Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na⁺ and Ca²⁺ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K⁺ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (₆₃TVGDGN₆₈) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (₆₃TVGYGD₆₈; Fig. 1 A and B), leading to K⁺ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K⁺-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na⁺ or K⁺ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.Although proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein–protein interactions (2–4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein’s structural dynamics link to its functional mechanisms (12–21).A case in point is the nitric oxide synthase (NOS) enzymes (22–24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached “oxygenase” domain, which then enables NO synthesis to begin (22, 25–27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly dynamic and flexible due to a connecting hinge that allows it to alternate between its electron-accepting (FAD→FMN) or closed conformation and electron-donating (FMN→heme) or open conformation (Fig. 1 A and B) (28, 30–36). In the electron-accepting closed conformation, the FMN domain interacts with the NADPH/FAD domain (FNR domain) to receive electrons, whereas in the electron donating open conformation the FMN domain has moved away to expose the bound FMN cofactor so that it may transfer electrons to a protein acceptor like the NOS oxygenase domain, or to a generic protein acceptor like cytochrome c. In this way, the reductase domain structure cycles between closed and open conformations to deliver electrons, according to a conformational equilibrium that determines the movements and thus the electron flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar conformational switching mechanism is thought to enable electron transfer through the FMN domain in the related flavoproteins NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42).Open in a separate windowFig. 1.(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.NOS enzymes also contain a calmodulin (CaM) binding domain that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). CaM binding to NOS enzymes increases electron transfer from NADPH through the reductase domain and also triggers electron transfer from the FMN domain to the NOS heme as is required for NO synthesis (31, 32). The ability of CaM, or similar signaling proteins, to regulate electron transfer reactions in enzymes is unusual, and the mechanism is a topic of interest and intensive study. It has long been known that CaM binding alters NOSr structure such that, on average, it populates a more open conformation (43, 44). Recent equilibrium studies have detected a buildup of between two to four discreet conformational populations in NOS enzymes and in related flavoproteins, and in some cases, have also estimated the distances between the bound FAD and FMN cofactors in the different species (26, 36, 37, 39, 40), and furthermore, have confirmed that CaM shifts the NOS population distribution toward more open conformations (34, 36, 45). Although valuable, such ensemble-averaged results about conformational states cannot explain how electrons transfer through these enzymes, or how CaM increases the electron flux in NOS, because answering these questions requires a coordinate understanding of the dynamics of the conformational fluctuations. Indeed, computer modeling has indicated that a shift toward more open conformations as is induced by CaM binding to nNOS should, on its own, actually diminish electron flux through nNOS and through certain related flavoproteins (38). Despite its importance, measuring enzyme conformational fluctuation dynamics is highly challenging, and as far as we know, there have been no direct measures on the NOS enzymes or on related flavoproteins, nor studies on how CaM binding might influence the conformational fluctuation dynamics in NOS.To address this gap, we used single-molecule fluorescence energy resonance transfer (FRET) spectroscopy to characterize individual molecules of nNOSr that had been labeled at two specific positions with Cyanine 3 (Cy3) donor and Cyanine 5 (Cy5) acceptor dye molecules, regarding their conformational states distribution and the associated conformational fluctuation dynamics, which in turn enabled us to determine how CaM binding impacts both parameters. This work provides a unique perspective and a novel study of the NOS enzymes and within the broader flavoprotein family, which includes the mammalian enzymes methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR), and reveals how CaM’s control of the conformational behaviors may regulate the electron transfer reactions of NOS catalysis.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

Tuning a riboswitch response through structural extension of a pseudoknot

Structural and dynamic features of RNA folding landscapes represent critical aspects of RNA function in the cell and are particularly central to riboswitch-mediated control of gene expression. Here, using single-molecule fluorescence energy transfer imaging, we explore the folding dynamics of the preQ₁ class II riboswitch, an upstream mRNA element that regulates downstream encoded modification enzymes of queuosine biosynthesis. For reasons that are not presently understood, the classical pseudoknot fold of this system harbors an extra stem–loop structure within its 3′-terminal region immediately upstream of the Shine–Dalgarno sequence that contributes to formation of the ligand-bound state. By imaging ligand-dependent preQ₁ riboswitch folding from multiple structural perspectives, we reveal that the extra stem–loop strongly influences pseudoknot dynamics in a manner that decreases its propensity to spontaneously fold and increases its responsiveness to ligand binding. We conclude that the extra stem–loop sensitizes this RNA to broaden the dynamic range of the ON/OFF regulatory switch.A variety of small metabolites have been found to regulate gene expression in bacteria, fungi, and plants via direct interactions with distinct mRNA folds (1–4). In this form of regulation, the target mRNA typically undergoes a structural change in response to metabolite binding (5–9). These mRNA elements have thus been termed “riboswitches” and generally include both a metabolite-sensitive aptamer subdomain and an expression platform. For riboswitches that regulate the process of translation, the expression platform minimally consists of a ribosomal recognition site [Shine–Dalgarno (SD)]. In the simplest form, the SD sequence overlaps with the metabolite-sensitive aptamer domain at its downstream end. Representative examples include the S-adenosylmethionine class II (SAM-II) (10) and the S-adenosylhomocysteine (SAH) riboswitches (11, 12), as well as prequeuosine class I (preQ₁-I) and II (preQ₁-II) riboswitches (13, 14). The secondary structures of these four short RNA families contain a pseudoknot fold that is central to their gene regulation capacity. Although the SAM-II and preQ₁-I riboswitches fold into classical pseudoknots (15, 16), the conformations of the SAH (17) and preQ₁-II counterparts are more complex and include a structural extension that contributes to the pseudoknot architecture (14). Importantly, the impact and evolutionary significance of these “extra” stem–loop elements on the function of the SAH and preQ₁-II riboswitches remain unclear.PreQ₁ riboswitches interact with the bacterial metabolite 7-aminomethyl-7-deazaguanine (preQ₁), a precursor molecule in the biosynthetic pathway of queuosine, a modified base encountered at the wobble position of some transfer RNAs (14). The general biological significance of studying the preQ₁-II system stems from the fact that this gene-regulatory element is found almost exclusively in the Streptococcaceae bacterial family. Moreover, the preQ₁ metabolite is not generated in humans and has to be acquired from the environment (14). Correspondingly, the preQ₁-II riboswitch represents a putative target for antibiotic intervention. Although preQ₁ class I (preQ₁-I) riboswitches have been extensively investigated (18–28), preQ₁ class II (preQ₁-II) riboswitches have been largely overlooked despite the fact that a different mode of ligand binding has been postulated (14).The consensus sequence and the secondary structure model for the preQ₁-II motif (COG4708 RNA) (Fig. 1A) comprise ∼80–100 nt (14). The minimal Streptococcus pneumoniae R6 aptamer domain sequence binds preQ₁ with submicromolar affinity and consists of an RNA segment forming two stem–loops, P2 and P4, and a pseudoknot P3 (Fig. 1B). In-line probing studies suggest that the putative SD box (AGGAGA; Fig. 1) is sequestered by pseudoknot formation, which results in translational-dependent gene regulation of the downstream gene (14).Open in a separate windowFig. 1.PreQ₁ class II riboswitch. (A) Chemical structure of 7-aminomethyl-7-deazaguanosine (preQ₁); consensus sequence and secondary structure model for the COG4708 RNA motif (adapted from reference 14). Nucleoside presence and identity as indicated. (B) S. pneumoniae R6 preQ₁-II RNA aptamer investigated in this study. (C) Schematics of an H-type pseudoknot with generally used nomenclature for comparison.Here, we investigated folding and ligand recognition of the S. pneumoniae R6 preQ₁-II riboswitch, using complementary chemical, biochemical, and biophysical methods including selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), mutational analysis experiments, 2-aminopurine fluorescence, and single-molecule fluorescence resonance energy transfer (smFRET) imaging. In so doing, we explored the structural and functional impact of the additional stem–loop element in the context of its otherwise “classical” H-type pseudoknot fold (29–32) (Fig. 1C). Our results reveal that the unique 3′-stem–loop element in the preQ₁-II riboswitch contributes to the process of SD sequestration, and thus the regulation of gene expression, by modulating both its intrinsic dynamics and its responsiveness to ligand binding.     

A family of starch-active polysaccharide monooxygenases

The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries.Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species (1–5). They have recently been found to oxidatively degrade chitin (6–8) and cellulose (8–14). PMOs have been shown to oxidize either the C1 or C4 atom of the β(1→4) glycosidic bond on the surface of chitin (6, 7) or cellulose (10–12, 14), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases. Several fungal PMOs were shown to significantly enhance the degradation of cellulose by hydrolytic cellulases (9), indicating that these enzymes can be used in the conversion of plant biomass into biofuels and other renewable chemicals.There are three families of PMOs characterized thus far: fungal PMOs that oxidize cellulose (9–12) (also known as GH61 and AA9); bacterial PMOs that are active either on chitin (6, 8) or cellulose (8, 13) (also known as CBM33 and AA10); and fungal PMOs that oxidize chitin (AA11) (7). Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel β-sandwich core and a highly conserved monocopper active site on a flat protein surface (Fig. 1A) (2, 6, 7, 9, 10, 15–17). Two histidine residues in a motif termed the histidine brace coordinate the copper center. The N-terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the N_ε position in fungal PMOs (Fig. 1A).Open in a separate windowFig. 1.(A) Representative overall and active site structures of fungal PMOs (PDB ID code 2YET) (10). (B) Structure of cellulose (18, 19). Chitin also contains β(1→4) linkages and has similar crystalline higher order structure to cellulose. (C) Model structure of amylopectin (23–25). Hydrogen bonds are shown with green dashed lines.Considering the conserved structural features, it is not surprising that the currently known PMOs act on substrates with similar structures. Cellulose and chitin contain long linear chains of β(1→4) linked glucose units and N-acetylglucosamine units, respectively (Fig. 1B). The polymer chains form extensive hydrogen bonding networks, which result in insoluble and very stable crystalline structures (18–21). PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the C1 or C4 position (6, 16, 22). Some bacterial chitin-binding proteins are cellulose-active PMOs (8, 13, 14), further suggesting that the set of PMO substrates is restricted to β(1→4) linked polymers of glucose and glucose derivatives.Here, we report on the identification of new families of PMOs that contain several key features of previously characterized PMOs, but act on substrates different from cellulose or chitin. A member of one of these novel families of PMOs, NCU08746, was shown to oxidatively cleave amylose, amylopectin, and starch. We designate the NCU08746 family as starch-active PMOs. Both amylose and amylopectin contain linear chains of α(1→4) linked glucose, whereas the latter also contains α(1→6) glycosidic linkages at branch points in the otherwise α(1→4) linked polymer. Unlike cellulose and chitin, amylose and amylopectin do not form microcrystals; instead, they exist in disordered, single helical, and double helical forms (23–27) (see Fig. 1C for example). Starch exists partially in nanocrystalline form, but lacks the flat molecular surfaces as those found in chitin and cellulose. The discovery of starch-active PMOs shows that this oxidative mechanism of glycosidic bond cleavage is more widespread than initially expected.     

Robust efficiency and actuator saturation explain healthy heart rate control and variability

The correlation of healthy states with heart rate variability (HRV) using time series analyses is well documented. Whereas these studies note the accepted proximal role of autonomic nervous system balance in HRV patterns, the responsible deeper physiological, clinically relevant mechanisms have not been fully explained. Using mathematical tools from control theory, we combine mechanistic models of basic physiology with experimental exercise data from healthy human subjects to explain causal relationships among states of stress vs. health, HR control, and HRV, and more importantly, the physiologic requirements and constraints underlying these relationships. Nonlinear dynamics play an important explanatory role––most fundamentally in the actuator saturations arising from unavoidable tradeoffs in robust homeostasis and metabolic efficiency. These results are grounded in domain-specific mechanisms, tradeoffs, and constraints, but they also illustrate important, universal properties of complex systems. We show that the study of complex biological phenomena like HRV requires a framework which facilitates inclusion of diverse domain specifics (e.g., due to physiology, evolution, and measurement technology) in addition to general theories of efficiency, robustness, feedback, dynamics, and supporting mathematical tools.Biological systems display a variety of well-known rhythms in physiological signals (1–6), with particular patterns of variability associated with a healthy state (2–6). Decades of research demonstrate that heart rate (HR) in healthy humans has high variability, and loss of this high HR variability (HRV) is correlated with adverse states such as stress, fatigue, physiologic senescence, or disease (6–13). The dominant approach to analysis of HRV has been to focus on statistics and patterns in HR time series that have been interpreted as fractal, chaotic, scale-free, critical, etc. (6–17). The appeal of time series analysis is understandable as it puts HRV in the context of a broad and popular approach to complex systems (5, 18), all while requiring minimal attention to domain-specific (e.g., physiological) details. However, despite intense research activity in this area, there is limited consensus regarding causation or mechanism and minimal clinical application of the observed phenomena (10). This paper takes a completely different approach, aiming for more fundamental rigor (19–24) and methods that have the potential for clinical relevance. Here we use and model data from experimental studies of exercising healthy athletes, to add simple physiological explanations for the largest source of HRV and its changes during exercise. We also present methods that can be used to systematically pursue further explanations about HRV that can generalize to less healthy subjects.Fig. 1 shows the type of HR data analyzed, collected from healthy young athletes (n = 5). The data display responses to changes in muscle work rate on a stationary bicycle during mostly aerobic exercise. Fig. 1A shows three separate exercise sessions with identical workload fluctuations about three different means. With proper sleep, hydration, nutrition, and prevention from overheating, trained athletes can maintain the highest workload in Fig. 1 for hours and the lower and middle levels almost indefinitely. This ability requires robust efficiency: High workloads are sustained while robustly maintaining metabolic homeostasis, a particularly challenging goal in the case of the relatively large, metabolically demanding, and fragile human brain.Open in a separate windowFig. 1.HR responses to simple changes in muscle work rate on a stationary bicycle: Each experimental subject performed separate stationary cycle exercises of ∼10 min for each workload profile, with different means but nearly identical square wave fluctuations around the mean. A typical result is shown from subject 1 for three workload profiles with time on the horizontal axis (zoomed in to focus on a 6-min window). (A) HR (red) and workload (blue); linear local piecewise static fits (black) with different parameters for each exercise. The workload units (most strenuous exercise on top of graph) are shifted and scaled so that the blue curves are also the best global linear fit. (B) Corresponding dynamics fits, either local piecewise linear (black) or global linear (blue). Note that, on all time scales, mean HR increases and variability (HRV) goes down with the increasing workload. Breathing was spontaneous (not controlled).Whereas mean HR in Fig. 1A increases monotonically with workloads, both slow and fast fluctuations (i.e., HRV) in HR are saturating nonlinear functions of workloads, meaning that both high- and low-frequency HRV component goes down. Results from all subjects showed qualitatively similar nonlinearities (SI Appendix). We will argue that this saturating nonlinearity is the simplest and most fundamental example of change in HRV in response to stressors (11, 12, 25) [exercise in the experimental case, but in general also fatigue, dehydration, trauma, infection, even fear and anxiety (6–9, 11, 12, 25)].Physiologists have correlated HRV and autonomic tone (7, 11, 12, 14), and the (im)balance between sympathetic stimulation and parasympathetic withdrawal (12, 26–28). The alternation in autonomic control of HR (more sympathetic and less parasympathetic tone during exercise) serves as an obvious proximate cause for how the HRV changes as shown in Fig. 1, but the ultimate question remains as to why the system is implemented this way. It could be an evolutionary accident, or could follow from hard physiologic tradeoff requirements on cardiovascular control, as work in other systems suggests (1). Here, the explanation of HRV similarly involves hard physiological tradeoffs in robust efficiency and employs the mathematical tools necessary to make this explanation rigorous in the context of large measurement and modeling uncertainties.     

Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A

Diverse mechanisms including activation of NMDA receptors, microglial activation, reactive astrogliosis, loss of descending inhibition, and spasticity are responsible for ∼40% of cases of intractable neuropathic pain after spinal cord injury (SCI). Because conventional treatments blocking individual mechanisms elicit only short-term effectiveness, a multimodal approach with simultaneous actions against major pain-related pathways may have value for clinical management of chronic pain. We hypothesize that [-]-huperzine A (HUP-A), an alkaloid isolated from the club moss Huperzia serrata, that is a potent reversible inhibitor of acetylcholinesterase and NMDA receptors, could mitigate pain without invoking drug tolerance or dependence by stimulating cholinergic interneurons to impede pain signaling, inhibiting inflammation via microglial cholinergic activation, and blocking NMDA-mediated central hypersensitization. We tested our hypothesis by administering HUP-A i.p. or intrathecally to female Sprague–Dawley rats (200–235 g body weight) after moderate static compression (35 g for 5 min) of T10 spinal cord. Compared with controls, HUP-A treatment demonstrates significant analgesic effects in both regimens. SCI rats manifested no drug tolerance following repeated bolus i.p. or chronic intrathecal HUP-A dosing. The pain-ameliorating effect of HUP-A is cholinergic dependent. Relative to vehicle treatment, HUP-A administration also reduced neural inflammation, retained higher numbers of calcium-impermeable GluR2-containing AMPA receptors, and prevented Homer1a up-regulation in dorsal horn sensory neurons. Therefore, HUP-A may provide safe and effective management for chronic postneurotrauma pain by reestablishing homeostasis of sensory circuits.Neuropathic pain is one of the most debilitating sequelae of neurotrauma and is an unmet clinical need for at least 40% of patients with spinal cord injury (SCI) (1). Administration of conventional drugs has shown only various degrees of short-term efficacy in reducing at- and/or below-injury-level hypersensitivity after SCI by acting on individual pathways to inhibit descending facilitation of pain-transmission neurons (2), activate inhibitory interneurons in the spinal cord (2, 3), mitigate firing of pain-transmission neurons (3–7), or impede inflammation triggered by the activation of astrocytes and microglial cells (8). The diversity of mechanisms and potential targets for intervention may be partially responsible for the difficulty in developing therapeutics that can provide long-term efficacy. Therefore we postulated that devising a multimodal treatment with potent, defined simultaneous effects on multiple pain-related pathways might be an effective concept for clinical management of neuropathic pain.Given the potential roles of cholinergic agonists (9, 10) and antagonists of NMDA-subtype glutamate receptors (10, 11) in the treatment of neuropathic pain, we hypothesized that [-]-huperzine A (HUP-A) (Fig. 1A), a naturally occurring Lycopodium alkaloid isolated from the Chinese club moss, Huperzia serrata (Fig. 1B) that has potent reversible inhibitory action on acetylcholinesterase (AChE) (Fig. 1C) (10, 12) and NMDA receptors (Fig. 1D) (13), might be an exceptional prospect for multimodal treatment of SCI-induced neuropathic pain. We tested whether HUP-A–derived NMDA blockade and the overall HUP-A–derived augmentation of cholinergic neurotransmission arising from AChE inhibition might be harnessed specifically to stage a multifocal intervention in post-SCI pain pathways because (i) antagonism of NMDA receptor prevents the post-SCI hyperexcitability of neurons in the dorsal horn (DH) of the spinal cord that feature a wide, dynamic range of pain transmission (7); (ii) activation of presynaptic α3β2 nicotinic ACh receptors (nAChR) minimizes release of glutamate from C-fiber terminals in the DH that are involved in nociceptive neurotransmission (14); (iii) activation of GABAergic interneurons via stimulation of their M2 and M4 muscarinic ACh receptors (mAChR), in turn, inhibits presynaptic release of glutamate from primary afferent axons (15, 16); (iv) activation of the α4β2 nAChR on GABAergic inhibitory interneurons mitigates the firing of secondary spinothalamic pain transmission neurons (17); and (v) stimulation of the α7 nAChR on microglial cells blocks their activation, ameliorating neuroinflammation (18, 19). The primary goal of our study is to develop a class of therapeutics for the management of chronic neuropathic pain that aims to restore homeostasis of the sensory neurocircuitry by simultaneous multimodal mechanisms without invoking drug tolerance and dependence or respiratory suppression. HUP-A has been used for centuries in herbal medicine to treat inflammatory and other diseases (10, 12), has demonstrated effectiveness in inhibiting hypersensitivity triggered by peripheral neuropathy (20), and has been studied in phase II clinical trials for Alzheimer’s disease in the United States (11). We tested whether HUP-A regimens might translate into an effective and safe therapy for neuropathic pain following SCI in adult female rats (21–23).Open in a separate windowFig. 1.HUP-A is a natural alkaloid extract. HUP-A''s molecular structure is presented in (A) from the club moss Huperzia serrata (B) that is a potent, highly specific, and reversible inhibitor of AChE (C) as well as a noncompetitive partial antagonist of the NMDA receptor (D). A reproducible moderate static-compression SCI model maintaining 35 g of static weight on the dorsal surface of T10 spinal cord for 5 min was used for the present study (E).     

Solid-phase peptide synthesis and solid-phase fragment coupling mediated by isonitriles

The synthesis of polypeptides on solid phase via mediation by isonitriles is described. The acyl donor is a thioacid, which presumably reacts with the isonitrile to generate a thio-formimidate carboxylate mixed anhydride intermediate. Applications of this chemistry to reiterative solid-phase peptide synthesis as well as solid-phase fragment coupling are described.Amide bond formations are arguably among the most important constructions in organic chemistry (1, 2). The centrality of the amide linkage, as found in polypeptides and proteins, in the maintenance of life hardly needs restatement. Numerous strategies, resulting in a vast array of protocols to synthesize biologically active polypeptides and proteins, have been demonstrated (3, 4). Central to reiterative polypeptide bond formations was the discovery and remarkable development of solid-phase peptide synthesis (SPPS) (5, 6). The extraordinary impact of SPPS in fostering enhanced access to homogeneous polypeptides is clear to everyone in the field.As we have described elsewhere, by classical, mechanistic reasoning, we were led to conjecture about some hitherto-unexplored possibilities relevant to the chemistry of isonitriles (7–14). It was anticipated that isonitriles might be able to mediate the acylation of amines, thus giving rise to amides (15). Early experiments focused on free carboxylic acids as the acylating agents. As our studies progressed, it was found that the combination of thioacids, amines, and isonitriles leads to the efficient formation of amide bonds under stoichiometric or near-stoichiometric conditions (7–13, 16, 17). Although there remain unresolved issues of detail and nuance, the governing mechanism for amide formation under these conditions involves reaction of the thioacid, 1, with an isonitrile, 2, to generate a thio-formimidate carboxylate mixed anhydride (thio-FCMA), 3, which is intercepted by the “acyl-accepting” amine to generate amide, 5, and thioformamide, 6 (Fig. 1). The efficiency of the amidation was further improved through the use of hydroxybenzotriazole (HOBt) (18), which could well give rise to HOBt ester 7, although this pathway has not been mechanistically proven.Open in a separate windowFig. 1.Isonitrile-mediated amidation; structure of OT.The potentialities of the isonitrile-mediated amidation method were foreshadowed via its application to the synthesis of cyclosporine (19). The power of the method was particularly well demonstrated in the context of our recent total synthesis of oxytocin (OT) (20), wherein isonitrile mediation was used in each of the peptide bond constructions, leading to the synthesis of the hormone in high yield and excellent purity. This nonapeptide is involved in a range of biological functions including parturition and lactation (21, 22). Signaling of OT to its receptor (OTR) is apparently an important factor in quality maintenance of various CNS functions (23). The ability to synthesize such modestly sized, but bio-impactful peptides in both native (wild-type) form, and as strategically modified variants, is one of the current missions of our laboratory, with the objective of possible applications to the very serious problem of autism (24–26).     

Biosynthetic investigation of phomopsins reveals a widespread pathway for ribosomal natural products in Ascomycetes

Production of ribosomally synthesized and posttranslationally modified peptides (RiPPs) has rarely been reported in fungi, even though organisms of this kingdom have a long history as a prolific source of natural products. Here we report an investigation of the phomopsins, antimitotic mycotoxins. We show that phomopsin is a fungal RiPP and demonstrate the widespread presence of a pathway for the biosynthesis of a family of fungal cyclic RiPPs, which we term dikaritins. We characterize PhomM as an S-adenosylmethionine–dependent α-N-methyltransferase that converts phomopsin A to an N,N-dimethylated congener (phomopsin E), and show that the methyltransferases involved in dikaritin biosynthesis have evolved differently and likely have broad substrate specificities. Genome mining studies identified eight previously unknown dikaritins in different strains, highlighting the untapped capacity of RiPP biosynthesis in fungi and setting the stage for investigating the biological activities and unknown biosynthetic transformations of this family of fungal natural products.Ascomycetes constitute the largest phylum of the fungal kingdom and are a prolific source of natural products that are of great significance to humankind (1–4). Beneficial ascomycetous natural products include many clinically important drugs and various food and industrial chemicals. Many other compounds produced by ascomycetes are toxic and are known as mycotoxins, which pose substantial threats to human food supplies and health (5). Major classes of natural products from Ascomycetes include alkaloids (6, 7), terpenoids (8), polyketides (PKs) (9–12), nonribosomal peptides (NRPs) (12–14), and PK-NRP hybrids (15, 16). However, ribosomally synthesized and posttranslationally modified peptides (RiPPs), a rapidly growing class of natural products (17), have rarely been found in Ascomycetes. It has been known since 2007 that amatoxins and phallotoxins are RiPPs produced by strains of another fungal phylum, Basidiomycetes (which, together with Ascomycetes, compose the subkingdom Dikarya) (17–19). It also was recently reported that the ustiloxins produced by Aspergillus flavus and Ustilaginoidea virens belong to the RiPP class (20, 21).Phomopsins are a group of hexapeptide mycotoxins produced by the pathogenic Ascomycetes Phomopsis leptostromiformis (class Sordariomycetes, order Diaporthales) that infects lupins (Lupinus). Lupin-based animal food contaminated with phomopsins causes lupinosis, a liver disease of sheep and cattle that is particularly problematic in Australia and South Africa but affects other countries as well (22). By targeting the vinca domain of tubulin (23), phomopsins exhibit potent antimitotic activity and thus also represent a potential lead for antitumor drug development. Phomopsins have a 13-member macrocyclic ring formed by an ether bridge linking an Ile residue to the phenyl ring of a Tyr (Fig. 1A). A similar Tyr-containing macrocycle is also present in ustiloxins (Fig. 1B). Unlike ustiloxins, however, phomopsins contain no proteinogenic amino acids and feature a set of dehydroamino acids found only in this group of compounds, including 2,3-dehydroisoleucine, 2,3-dehydroaspartic acid, 3,4-dehydroproline, and 3,4-dehydrovaline (Fig. 1A).Open in a separate windowFig. 1.Chemical structures of phomopsins (A) and ustiloxins (B). The compounds labeled in red are previously unknown N,N-dimethylated dikaritins identified in this study.Early isotopic labeling studies showed that Ile, Pro, and Phe were all incorporated into the phomopsin scaffold (24). Because a Phe hydroxylase that can convert Phe to Tyr has not been found in Ascomycetes, the labeling study seems incongruent with a ribosomal route to phomopsins, in which Tyr, not Phe, would need to be incorporated into the phomopsin structure via a tRNA-dependent pathway. Here we report our investigation of the biosynthetic pathway of phomopsins, which explains the paradoxical observations in the early labeling studies and unequivocally demonstrates that phomopsins belong to the family of RiPP natural products. Moreover, our study shows that RiPPs are widespread in fungi, paving the way for a study of their biological activities and biosynthesis.     

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.Muscle contraction is driven by the relative sliding of the actin-containing thin filaments along the myosin-containing thick filaments arranged in a parallel array in the muscle sarcomere (Fig. 1A). Filament sliding in turn is driven by a structural change in the myosin head domains (Fig. 1B) while they are bound to actin, coupled to the hydrolysis of ATP (1). Contraction of skeletal and cardiac muscle is triggered by calcium binding to troponin in the thin filaments, accompanied by a change in the structure of the thin filaments that permits myosin head binding (2). However, the strength and dynamics of contraction are modulated by posttranslational modifications in other sarcomeric proteins, including the myosin regulatory light chain (RLC) (3), which is part of the myosin head, and myosin binding protein-C (4–6) (MyBP-C) (Fig. 1B). In an emerging concept of thick filament regulation in striated muscle that is analogous to myosin-linked regulation in smooth muscle (7–11), RLC and MyBP-C are thought to modulate contraction by controlling the conformation of the myosin heads.Open in a separate windowFig. 1.Sarcomere location and domain architecture of MyBP-C. (A) C-zone (green) of the thick filament in relation to its proximal (P) and distal (D) regions and the thin filament (gray). (B) Cartoon representation of MyBP-C (green) anchored to the thick filament backbone (purple) via its C-terminal domains; myosin heads are pink and troponin is yellow. (C) Domain organization and interactions of MyBP-C.According to this concept, the thick filament has an OFF state in which the myosin heads are folded back against its surface (Fig. 1B), rendering them unavailable for interaction with actin, and an ON state in which the heads are released from the thick filament surface and made available for actin binding. The physiological and pathological significance of thick filament regulation and its relationship to the well-studied thin filament mechanisms remain poorly understood, but much recent attention has focused on MyBP-C for two main reasons. First, mutations in the cardiac MYBPC3 gene are commonly associated with hypertrophic cardiomyopathy (12, 13), and this association has driven a wide range of studies at the molecular, cellular, and whole-animal levels aimed at understanding the etiology of MYBPC3-linked disease. Second, although MyBP-C is a constitutive component of the thick filament, there is a large body of evidence that it can also bind the thin filaments (14, 15), raising the possibility that one role of MyBP-C may be to synchronize the regulatory states of the thin and thick filaments (11, 15–17).MyBP-C is localized to the central region or “C-zone” of each half-thick filament (Fig. 1A), appearing in nine transverse stripes with a 43-nm periodicity closely matching that of the myosin heads (Fig. 1B) (10). MyBP-C has 11 Ig-like or fibronectin-like domains (Fig. 1C) denoted C0–C10, with additional linking sequences, notably the MyBP-C “motif” or “m” domain between C1 and C2 and the proline/alanine-rich (P/A) linker between C0 and C1. The m domain has multiple phosphorylation sites (4–6). Constitutive binding to the thick filament is mediated by interactions of domains C8–C10 with myosin and titin. The C1mC2 region binds to the coiled-coil subfragment-2 (S2) domain of myosin adjacent to the myosin heads, and this interaction is abolished by MyBP-C phosphorylation (5); the C0 domain binds to the RLC in the myosin head itself (18). The N-terminal domains of MyBP-C also bind to actin in a phosphorylation-dependent manner (14, 15) (Fig. 1B), and EM and X-ray studies on intact sarcomeres of skeletal muscle suggest that MyBP-C binds to thin filaments under relaxing conditions (10, 11).The function of MyBP-C and the mechanisms underlying its modulation in cardiomyopathy remain poorly understood, however. Ablation of MyBP-C in a knockout mouse model leads to a hypertrophic phenotype associated with impaired contractile function (19), but cardiomyocytes isolated from these mice exhibit increased power output during working contractions (20). A range of studies at the isolated protein and cellular levels have led to the concept that MyBP-C exerts a predominantly inhibitory effect on contractility mediated through two distinct mechanisms (15, 16, 21). MyBP-C may tether myosin heads to the surface of the thick filament, preventing their interaction with actin, and its N terminus may bind to thin filaments, inhibiting interfilament sliding at low load. Other studies, however, have demonstrated an activating effect of MyBP-C mediated by binding of its N-terminal domains to the thin filament. N-terminal fragments of MyBP-C enhance force production in skinned cardiac muscle cells and motility in isolated filament preparations at zero or submaximal calcium concentrations (22–25). The same effect is observed in cardiomyocytes from MyBP-C knockout mice (22), suggesting that the activating effect is not due to competitive removal of an inhibitory effect of native MyBP-C.To resolve these apparently contradictory hypotheses about the physiological function of the N-terminal domains of MyBP-C, we determined the structural changes in the thick and thin filaments of intact sarcomeres in heart muscle cells induced by N-terminal MyBP-C fragments using bifunctional rhodamine probes on RLC and troponin C (TnC) (26). These probes allowed the structural changes in both types of filament to be directly compared with those associated with calcium activation and myosin head binding in the native environment of the cardiac muscle sarcomere. The results lead to a model for the physiological function of MyBP-C that integrates the regulatory roles of the thin and thick filaments and the inhibitory and activating effects of MyBP-C at the level of the intact sarcomere.     

Remote loading of preencapsulated drugs into stealth liposomes

Loading drugs into carriers such as liposomes can increase the therapeutic ratio by reducing drug concentrations in normal tissues and raising their concentrations in tumors. Although this strategy has proven advantageous in certain circumstances, many drugs are highly hydrophobic and nonionizable and cannot be loaded into liposomes through conventional means. We hypothesized that such drugs could be actively loaded into liposomes by encapsulating them into specially designed cyclodextrins. To test this hypothesis, two hydrophobic drugs that had failed phase II clinical trials because of excess toxicity at deliverable doses were evaluated. In both cases, the drugs could be remotely loaded into liposomes after their encapsulation (preloading) into cyclodextrins and administered to mice at higher doses and with greater efficacy than possible with the free drugs.There is currently wide interest in the development of nanoparticles for drug delivery (1–7). This area of research is particularly relevant to cancer drugs, wherein the therapeutic ratio (dose required for effectiveness to dose causing toxicity) is often low. Nanoparticles carrying drugs can increase this therapeutic ratio over that achieved with the free drug through several mechanisms. In particular, drugs delivered by nanoparticles are thought to selectively enhance the concentration of the drugs in tumors as a result of the enhanced permeability and retention (EPR) effect (8–18). The enhanced permeability results from a leaky tumor vascular system, whereas the enhanced retention results from the disorganized lymphatic system that is characteristic of malignant tumors.Much current work in this field is devoted to designing novel materials for nanoparticle generation. This new generation of nanoparticles can carry drugs—particularly those that are insoluble in aqueous medium—that are difficult to incorporate into conventional nanoparticles such as liposomes. However, the older generation of nanoparticles has a major practical advantage in that they have been extensively tested in humans and approved by regulatory agencies such as the Food and Drug Administration in the United States and the European Medicines Agency in Europe. Unfortunately, many drugs cannot be easily or effectively loaded into liposomes, thereby compromising their general use.In general, liposomal drug loading is achieved by either passive or active methods (9, 19–22). Passive loading involves dissolution of dried lipid films in aqueous solutions containing the drug of interest. This approach can only be used for water-soluble drugs, and the efficiency of loading is often low. In contrast, active loading can be extremely efficient, resulting in high intraliposomal concentrations and minimal wastage of precious chemotherapeutic agents (9, 23, 24). In active loading, drug internalization into preformed liposomes is typically driven by a transmembrane pH gradient. The pH outside the liposome allows some of the drug to exist in an unionized form, able to migrate across the lipid bilayer. Once inside the liposome, the drug becomes ionized due to the differing pH and becomes trapped there (Fig. 1A). Many reports have emphasized the dependence of liposome loading on the nature of the transmembrane pH gradient, membrane–water partitioning, internal buffering capacity, aqueous solubility of the drug, lipid composition, and other factors (25–28). As described in a recent model (28), the aqueous solubility of the drug is one of the requirements for efficient active loading. Another key element for the success of remote loading is the presence of weakly basic functional groups on the small molecule.Open in a separate windowFig. 1.Schematic representation of active loading of a liposome. (A) Remote loading of an ionizable hydrophilic drug using a transmembrane pH results in efficient incorporation. (B) A poorly soluble hydrophobic drug results in meager incorporation into preformed liposomes under similar conditions. (C) Encapsulation of a poorly soluble drug into an ionizable cyclodextrin (R = H, ionizable alkyl or aryl groups) enhances its water solubility and permits efficient liposomal loading via a pH gradient.Only a small fraction of chemotherapeutic agents possesses the features required for active loading with established techniques. Attempts at active loading of such nonionizable drugs into preformed liposomes result in poor loading efficiencies (Fig. 1B). One potential solution to this problem is the addition of weakly basic functional groups to otherwise unloadable drugs, an addition that would provide the charge necessary to drive these drugs across the pH gradient. However, covalent modification of drugs often alters their biological and chemical properties, and is not desirable in many circumstances. We therefore attempted to develop a general strategy that would allow loading of unmodified hydrophobic chemotherapeutics devoid of weakly basic functional groups. For this purpose, we used modified β-cyclodextrins to facilitate the loading of such drugs into liposomes (Fig. 1C).Cyclodextrins are a family of cyclic sugars that are commonly used to solubilize hydrophobic drugs (29–32). They possess a hydrophobic cavity and a hydrophilic surface and are known to stably encapsulate a large variety of hydrophobic organic molecules in aqueous media. Cyclodextrins bind to their cargos strongly enough to form relatively stable complexes, but allow a slow efflux of the entrapped drug and consequent steady concentrations of free drug once administered in vivo (33, 34). Cyclodextrins are nontoxic and biologically inert, and several have been approved for use in humans (35–38).In this work, we synthesized cyclodextrins with multiple weakly basic or weakly acidic functional groups on their solvent-exposed surfaces. We hypothesized that these modified cyclodextrins would still be able to encapsulate chemotherapeutic agents and that the cyclodextrin–drug complexes could then be remotely loaded into liposomes using pH gradients that exploited ionizable groups on the cyclodextrins rather than on the drugs themselves.