who was a longtime collaborator and friend, and his former postdoctoral advisor, the late Gene Kennedy, on the occasion of Kennedy''s 90th birthday in 2009 (photo courtesy of William Dowhan).The first paper, published in 1990, documented the importance of phosphatidylinositol/phosphatidylcholine transfer proteins in vivo. Dowhan''s group, which has been based at the University of Texas Medical School since 1972, used a combination of biochemistry and genetics to clone the protein''s gene. Dowhan had first heard of phospholipid transfer proteins in 1969, when he began his postdoctoral training with Eugene (Gene) Kennedy at Harvard Medical School. At his very first Kennedy lab meeting, the discussion centered around a publication that had just come out (1). The paper described “one of the first observations of proteins in the soluble phase that transferred lipids between bilayers,” recalls Dowhan. “No one could figure out what these proteins really did in vivo, but they knew the proteins had this function” of transferring lipids between membranes.As he moved through his career, Dowhan focused on cloning and characterizing genes and purifying enzymes responsible for phospholipid metabolism in Escherichia coli. Then came a sabbatical in 1983 with Gottfried (Jeff) Schatz at the Biozentrum of the University of Basel in Switzerland, that expanded Dowhan''s research directions into yeast genetics. He says the opportunity to work with Schatz “got me into the possibility of looking for this phosphatidylinositol/phosphatidylcholine transfer protein (PI-TP) in yeast, which I probably would have never done if I hadn''t taken this sabbatical.”Fresh from his sabbatical, Dowhan started tracking down the protein and its gene in vivo. “I submitted a grant at that time with some preliminary data that we had begun to purify to homogeneity the PI-TP from yeast, which had never been done before. Fortunately, we got the grant,” he says.The Dowhan group managed to purify PI-TP from yeast. “The most important part was using basic biochemistry and understanding how to purify proteins before the advent of genetically tagging proteins for affinity chromatography,” explains Dowhan.For the next step in the process of finding the gene for the protein, Dowhan and colleagues had to apply reverse genetics because the yeast genome was not available in the late 1980s. They sequenced the amino terminus of the protein, made the corresponding oligonucleotide probes, tested yeast cDNA libraries with those probes, and pulled out the gene. “We still didn''t know the role PI-TP played in cell function. But now we had the sequence of the gene and the knock-out mutant was not viable,” notes Dowhan. “So we published” the findings.At the same time, Vytas Bankaitis, now at the University of North Carolina, had been working on cloning the SEC14 gene in yeast, which is necessary for vesicular transport. “It turns out we had missed the DNA sequence,” Dowhan says. From Bankaitis'' work, it was obvious that “PI-TP was the product of the SEC14 gene. It all came together in a joint report in Nature. Now we had a function associated with the SEC14 gene, which we didn''t have before,” Dowhan explains (2). “We had a phenotype of a mutant lacking this phospholipid transfer protein, which then stopped vesicular transport.”This initial link between phospholipid metabolism and vesicular transport opened up the field to characterization of the Sec14 protein superfamily in a broad range of biological systems. These proteins contain lipid-binding domains, which sense membrane lipid composition and integrate lipid metabolism and lipid-mediated signaling with an array of cellular processes.The second JBC Classic focused on a different feature of phospholipids: their role in protein folding. Dowhan was fascinated by membrane proteins ever since he was a graduate student and had gone to the Kennedy laboratory as a postdoctoral fellow, intending to purify the membrane component expressed by the lac operon for lactose transport in E. coli. He was unsuccessful because, at that time, the necessary detergents were not available. Once the lactose permease was purified (3), Dowhan noticed in the literature that other researchers mentioned that when the protein was reconstituted in liposomes missing phosphatidylethanolamine, the protein was defective in energy-dependent uphill transport. Dowhan recalls that he wondered, “Was that an artifact of the liposome system or was that also true in vivo?”To get to the bottom of this observation, Dowhan''s group used E. coli to generate null mutants of what were considered to be absolutely essential genes for phospholipid synthesis and cell viability. They created a null mutant of the pssA gene, which encodes the committed step to the synthesis of the major phospholipid, phosphatidylethanolamine. By establishing conditions in which bacterial cells lacking phosphatidylethanolamine remained viable, the investigators were able to identify and characterize different cell phenotypes caused by the missing phospholipid both in vivo and in vitro. In collaboration with Ronald Kaback at UCLA, Dowhan''s group showed that phosphatidylethanolamine was essential for the proper folding of an epitope of lactose permease that was also necessary to support the energy-dependent uphill transport of lactose. “Studies by others have since shown a similar chaperone role for lipids in other bacteria, plants and mammalian cells,” notes Simoni.To obtain their data, the investigators developed a new technique, the Eastern-Western blot. In this method, membrane proteins were delipidated and partially denatured by SDS. The proteins underwent gel electrophoresis and then were transferred to a solid support by Western blotting. A series of individual lipids were then laid over the proteins at a 90° angle so that the investigators could see, after incubating with conformation-specific antibodies, which lipids helped which membrane proteins regain proper conformation.This technique was used to establish that phosphatidylethanolamine was necessary in a late step of folding of lactose permease, but was not necessary to maintain the final folded state. This observation suggested that lipids act as molecular chaperones in helping protein maturation. “This paper set the stage for understanding how lipids affect the topological organization of wild-type proteins in the membrane,” notes Dowhan.Dowhan and his collaborator Mikhail Bogdanov have continued using bacterial mutants in phospholipid metabolism to systematically manipulate the native membrane lipid compositions during the cell cycle. They have analyzed the transmembrane domain orientation of membrane proteins to establish the molecular basis for lipid-dependent organization of lactose permease and other secondary transporters (4).Dowhan says his work has two take-home messages. One is that “Lipids aren''t just glorified biological detergents,” he says. “They have specific roles” in the cell. The other message is in the power of numbers. Dowhan says the more techniques applied to solve a biological mystery, the more likely the mystery will be successfully solved.     

Synthetic crowding agent dextran causes excluded volume interactions exclusively to tracer protein apoazurin

To understand protein biophysics in crowded cellular environments, researchers often use synthetic polymers as ‘crowding agents’ in vitro. The idea is that these agents will occupy space and reproduce the in vivo scenario in terms of excluded volume. However, recent work has challenged this concept and pointed out that attractive interactions between protein and crowding agent will provide an enthalpic contribution to the overall effect on protein thermodynamics. Here we use a typical synthetic crowding agent and a well-studied model protein to demonstrate in a window of 50 K that the presence of dextran 20 affects apoazurin by steric repulsion.     

Protein folding in confined and crowded environments

Confinement and crowding are two major factors that can potentially impact protein folding in cellular environments. Theories based on considerations of excluded volumes predict disparate effects on protein folding stability for confinement and crowding: confinement can stabilize proteins by over 10k_BT but crowding has a very modest effect on stability. On the other hand, confinement and crowding are both predicted to favor conformations of the unfolded state which are compact, and consequently may increase the folding rate. These predictions are largely borne out by experimental studies of protein folding under confined and crowded conditions in the test tube. Protein folding in cellular environments is further complicated by interactions with surrounding surfaces and other factors. Concerted theoretical modeling and test-tube and in vivo experiments promise to elucidate the complexity of protein folding in cellular environments.     

Lipid Protein Interactions Couple Protonation to Conformation in a Conserved Cytosolic Domain of G Protein-coupled Receptors

The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic “proton switch.” Infrared and fluorescence spectroscopic pK_a determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pK_a into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligand-specific interactions in class I G protein-coupled receptors.G protein-coupled receptors (GPCRs)² are hepta-helical membrane proteins that couple a large variety of extracellular signals to cell-specific responses via activation of G proteins. In the visual photoreceptor rhodopsin, a prototypical class I GPCR (1, 2), molecular activation processes can be monitored in real time by spectroscopic assays and analyzed in the context of several crystal structures (3–8). The primary signal for rhodopsin is the 11-cis to all-trans photoisomerization of retinal covalently bound to the apoprotein opsin through a protonated Schiff base to Lys²⁹⁶. Current models converge toward a picture in which “microdomains” act as conformational switches that are coupled to different degrees to the primary activation process. Two activating “proton switches” have been identified (9) as follows: breakage of an intramolecular salt bridge (10) by transfer of the Schiff base proton to its counter ion Glu-113 (11) is followed by movement of helix-6 (H6) (12, 13) in the metarhodopsin II_a (MII_a) to MII_b transition. The MII_b state takes up a proton at Glu-134 (14) in the class-conserved D(E)RY motif at the C-terminal end of helix-3 (H3) leading to the MII_bH⁺ intermediate (15, 16), which activates transducin (G_t), the G protein of the photoreceptor cell. Glu-134 regulates the pH sensitivity of receptor signaling (17) in membranes as reviewed previously (18), and in complex with G_t the protonated state of the carboxyl group becomes stabilized (19). This charge alteration is linked to the release of an “ionic lock,” originally described for the β₂-adrenergic receptor (20), which also in rhodopsin stabilizes the inactive state (16) through interactions between the cytosolic ends of H3 and H6 (21).In the absence of a lipidic bilayer, proton uptake and H6 movement become uncoupled (15). Lipidic composition affects MII formation, rhodopsin structure, and oligomerization (22–24) and differs at the rhodopsin membrane interface from the bulk lipidic phase (25). Likewise, MII formation specifically affects lipid structure (26). Although of fundamental importance for GPCR activation, the potential implication of lipid protein interactions in “proton switching” is not clear. A functional role of Glu-134 in lipid interactions has been originally derived from IR spectra where E134Q replacement abolished changes of lipid headgroup vibrations in the MIIG_t complex (19). Computational approaches emphasized the “strategic” location of the D(E)RY motif (27), and the Glu-134 carboxyl pK_a may critically depend on the lipid protein interface (28). However, the implications for proton switching are not evident, and the theoretical interest is contrasted by the lack of experimental data addressing the effect of the lipidic phase on side chain protonation, secondary structure, and membrane topology of the D(E)RY motif.We have studied the coupling between conformation and protonation in single transmembrane segments derived from H3 of bovine rhodopsin. We have assessed the “modular” function of the D(E)RY motif by determining parameters not evident from the crystal structures, i.e. the pK_a of the conserved carboxyl, its linkage to helical structure, and the effect of protonation on side chain to lipid headgroup distance. We show that the D(E)RY motif encodes an autonomous “proton switch” controlling side chain exposure and helix formation in the low dielectric of a lipidic phase. The data ascribe a functional role to lipid protein interactions that couple the chemical potential of protons to an activity-promoting GPCR conformation in a ligand-independent manner.     

A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris

Background

Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers.

Methodology/Principal Findings

Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigorously analyzed for differences in function or solution structure. Yeast expression levels and yield of purified protein from P. pastoris (∼130 mg per kg cells) were about three-fold higher for Opti-Pgp than for wild-type protein. Opti-Pgp conveyed full in vivo drug resistance against multiple anticancer and fungicidal drugs. ATP hydrolysis by purified Opti-Pgp was strongly stimulated ∼15-fold by verapamil and inhibited by cyclosporine A with binding constants of 4.2±2.2 µM and 1.1±0.26 µM, indistinguishable from wild-type Pgp. Maximum turnover number was 2.1±0.28 µmol/min/mg and was enhanced by 1.2-fold over wild-type Pgp, likely due to higher purity of Opti-Pgp preparations. Analysis of purified wild-type and Opti-Pgp by CD, DSC and limited proteolysis suggested similar secondary and ternary structure. Addition of lipid increased the thermal stability from T_m ∼40°C to 49°C, and the total unfolding enthalpy. The increase in folded state may account for the increase in drug-stimulated ATPase activity seen in presence of lipids.

Conclusion

The significantly higher yields of protein in the native folded state, higher purity and improved function establish the value of our gene optimization approach, and provide a basis to improve production of other membrane proteins.     

KSR Int'l Co. v. Teleflex, Inc.: no obvious changes for the biotechnology market

With the advent of molecular biology, genomics, and proteomics, the intersection between science and law has become increasingly significant. In addition to the ethical and legal concerns surrounding the collection, storage, and use of genomic data, patent disputes for new biotechnologies are quickly becoming part of mainstream business discussions. Under current patent law, new technologies cannot be patented if they are “obvious” changes to an existing patent. The definition of “obvious,” therefore, has a huge impact on determining whether a patent is granted. For example, are modifications to microarray protocols, popular in diagnostic medicine, considered “obvious” improvements of previous products? Also, inventions that are readily apparent now may not have been obvious when discovered. Polymerase chain reaction, or PCR, is now a common component of every biologist’s toolbox and seems like an obvious invention, though it clearly was not in 1983. Thus, there is also a temporal component that complicates the interpretation of an invention’s obviousness. The following article discusses how a recent Supreme Court decision has altered the definition of “obviousness” in patent disputes. By examining how the obviousness standard has changed, the article illuminates how legal definitions that seem wholly unrelated to biology or medicine could still potentially have enormous effects on these fieldsJust what is obvious or not is a question that has provoked substantial litigation in the Federal Circuit, the appellate court with special jurisdiction over patent law disputes. Under U.S. patent law, an inventor may not obtain a patent, which protects his invention from infringement by others, if the differences between the subject matter sought to be patented and the prior art are such that “the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill” in the patent’s subject matter area [1]. However, what was “obvious” at the time of invention to a person of ordinary skill is hardly clear and is, in effect, a legal fiction designed to approximate objectivity. As illustrated by Chief Justice John Roberts of the Supreme Court in a moment of levity, “Who do you get to ... tell you something’s not obvious … the least insightful person you can find?” [2] Despite the apparent objectivity provided by a “person of ordinary skill” obviousness standard, the difficulty lies in that such a standard is still susceptible to multiple interpretations, depending on the point of view and knowledge ascribed to the “ordinary person.” As such, how obviousness is defined and interpreted by the courts will have important implications on biotechnology patents and the biotechnology business.The issue of obviousness arose in April 2007 when the Supreme Court handed down its decision in KSR Int’l Co. v. Teleflex, Inc. [3] The facts of the case were anything but glamorous; in the suit, Teleflex, a manufacturer of adjustable pedal systems for automobiles, sued KSR, its rival, for infringement of its patent, which “describe[d] a mechanism for combining an electronic sensor with an adjustable automobile pedal so that the pedal’s position can be transmitted to a computer that controls the throttle in the vehicle’s engine.” [4] Teleflex believed that KSR’s new pedal design was too similar to its own patented design and therefore infringed upon it [5]. In defense, KSR argued that Teleflex’s patent was merely the obvious combination of two pre-existing elements and, thus, the patent, upon which Teleflex’s infringement claim was based, was invalid.Patent law relies on the concept of obviousness to distinguish whether new inventions are worthy of being protected by a patent. If a new invention is too obvious, it is not granted a patent and is therefore not a legally protected property interest. However, if an invention is deemed not obvious and has met the other patentability requirements, a patent will be granted, thereby conferring exclusive use of the invention to the patent holder. This exclusive right prohibits others from making, using, selling, offering to sell, or importing into the United States the patented invention [6]. Essentially, the definition of obviousness sets the balance between rewarding new inventions with exclusive property rights and respecting old inventions by not treating minor variations of existing patents as new patents. In this manner, the law seeks to provide economic incentives for the creation of new inventions by ensuring that the property right conferred by the patent will be protected against insignificant variations. The importance of where the line for obviousness is drawn and how clearly it is drawn is especially important in the biotechnology industry. Studies have shown that the development of a new pharmaceutical therapy can take up to 14 years with costs exceeding $800 million [7]. Such an enormous investment of time and money would not be practical if it did not predictably result in a legally enforceable property right.The standard for what constitutes a patentable discovery has evolved over the last 150 years. In 1851, the Supreme Court held in Hotchkiss v. Greenwood that a patentable discovery required a level of ingenuity above that possessed by an ordinary person [8]. Lower courts treated the Hotchkiss standard as a subjective standard, whereby courts sought to determine “what constitute[d] an invention” [9] and a “flash of creative genius” [10]. However, the attempts at imposing the Hotchkiss standard proved unworkable, and in 1952, Congress overrode the case law with the Patent Act, “mandat[ing] that patentability be governed by an objective nonobviousness standard.” [11] This new statutory standard moved the courts away from subjective determinations and toward a more workable, objective obviousness standard.While the Patent Act laid the foundation for the current obviousness standard, the Supreme Court in Graham v. John Deere Co. interpreted the statutory language in an attempt to provide greater clarity as to what exactly “obvious” meant [12]. The Supreme Court determined that the objective analysis would require “the scope and content of the prior art ... to be determined; differences between the prior art and the claims at issue ... to be ascertained; and the level of ordinary skill in the pertinent art resolved.” [13] In addition to analysis under this three-part framework, the Supreme Court called for several secondary considerations to be weighed, including “commercial success, long felt but unresolved needs, [and the] failure of others [to solve the problem addressed].” [13]Unsurprisingly, lower courts were unsatisfied with the Supreme Court’s attempts to clarify the obviousness standard and sought to provide “more uniformity and consistency” to their evaluation of obviousness than the Supreme Court’s jumble of factors provided [14]. In search of consistency, the Federal Circuit created the “teaching, suggestion, or motivation” test (TSM test) “under which a patent is only proved obvious if ‘some motivation or suggestion to combine prior art teachings’ can be found in the prior art, the nature of the problem, or the knowledge of a person having ordinary skill in the art.” [14] Through implementation of the TSM test, the Federal Circuit sought to maintain the flexibility envisioned by the Supreme Court in Graham, while at the same time providing more certainty and predictability to obviousness determinations.The issue before the Supreme Court in KSR Int’l Co. v. Teleflex, Inc. was whether the Federal Circuit’s elaboration on the statutory language of the Patent Act, the TSM test, was consistent with the terms of the Patent Act itself and the Supreme Court’s own analysis in Graham. The Supreme Court determined that while the TSM test was, on its terms, consistent with the framework set out in Graham, the rigid manner in which the Federal Circuit had taken to applying that standard was inconsistent with the flexible approach established by Graham [15]. More generally, it appears the Supreme Court was mainly interested in restoring a more rounded, thorough inquiry to the evaluation of obviousness: “Graham set forth a broad inquiry and invited courts, where appropriate, to look at any secondary considerations that would prove instructive.” [16] As stated by the Supreme Court, “[r]igid preventative rules that deny factfinders recourse to common sense, however, are neither necessary under our case law nor consistent with it.” [17] As such, the Supreme Court reversed the findings of the Federal Circuit, which had found the Teleflex patent valid, and remanded the case back to the lower court with directions to analyze, without rigid adherence to the TSM test, whether the Teleflex patent was obvious [18].The Supreme Court’s ruling in KSR Int’l Co. v. Teleflex, Inc. that the Federal Circuit apply its TSM test less rigidly may have implications for those seeking biotechnology patents in the future. As discussed above, the large investments necessary to develop a marketable biotechnology product demand that entrepreneurs making those investments be reasonably assured that they can predict any future legal hurdles in patenting their invention and in ultimately protecting their patent. As explained by the Biotechnology Industry Organization in its amicus curiae brief in KSR Int’l Co. v. Teleflex, Inc., “[i]nvestment thus is predicated on an expected return on investment in the form of products or services that are protected by patents whose validity can be fairly determined.” [19] Therefore, the Supreme Court’s insistence that the Federal Circuit no longer rigidly rely on the TSM test could increase uncertainty in the grant of future patents. However, the Supreme Court’s refusal to completely dismiss the TSM test, while in fact endorsing its continued use, albeit on a less rigid basis, has to be viewed as a profound victory for an industry with a significant stake in maintaining the status quo. Moreover, it is unclear how much the Supreme Court’s holding in KSR Int’l Co. v. Teleflex, Inc. will truly change the legal analysis of the lower courts, given the evidence that lower courts already were independently shifting away from rigid adherence to the TSM test before the Supreme Court’s ruling [20].More importantly, several aspects of the Supreme Court’s reasoning in KSR Int’l Co. v. Teleflex, Inc. seem to directly address relevant concerns of the biotechnology market in favorable ways. First, the Supreme Court made clear that though a product is the result of a combination of elements that were “obvious to try,” it is not necessarily “obvious” under the Patent Act. Retaining the possibility that “obvious to try” inventions still may be patentable is extremely important to the biotechnology industry in particular because “many patentable inventions in biotechnology spring from known components and methodologies found in [the] prior art.” [21] Rather than foreclosing all “obvious to try” inventions as being obvious, and therefore not patentable, the Supreme Court instead explained that where there is “a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions,” it is more likely that a person of ordinary skill would find it obvious to pursue “known options.” [22] Thus, the proper inquiry, as stated by the Supreme Court, is “whether the improvement is more than the predictable use of prior art elements according to their established functions.” [23] While this reasoning may prevent some “obvious to try” inventions from being patented, it is unlikely to have a substantial effect on inventions in the biotechnology market because “most advances in biotechnology are only won through great effort and expense, and with only a low probability of success in achieving the claimed invention at the outset.” [24] In other words, it would be hard to characterize the use of prior art in the biotechnology context as predictable based on the inherent unpredictability of obtaining favorable results. As such, most biotechnology inventions would presumably fall outside the Supreme Court’s “obvious to try” reasoning due to the very nature of the industry, meaning they would remain patentable under the Supreme Court’s KSR Int’l Co. v. Teleflex, Inc. decision.Second, the Supreme Court recognized the “distortion caused by hindsight bias” and the importance of avoiding “arguments reliant upon ex post reasoning,” though it lessened the Federal Circuit’s rigid protection against hindsight bias [24]. Hindsight bias requires that obviousness be viewed at the time the invention was made, because what may seem revolutionary at the time of invention may, upon the passage of time, seem “obvious.” Cognizance of hindsight bias is crucial for biotechnology patents because “there often is a long ‘passage of time between patent application filing and litigation with biotechnology inventions [that] can exacerbate the problem’ of hindsight bias.” [25] The problem is further exacerbated by the “significantly longer durations of commercial utility” biotechnology inventions enjoy as compared to those in other fields [25]. The more time between the filing of a patent and the subsequent litigation over its validity, the greater the risk that “reliable accounts of [the] context” in which the discovery is made will no longer exist [26]. As such, inventions that were not obvious when they were created will be inescapably colored by the passage of time and by new knowledge and discoveries; the likelihood of this occurrence is higher the further removed the litigation is from the patent filing date. Once again, however, it seems clear that despite the Supreme Court’s abandonment of the TSM test’s rigidity, strong protections against hindsight bias still were emphasized in the Supreme Court’s KSR Int’l Co. v. Teleflex, Inc. decision. In fact, lower courts applying KSR Int’l Co. v. Teleflex, Inc. acknowledge they are “cautious” to avoid “using hindsight” in biotechnology obviousness determinations [27].Finally, the Supreme Court seems to believe that the imposition of a more flexible approach will be more likely to benefit markets not directly at issue in KSR Int’l Co. v. Teleflex, Inc. The Supreme Court asserted, “[t]he diversity of inventive pursuits and of modern technology counsels against limiting the analysis” to the rigid TSM test of the Federal Circuit [28]. This language suggests that the Supreme Court expects lower courts to take into consideration the special considerations facing unique markets, such as the biotechnology market. As such, the specific concerns of the biotechnology market discussed above may receive more attention under the flexible framework asserted by the Supreme Court in KSR Int’l Co. v. Teleflex, Inc.Leading up to the oral argument in KSR Int’l Co. v. Teleflex, Inc., there was widespread speculation that the case could result in a watershed moment, significantly altering the definition of obviousness in patent law. For many, including those in the biotechnology industry, there was ample reason to be concerned. Any change in the definition of obviousness would effectively shift property rights from new patent holders to old, or vice versa. However, the Supreme Court acted with restraint. While the decision purports to make substantial changes by doing away with the Federal Circuit’s TSM test, the opinion seems more like a mild-mannered rebuke of lower courts that had become too complacent in the implementation of their beloved test. If anything, the Supreme Court’s insistence on a more flexible formula is simply a call for lower courts to employ common sense, in addition to considering the factors from Graham and the TSM test. Accordingly, the Supreme Court’s opinion in KSR Int’l Co. v. Teleflex, Inc. is unlikely to have a pronounced effect on the biotechnology market, despite the widespread concern generated before the actual decision was handed down.     

DETC induces Leishmania parasite killing in human in vitro and murine in vivo models: a promising therapeutic alternative in Leishmaniasis

Background

Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. However, currently available drugs present serious problems regarding side-effects, variable efficacy, and cost. Affordable and less toxic drugs are urgently needed for leishmaniasis.

Methodology/Principal Findings

We demonstrate, by microscopy and viability assays, that superoxide dismutase inhibitor diethyldithiocarbamate (DETC) dose-dependently induces parasite killing (p<0.001) and is able to “sterilize” Leishmania amazonensis infection at 2 mM in human macrophages in vitro. We also show that DETC-induced superoxide production (p<0.001) and parasite destruction (p<0.05) were reverted by the addition of the antioxidant N-acetylcysteine, indicating that DETC-induced killing occurs through oxidative damage. Furthermore, ultrastructural analysis by electron microscopy demonstrates a rapid and highly selective destruction of amastigotes in the phagosome upon DETC treatment, without any apparent damage to the host cell, including its mitochondria. In addition, DETC significantly induced parasite killing in Leishmania promastigotes in axenic culture. In murine macrophages infected with Leishmania braziliensis, DETC significantly induced in vitro superoxide production (p = 0.0049) and parasite killing (p = 0.0043). In vivo treatment with DETC in BALB/C mice infected with Leishmania braziliensis caused a significant decrease in lesion size (p<0.0001), paralleled by a 100-fold decrease (p = 0.0087) in parasite burden.

Conclusions/Significance

Due to its strong leishmanicidal effect in human macrophages in vitro, its in vivo effectiveness in a murine model, and its previously demonstrated in vivo safety profile in HIV treatment, DETC treatment might be considered as a valuable therapeutic option in human leishmaniasis, including HIV/Leishmania co-infection.     

A Refined Model of the Prototypical Salmonella SPI-1 T3SS Basal Body Reveals the Molecular Basis for Its Assembly

The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a “basal body”, a lock-nut structure spanning both bacterial membranes, and a “needle” that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.     

The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression

Background

ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines.

Findings

Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the “cell activation”, “immune response”, “apoptosis”, “signal transduction” and “development and differentiation” categories, whereas in the E/R KD-DOWN set only the “PI3K/AKT/mTOR signaling” and “hematopoietic stem cells” categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories “stem cell properties”, “B-cell differentiation”, “immune response”, “cell adhesion” and “DNA damage” with RT-qPCR.

Conclusion

Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.     

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ∼150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the “W” pocket) and the sialic acid-binding site (the “R” pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.Tetanus is an acute, often fatal disease of humans that was first described by Hippocrates over 24 centuries ago (1). Tetanus is characterized by generalized increased rigidity and convulsive spasms of skeletal muscles. Tetanus is caused by exposure to tetanus neurotoxin (TeNT)³ produced by the spore-forming bacterium Clostridium tetani. TeNT is delivered from the bloodstream to the peripheral nervous system, from where TeNT traffics to the central nervous system to cleave vesicle-associated membrane protein-2 (VAMP2), which inhibits neurotransmitter release and elicits spastic paralysis (2). Although prevented by vaccination, tetanus is responsible for hundreds of thousands of deaths per year in countries where vaccination is not common (3).TeNT is produced as a ∼150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (∼50 kDa) and a C-terminal heavy chain domain (∼100 kDa) linked through a single disulfide bond (4). TeNT light chain is a zinc metalloprotease that cleaves the neuronal SNARE protein VAMP2 (2). The TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptor-binding domain (HCR/T, ∼50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of α-motor neurons. Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5–9). Current models implicate a dual receptor mechanism for the binding of the clostridial neurotoxins to neurons, which includes a ganglioside-binding component (10). Complex gangliosides are sialic acid-containing glycosphingolipids that are located on the outer leaflet of cell membranes and contain a common “core” (GA1) consisting of Gal(β1–3)GalNAc(β1–4)Gal(β1–4)Glc(β1–1)Cer to which one or more N-acetylneuraminic acids (sialic acids) are bound, yielding “a” and “b” series gangliosides (11, 12). Numerous structural and biochemical studies have established that HCR/T contains two carbohydrate-binding sites: a lactose-binding site and a sialic acid-binding site (13). Previous studies showed that Trp¹²⁸⁹ is the key residue for the lactose-binding site, and Arg¹²²⁶ is the key residue for the sialic acid-binding site (14). In this study, we denote the lactose-binding site as the “W” pocket and the sialic acid-binding site as the “R” pocket. Binz and co-workers (14) showed that functional R and W binding sites were required for TeNT toxicity (7). These biochemical and cellular studies were supported by a co-crystal structure of HCR/T bound to a GT1b-β anomer analog, which showed that the W and R carbohydrate-binding pockets were located at different regions of TeNT (7). We recently reported that the W pocket binds gangliosides via the GA1 core structure, whereas the R pocket binds gangliosides via di- or tri-sialic acid moieties (15) where simultaneous binding of TeNT to two gangliosides was synergistic (see Fig. 1a). In the current study, gangliosides were identified that bound exclusively to either the W pocket or R pocket, which allowed the characterization of the role of ganglioside binding to the W and R pockets as dual receptors for TeNT entry into neurons.Open in a separate windowFIGURE 1.Interaction of the HCR domain of TeNT with its putative cellular receptor. a, HCR/T has two ganglioside-binding sites. The W pocket binds to the terminal GalNAc-Gal of the ganglioside (illustrated by GM1a). The R pocket binds to the di-sialic acid of the ganglioside (illustrated by GD3). b, alternating lanes of molecular mass marker proteins and cortical neuron lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was stained for protein with Ponceau S (bottom panel), and then the membrane strips were incubated with 10 nm of the indicated HCR/T (HCR/T wild type (wt), HCR/T (R+, W−), HCR/T (R−, W+), or HCR/T (R−, W−)) followed by HRP-conjugated α-FLAG antibody. The bands were visualized with SuperSignal; exposed film is shown (upper panel). The asterisk denotes the position of purified gangliosides resolved under identical conditions. Migration of the molecular mass marker proteins is indicated (kDa) in the left-most lane in the upper panel.     

Wasp-waist interactions in the North Sea ecosystem

Background

In a “wasp-waist” ecosystem, an intermediate trophic level is expected to control the abundance of predators through a bottom-up interaction and the abundance of prey through a top-down interaction. Previous studies suggest that the North Sea is mainly governed by bottom-up interactions driven by climate perturbations. However, few studies have investigated the importance of the intermediate trophic level occupied by small pelagic fishes.

Methodology/Principal Findings

We investigated the numeric interactions among 10 species of seabirds, two species of pelagic fish and four groups of zooplankton in the North Sea using decadal-scale databases. Linear models were used to relate the time series of zooplankton and seabirds to the time series of pelagic fish. Seabirds were positively related to herring (Clupea harengus), suggesting a bottom-up interaction. Two groups of zooplankton; Calanus helgolandicus and krill were negatively related to sprat (Sprattus sprattus) and herring respectively, suggesting top-down interactions. In addition, we found positive relationships among the zooplankton groups. Para/pseudocalanus was positively related to C. helgolandicus and C. finmarchicus was positively related to krill.

Conclusion/Significance

Our results indicate that herring was important in regulating the abundance of seabirds through a bottom-up interaction and that herring and sprat were important in regulating zooplankton through top-down interactions. We suggest that the positive relationships among zooplankton groups were due to selective foraging and switching in the two clupeid fishes. Our results suggest that “wasp-waist” interactions might be more important in the North Sea than previously anticipated. Fluctuations in the populations of pelagic fish due to harvesting and depletion of their predators might accordingly have profound consequences for ecosystem dynamics through trophic cascades.     

Heterogeneous Ribonucleoprotein K (hnRNP K) Binds miR-122, a Mature Liver-Specific MicroRNA Required for Hepatitis C Virus Replication

Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5′ untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing ∼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3′ portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.MicroRNAs (miRNAs) are a class of noncoding RNA of ∼22-nucleotides in length that can regulate gene expression by either targeting RNA for degradation or suppressing their translation through base pairing to the RNAs (1). Since their discovery in 1993 in Caenorhabditis elegans, miRNAs have been found in many species and are involved in the regulation of proliferation, differentiation, apoptosis, and development (1, 2). Moreover, miRNAs are also critical factors in the development of cancers, neurodegenerative diseases, and infectious diseases (3).MiR-122 is a highly abundant RNA in hepatocytes that regulates lipid metabolism, regeneration, and neoplastic transformation (4–6). In addition, miR-122 is required for the replication of the hepatitis C virus (HCV), a positive-strand RNA virus that infects over 170 million people worldwide (7–9). MiR-122 binds to a conserved sequence in the 5′ untranslated region (UTR) of the HCV RNA to increase the stability of the HCV RNA (10). Silencing of miR-122 can abolish HCV RNA accumulation in non-human primates (11). The expression of human miR-122 in non-hepatic cells can confer the ability to replicate HCV RNA (12). MiR-122 is one of the most critical host factors for HCV replication.We previously reported that the HCV RNA sequence that anneals to miR-122 is recognized by the heterogeneous ribonucleoprotein K (hnRNP K), a multifunctional RNA-binding protein known to be involved in RNA processing, translation, and the replication of several RNA viruses (13–15). In an unbiased screen for proteins from human proteome chips containing over 17,000 proteins, we identified 40 proteins that bind mature miR-122, including hnRNP K. Recombinant hnRNP K recognizes short pyrimidine sequences in miR-122 in vitro and a similar sequence in the HCV 5′ UTR. In hepatocytes endogenous hnRNP K can form a coprecipitable complex with miR-122, whether or not the cells contain replicating HCV. HnRNP K is thus a protein that binds a mature microRNA.