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.     

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.     

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.     

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.     

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).     

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.     

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.     

Thermodynamic origins of protein folding,allostery, and capsid formation in the human hepatitis B virus core protein

HBc, the capsid-forming “core protein” of human hepatitis B virus (HBV), is a multidomain, α-helical homodimer that aggressively forms human HBV capsids. Structural plasticity has been proposed to be important to the myriad functions HBc mediates during viral replication. Here, we report detailed thermodynamic analyses of the folding of the dimeric HBc protomer under conditions that prevented capsid formation. Central to our success was the use of ion mobility spectrometry–mass spectrometry and microscale thermophoresis, which allowed folding mechanisms to be characterized using just micrograms of protein. HBc folds in a three-state transition with a stable, dimeric, α-helical intermediate. Extensive protein engineering showed thermodynamic linkage between different structural domains. Unusual effects associated with mutating some residues suggest structural strain, arising from frustrated contacts, is present in the native dimer. We found evidence of structural gatekeepers that, when mutated, alleviated native strain and prevented (or significantly attenuated) capsid formation by tuning the population of alternative native conformations. This strain is likely an evolved feature that helps HBc access the different structures associated with its diverse essential functions. The subtle balance between native and strained contacts may provide the means to tune conformational properties of HBc by molecular interactions or mutations, thereby conferring allosteric regulation of structure and function. The ability to trap HBc conformers thermodynamically by mutation, and thereby ablate HBV capsid formation, provides proof of principle for designing antivirals that elicit similar effects.The “protein-folding problem” describes how a polypeptide sequence contains all the information needed for it to adopt a specific 3D structure spontaneously (1). The chemistry and thermodynamic code that causes proteins to fold also underpins protein–protein interactions, allostery, and supramolecular assembly. An emerging trend has been the study of model proteins free from kinetic traps, aggregation, or metal binding, features that can confound experimental execution and data interpretation (2, 3). Consequently, model proteins are small (typically <130 residues), soluble monomers with few proline or cysteine residues and no prosthetic groups (2, 3).Although model proteins have been instrumental in taking the field to its current zenith, there is a paucity of experimental insights into the conformational dynamics of larger, oligomeric proteins, especially those implicated in diseases (3). Such proteins usually have complex behavior refractory to detailed experimental studies. However, the connection between sequence, structure, dynamics, and allostery makes studies of larger proteins central to understanding biological function and aiding drug design (vide infra) (4). One such protein is HBc, the capsid-forming “core protein” of human hepatitis B virus (HBV), a major pathogen that kills 600,000 people annually (5). Although excellent vaccines exist, there are no effective cures for extant chronic infections (5, 6). In addition to capsid formation, HBc plays many essential roles in HBV replication (7–9), making it an attractive drug target (10–15).WT HBc is a 183-residue polypeptide comprising a structured capsid-forming region (residues 1–149; Fig. 1A) and a basic, nucleic acid-binding domain (residues 150–183) (16–18). The structured N-terminal region (hereafter HBc_1–149) spontaneously self-assembles in vitro and in vivo to form icosahedral capsid-like particles (CLPs) identical to nucleocapsids isolated from patient serum (19, 20). X-ray crystallography and cryo-EM have characterized the structure of HBc_1–149 within the context of CLPs, virions, and hexamers (16, 19–23). HBc homodimers comprise two structural domains (Fig. 1A): Helices α₃ and α₄ from opposing monomers pack together and form a disulfide-linked, four-helix bundle dimerization interface (visible as protrusions on the capsid exterior; Fig. 1B), whereas α₁, α₂, and α₅ pack together and around the base of the four-helix bundle to create the hydrophobic core of “contact” domains (19). Weak interdimer interactions between contact domains stabilize HBV capsids (19, 24) (Fig. 1B).Open in a separate windowFig. 1.HBc_1–149 dimer structure within HBV capsids. (A) Four-helix bundle dimerization interface (black) is flanked by contact domains (orange and red). Helices are numbered, and the N and C termini of one monomer are indicated. The disulfide link between C61 of each monomer is indicated (cyan). (B) Exterior surface of a T = 4 capsid HBc_1–149 (PDB ID code 1QGT) (19). Dimers around the threefold and fivefold axes are indicated in blue/green and purple/orange, respectively. (Inset) Interacting quasiequivalent HBc_1–149 dimers from the fivefold (purple and orange) and threefold (blue and green) axes are shown. Hydrophobic contacts between contact domains stabilize capsids. Residues that perturb capsid formation when mutated are indicated.Multiple studies show clearly that HBc has a very malleable structure, with this structural plasticity argued to be functionally important (22, 23). This hypothesis accords well with antivirals that modulate HBc structure (11–15, 22, 23). Studies of HBV capsid assembly have inferred the existence of assembly-active (HBc^Ass) and assembly-incompetent (HBc^Inc) HBc conformations (12, 13, 21, 24, 25). However, there are few detailed insights on the thermodynamic origins of structure, allostery, and dynamics for the dimeric HBc_1–149 protomer, where structural plasticity must originate. This arises from dimeric HBc_1–149 being very challenging to study in vitro (compared with the model proteins described above) because it is a 298-residue disulfide-linked homodimer (containing 6 cysteine and 24 proline residues) that aggregates aggressively and forms capsids.Here, we report detailed folding and stability studies of dimeric HBc_1–149. These show HBc_1–149 folds in a three-state transition with a populated, dimeric, α-helical intermediate. Of 29 “chemically conservative” mutants used to probe folding energetics (26), many had similar effects on the stability of the intermediate and native ensembles. The distribution of these mutations was consistent with the intermediate being stabilized by a significant native-like structure. However, some mutations destabilized the native state (N) much less than the intermediate state (I) relative to the denatured state (D), or significantly increased the free energy of unfolding (ΔG_D–N) relative to WT HBc_1–149. This suggests HBc_1–149 contains structural strain arising from frustrated contacts (27, 28). We found evidence of HBc_1–149 adopting multiple native conformers, where capsid assembly-competent conformers were less stable than those incapable of, or attenuated in, capsid formation. Frustrated regions likely contain structural gatekeepers that (28), when mutated, subtly tuned the folding energy landscape and altered capsid assembly. The presence of multiple native conformations and frustrated regions may explain the origins of allostery reported for HBc. Frustration is likely an evolved tradeoff that balances the conflicting requirements of HBc folding with allosteric regulation of native structure, capsid formation, and diverse functions of different conformers (29). The ability to trap HBc conformers thermodynamically by mutation and ablate capsid formation provides a proof of principle for designing antivirals that elicit similar effects.     

From the Cover: Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into “build–couple–pair” combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.Encompassing one of the largest classes of structurally diverse small molecule natural products, polyketides have provided multiple clinically useful drug classes that save lives (1–3). Natural polyketides from diverse microorganisms also deliver novel scaffolds that can be exploited in drug discovery programs by semisynthetic modification and combined chemical and biosynthetic approaches (4, 5) and as inspiration for total synthesis and combinatorial chemistry (6, 7). A complementary approach is combinatorial biosynthesis, which strives to reengineer biosynthetic pathways to generate novel polyketide scaffolds by one-pot, one-step synthesis via fermentation of recombinant microorganisms.Among fungal polyketides, benzenediol lactones (BDLs) offer rich pharmacophores with an extraordinary range of biological activities (8). They are defined by a 1,3-benzenediol moiety bridged by a macrocyclic lactone ring (9). Among BDLs, resorcylic acid lactones (RALs) display a C2–C7 connectivity, whereas dihydroxyphenylacetic acid lactones (DALs) feature a C3–C8 bond. Monocillin II (1; Fig. 1) and their congeners (radicicol and the pochonins) are RALs with 14-membered rings (RAL₁₄) that are specific inhibitors of heat shock protein 90 (Hsp90) (9, 10). Inhibition of Hsp90 promotes the degradation of oncogenic client proteins and leads to the combinatorial blockade of multiple cancer-causing pathways. Resorcylides and lasiodiplodins are RALs with 12-membered macrocycles (RAL₁₂): these phytotoxins display mineralocorticoid receptor antagonist and prostaglandin biosynthesis inhibitory activities in animals. 10,11-dehydrocurvularin (7; Fig. 1) is a DAL with a 12-membered ring (DAL₁₂) that modulates the heat shock response and the immune system (8, 9).Open in a separate windowFig. 1.Biosynthesis of natural benzenediol lactones. Biosynthetic assembly of monocillin II, trans-resorcylide, desmethyl-lasiodiplodin, and 10,11-dehydrocurvularin (the “on-program” main metabolites) in recombinant S. cerevisiae BJ5464-NpgA (24, 40) strains by native BDLSs (12, 14, 25). R indicates the hrPKS-generated priming unit that is elaborated by the nrPKS. Numerals in the colored spheres indicate the number of malonate-derived C₂ units (C−C bonds shown in bold) incorporated into the polyketide chain by the hrPKS vs. the nrPKS (“division of labor” or “split” by the BDLS: e.g., 5+4 indicates a pentaketide extended by four more malonate units). Stutter products are minor metabolites produced by extra extension cycles resulting in irregular “splits” (25).BDL scaffolds are biosynthesized by pairs of collaborating, sequentially acting iterative polyketide synthases (iPKSs) (3) forming quasi-modular BDL synthases (BDLSs) (Fig. 1) (11–14). Each of the BDLS subunits catalyze recursive, decarboxylative Claisen condensations of malonyl-CoA using a single core set of ketoacyl synthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains. BDL assembly initiates on a highly reducing iPKS (hrPKS) that produces a short chain carboxylic acid priming unit for a second, nonreducing iPKS (nrPKS). The length of the priming unit is set by the KS domain of the hrPKS, whereas the distinctive redox pattern and the configuration at each stereocenter is determined by the ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) domains that reduce the nascent β-keto groups after each condensation step according to a cryptic biosynthetic program (3). A direct handover of the priming unit from the hrPKS is catalyzed by the starter unit:ACP transacylase (SAT) domain of the nrPKS (15). After a set number of further elongation cycles without reduction, the highly reactive polyketide intermediate is guided by the product template (PT) domain of the nrPKS toward a programmed, regiospecific first ring closure (16). This aldol condensation yields a resorcylic acid moiety in the C2–C7 register or a dihydroxyphenylacetic acid group in the C8–C3 register (16–18). The last step of BDL scaffold biosynthesis is the release of the product, catalyzed by an O–C bond-forming thioesterase (TE) domain of the nrPKS. TE domains form the BDL macrolactone using the ω-1 alcohol as a nucleophile, but may use alternative nucleophiles such as the C9 enol to yield α-pyrones or water or alcohols from the media to form acyl resorcylic acids (ARAs), acyl dihydroxyphenylacetic acids (ADAs), and their esters (18–22). iPKSs that produce BDLs in the RAL₁₄, RAL₁₂, and DAL₁₂ subclasses have been characterized and reconstituted both in vivo by heterologous expression in yeast and in vitro using isolated recombinant iPKS enzymes (11–14, 23–25). Domain exchanges among different BDLSs were used to decipher some of the programming rules of these enzymes and yielded a limited number of structurally diverse unnatural products (3, 18, 20, 21, 26, 27).Combinatorial biosynthesis of polyketides is still in its infancy. Mix and match combinations of small, discrete type II PKSs from bacteria have yielded some novel scaffolds, but the outcome of the reactions proved difficult to predict and conceptualize. Engineering targeted changes in modular type I PKSs of prokaryotes generated small, focused libraries of conservative variants of selected bioactive scaffolds. However, these pioneering works with prokaryotic PKSs also yielded many unproductive combinations, suffered from greatly reduced product yields, and realized only a small fraction of the potential chemical space (28–31).Large-scale combinatorial biosynthesis with fungal iPKSs has not yet been described. Nevertheless, fungal BDLs provide a unique opportunity for diversity-oriented combinatorial biosynthesis. This is because BDLSs from different organisms are orthologous enzymes that are nonetheless programmed to generate nonequivalent, structurally complex macrocyclic products using malonyl-CoA as their sole, shared precursor. In this work, we considered whether BDL biosynthesis may be refactored to a modular, parallel synthetic scheme in which individually programmed biosynthons are freely coupled and released after intramolecular cyclization, in analogy to a build–couple–pair strategy in combinatorial chemistry (7, 32). Further, we explored whether BDLS subunit heterocombinations also reveal differences in iPKS promiscuity and plasticity.     

Aptamer photoregulation in vivo

The in vivo application of aptamers as therapeutics could be improved by enhancing target-specific accumulation while minimizing off-target uptake. We designed a light-triggered system that permits spatiotemporal regulation of aptamer activity in vitro and in vivo. Cell binding by the aptamer was prevented by hybridizing the aptamer to a photo-labile complementary oligonucleotide. Upon irradiation at the tumor site, the aptamer was liberated, leading to prolonged intratumoral retention. The relative distribution of the aptamer to the liver and kidney was also significantly decreased, compared to that of the free aptamer.Aptamers are single-stranded nucleic acids that have emerged as a promising class of therapeutics owing to their relative ease of synthesis and high affinity and selectivity toward a range of targets including small molecules, proteins, viral particles, and living cells (1–6). Aptamers can fold into well-defined conformations and are more resistant to enzymatic degradation than other oligonucleotides (7–9). Aptamers have been suggested for imaging applications because their relatively small size and molecular mass (∼10 kDa) allow fast tissue penetration and clearance from blood (10, 11). The same characteristics make aptamers promising for effective delivery of diagnostic and therapeutic agents to tissues or organs. However, nonspecific accumulation of aptamers in normal tissues is undesirable (12–15) because it diminishes the proportion of aptamer that targets the desired tissue. This can adversely affect the therapeutic index of the aptamer; this may be particularly true if the aptamer is conjugated to a drug or drug delivery device. Moreover, aptamers themselves can have nonspecific toxic effects (16, 17). Ideally aptamers would achieve a high concentration in a pathological tissue of interest while maintaining low levels elsewhere. The activity of aptamers can be modulated in vivo by binding to polymers or complementary oligonucleotide sequences (18–20), but spatiotemporal regulation of aptamer activity in vivo has not been achieved, whereby activity would be enhanced in target tissues and not others. Here, we report a strategy to provide light-triggered control of aptamer function and distribution in vivo.Light is an excellent means of providing external spatiotemporal control of biological systems (21–26). Many strategies have been developed to incorporate photosensitive groups in nucleotides that can control cellular function or affect biological pathways or gene expression by light (24–29). Of particular interest, such approaches can be used to provide spatiotemporal control of gene activation (24). Here we hypothesized that light triggering can be used to achieve spatiotemporal control of binding of an aptamer injected systemically to its target tissue in vivo, which would have implications for control of delivery of therapeutic aptamers and/or conjugated drugs or drug delivery systems. We designed a photo-triggerable system whereby the aptamer of interest is inactivated by hybridization to a photo-labile complementary oligonucleotide. Upon irradiation, the complementary sequence breaks down, releasing the functional aptamer (Fig. 1). The aptamer of interest is the single-stranded DNA 26-mer aptamer AS1411 (A₁₄₁₁; sequence: 5′-GGT GGT GGT GGT TGT GGT GGT GGT GG-3′) that binds with high affinity and selectivity to nucleolin (30–32), which is overexpressed on the cell membrane of several types of cancer cells, including the 4T1 breast cancer cells used here (33–35). A₁₄₁₁ has been used for cancer targeting in vitro and in vivo (35, 36). A complementary photo-triggerable inhibitory oligonucleotide (OliP) was designed [sequence: 5′-CCA CCA//CCA CCA//CAA CCA C-3′, where // indicates photo-labile 1-(2-nitrophenyl)ethyl bonds (37); Scheme S1].Open in a separate windowFig. 1.The AS1411 aptamer (A₁₄₁₁, red DNA strand) is hybridized to a complementary oligonucleotide (OliP, green DNA strand) containing photo-cleavable bonds (black dots). The hybridized complex cannot bind cells. The A₁₄₁₁ can be released by light-triggered breakage of the OliP, allowing binding to cell surfaces.     

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.     

Excess cholesterol induces mouse egg activation and may cause female infertility

The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-β-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.Scavenger receptor, class B type I (SR-BI) is an HDL receptor that transports unesterified cholesterol (UC) and its esters between lipoproteins and cells (1–3) and functions as a signaling receptor (4). SR-BI controls the structure and composition of plasma HDL and the amounts and fates of HDL cholesterol (2, 3, 5, 6). Homozygous null SR-BI knockout (KO) mice exhibit hypercholesterolemia with unusually large and UC-enriched HDL particles [abnormally high UC to total cholesterol (TC) ratio] (5, 7, 8). This unusual hypercholesterolemia apparently induces a deleterious accumulation of UC in RBCs and platelets that influences their maturation, lifetime in the bloodstream, structure, and/or function (7–13).Female but not male SR-BI KO mice exhibit fully penetrant infertility, despite their essentially normal ovarian histology (6, 14). Several lines of surgical transplantation, genetic, histologic, and pharmacologic evidence indicate that the abnormal structure and composition of circulating HDL in SR-BI KO mice contribute to their female infertility (Discussion). This infertility can be effectively corrected by altering (through a variety of approaches) the structure and abundance of the circulating HDL to which the ovaries are exposed (7, 14, 15). This evidence also suggests that the infertility is likely caused by defects in oocytes/eggs manifested during the periovulatory period without affecting the primordial follicle pool. Indeed, ∼19% of the ovulated eggs harvested ∼16 h after hormone-induced superovulation are dead (6, 14) (see below). The remaining oocytes are unable to be fertilized or develop into viable pups. Here, we examined SR-BI KO oocyte growth and progression through the peri- and postovulatory stages of meiosis (Fig. 1) and investigated the possibility that high levels of UC in HDL might result in excess cholesterol deposition in oocytes or eggs that might influence their growth, meiotic progression, and/or viability.Open in a separate windowFig. 1.Meiotic resumption and egg activation in the mouse. Schematic representation of meiotic resumption and egg activation by either fertilization or chemical agents. The stages at which primary (prophase I) and secondary (MII) arrest occur are indicated. Oocytes initiate meiosis during fetal development and enter primary arrest at the diplotene stage of prophase I around the time of birth (not illustrated here). Prophase I arrest is maintained during oocyte growth and differentiation. Prior to ovulation, meiosis resumes, the nuclear envelope of the germinal vesicle (GV) dissassembles [GV breakdown (GVBD)], and chromosomes condense and align on the first metaphase plate. During the first division, homologous chromosomes segregate and the first polar body (PB) is extruded (first PB in anaphase/telophase I). The remaining chromosomes realign on the second metaphase plate (MII) followed by secondary arrest. During MII arrest, the first PB often degrades. Eggs that are ovulated can exit MII arrest and complete meiosis when activated by a fertilizing sperm (upper right) or chemical agents, such as SrCl₂ or ethanol (lower right), which induce a spike (ethanol) or oscillations (sperm and SrCl₂) in cytoplasmic Ca²⁺ levels and suppress MAPK and MPF activities. On activation, sister chromatids segregate, and the second PB is extruded (second PB in anaphase/telophase II). The activated egg then progresses to pronuclear stage as the nuclear envelope reforms. On chemical activation, the egg can alternatively progress to metaphase III, segregate the remaining chromatids randomly during anaphase III, and extrude a third PB (anaphase/telophase III).Normally, after the luteinizing hormone surge of the estrous cycle and just before ovulation, mammalian oocytes complete the first meiotic division [meiosis I (MI)] and immediately thereafter proceed to metaphase II (MII), a stage at which they arrest (Fig. 1). A complex network of proteins and intracellular signals establishes and maintains MII arrest [e.g., elevated MAPK and maturation promoting factor (MPF; or cdk1/cyclin B) kinase activities] (16, 17) and subsequently permits exit from this stage after fertilization (e.g., reduction in MAPK and MPF activities) (16, 17). Productive fertilization of MII-arrested eggs leads to inositol triphosphate-induced oscillations in intracellular calcium concentration ([Ca²⁺]_i) that activate the eggs [exit from MII arrest, second polar body (PB) extrusion, formation of pronuclei, etc.] (Fig. 1) (18, 19). Fertilization-induced changes in intracellular zinc levels also may influence activation (19). The parthenogenetic stimulant SrCl₂ activates normal eggs (20) by inducing oscillations in [Ca²⁺]_i (21, 22) and suppressing MAPK and MPF kinase activities (23) that mimic those that occur after fertilization (21, 22). Brief exposure to ethanol induces a single spike in [Ca²⁺]_i and activates eggs (24, 25). Unlike fertilized eggs, which receive the paternal chromosome complement from the sperm, SrCl₂- or ethanol-activated eggs are haploid (Fig. 1) and cannot support proper embryogenesis (22).Here, we found that, within follicles of hormone-treated SR-BI KO females, oocytes apparently resumed meiosis normally. However, these cells spontaneously escaped MII arrest, exhibited reduced MPF and MAPK activities, and aberrantly progressed to haploid pronuclear, metaphase III, and anaphase/telophase III stages, similar to normal ovulated eggs after chemical (e.g., SrCl₂) (22, 25) activation (Fig. 1). Compared with control eggs, the eggs of SR-BI KO females accumulated excess cholesterol. Strikingly, we could recapitulate the apparently spontaneous activation of ovulated SR-BI KO eggs in eggs ovulated from fertile control mice (WT or SR-BI^+/−) by treating those eggs with a cholesterol/methyl-β-cyclodextrin (MβCD) complex to load them with excess cholesterol (26). In addition, cholesterol loading induced a single spike in [Ca²⁺]_i reminiscent of that seen in ethanol-activated eggs (24, 25). These results establish that excess cholesterol loading provides a new approach to activating mammalian MII-arrested eggs and suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol acting directly on the egg to induce premature activation. Analysis of SR-BI KO female infertility also raises the possibility that abnormalities in cholesterol metabolism might underlie some forms of human female infertility of unknown etiology.