Processing and classification of chemical data inspired by insect olfaction

The chemical sense of insects has evolved to encode and classify odorants. Thus, the neural circuits in their olfactory system are likely to implement an efficient method for coding, processing, and classifying chemical information. Here, we describe a computational method to process molecular representations and classify molecules. The three-step approach mimics neurocomputational principles observed in olfactory systems. In the first step, the original stimulus space is sampled by "virtual receptors," which are chemotopically arranged by a self-organizing map. In the second step, the signals from the virtual receptors are decorrelated via correlation-based lateral inhibition. Finally, in the third step, olfactory scent perception is modeled by a machine learning classifier. We found that signal decorrelation during the second stage significantly increases the accuracy of odorant classification. Moreover, our results suggest that the proposed signal transform is capable of dimensionality reduction and is more robust against overdetermined representations than principal component scores. Our olfaction-inspired method was successfully applied to predicting bioactivities of pharmaceutically active compounds with high accuracy. It represents a way to efficiently connect chemical structure with biological activity space.     

Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation

The TIM22 protein import pathway mediates the import of membrane proteins into the mitochondrial inner membrane and consists of two intermembrane space chaperone complexes, the Tim9-Tim10 and Tim8-Tim13 complexes. To facilitate mechanistic studies, we developed a chemical-genetic approach to identify small molecule agonists that caused lethality to a tim10-1 yeast mutant at the permissive temperature. One molecule, MitoBloCK-1, attenuated the import of the carrier proteins including the ADP/ATP and phosphate carriers, but not proteins that used the TIM23 or the Mia40/Erv1 translocation pathways. MitoBloCK-1 impeded binding of the Tim9-Tim10 complex to the substrate during an early stage of translocation, when the substrate was crossing the outer membrane. As a probe to determine the substrate specificity of the small Tim proteins, MitoBloCK-1 impaired the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex mediates the import of a subset of inner membrane proteins. MitoBloCK-1 also inhibited growth of mammalian cells and import of the ADP/ATP carrier, but not TIM23 substrates, confirming that MitoBloCK-1 can be used to understand mammalian mitochondrial import and dysfunction linked to inherited human disease. Our approach of screening chemical libraries for compounds causing synthetic genetic lethality to identify inhibitors of mitochondrial protein translocation in yeast validates the generation of new probes to facilitate mechanistic studies in yeast and mammalian mitochondria.     

Visualizing cellular interactions with a generalized proximity reporter

Interactions among neighboring cells underpin many physiological processes ranging from early development to immune responses. When these interactions do not function properly, numerous pathologies, including infection and cancer, can result. Molecular imaging technologies, especially optical imaging, are uniquely suited to illuminate complex cellular interactions within the context of living tissues in the body. However, no tools yet exist that allow the detection of microscopic events, such as two cells coming into close proximity, on a global, whole-animal scale. We report here a broadly applicable, longitudinal strategy for probing interactions among cells in living subjects. This approach relies on the generation of bioluminescent light when two distinct cell populations come into close proximity, with the intensity of the optical signal correlating with relative cellular location. We demonstrate the ability of this reporter strategy to gauge cell–cell proximity in culture models in vitro and then evaluate this approach for imaging tumor–immune cell interactions using a murine breast cancer model. In these studies, our imaging strategy enabled the facile visualization of features that are otherwise difficult to observe with conventional imaging techniques, including detection of micrometastatic lesions and potential sites of tumor immunosurveillance. This proximity reporter will facilitate probing of numerous types of cell–cell interactions and will stimulate the development of similar techniques to detect rare events and pathological processes in live animals.     

Superoxide dismutase 1 (SOD1) is a target for a small molecule identified in a screen for inhibitors of the growth of lung adenocarcinoma cell lines

We previously described four small molecules that reduced the growth of lung adenocarcinoma cell lines with either epidermal growth factor receptor (EGFR) or KRAS mutations in a high-throughout chemical screen. By combining affinity proteomics and gene expression analysis, we now propose superoxide dismutase 1 (SOD1) as the most likely target of one of these small molecules, referred to as lung cancer screen 1 (LCS-1). siRNAs against SOD1 slowed the growth of LCS-1 sensitive cell lines; conversely, expression of a SOD1 cDNA increased proliferation of H358 cells and reduced sensitivity of these cells to LCS-1. In addition, SOD1 enzymatic activity was inhibited in vitro by LCS-1 and two closely related analogs. These results suggest that SOD1 is an LCS-1-binding protein that may act in concert with mutant proteins, such as EGFR and KRAS, to promote cell growth, providing a therapeutic target for compounds like LCS-1.     

Switchable assembly and function of antibody complexes in vivo using a small molecule

The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody–drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody’s intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine’s half-life, and activation of bispecific, T cell–engaging antibodies.

Antibodies are multidomain proteins that have become an important therapeutic platform for a wide range of diseases (1). The key feature of antibodies that enables broad therapeutic application is their ability to couple selective molecular targeting to a functional output in a single molecule with long serum half-life. In the example of natural antibodies, targeting of pathogen-associated antigens is coupled to functions that facilitate an immune response, such as antibody-dependent cellular cytotoxicity via the recruitment of natural killer cells. In the case of antibody-based therapeutics, targeting of disease-specific antigens can be linked in a modular way to a myriad of desired natural or synthetic effector domains. These can include radionuclides, cytotoxic payloads, immune cell engagers, cytokines, and engineered cells (2–6).Historically, genetic and chemical techniques have been used to generate synthetic combinations of multiple domains that collectively impart both targeting and function into a single therapeutic molecule. This is exemplified by the wide array of antibody and protein fusion formats with unique specificities and therapeutic mechanisms that have entered the clinic to date (3–5, 7, 8). Despite the diversity of formats, the functional properties of these synthetic proteins are intrinsically defined by their chemical structures (Fig. 1A). Thus, once an antibody-based drug is infused into a patient, the clinician relinquishes any control over its activity, potentially for a period of weeks. The long half-lives can make it challenging to manage toxicities or to rapidly adjust drug activity in response to efficacy or pharmacodynamic biomarkers.Open in a separate windowFig. 1.LITE enables antibody complexes with switchable assembly and activity. (A) Most biologic drugs use a targeting domain to localize a functional, therapeutic moiety, but these molecular components are inextricably linked. (B) LITE is a technology to enable switchable assembly of individual antibody components into an active complex.Several classes of antibody-based drugs could benefit from a method of rapidly controlling drug activity and exposure. One such class is therapeutically active proteins that have been fused to the Fc-region of human IgG, which imparts a longer serum half-life (5, 8). The Fc fusion strategy has extended the half-lives of diverse proteins, including cytokine receptors (e.g., etanercept and aflibercept), hormones (e.g., dulaglutide), and cytokine mimetics (e.g., romiplostim) but at the risk of prolonging their potentially toxic activity for weeks after injection. Another class of antibody-based drugs, bispecific T cell–engaging antibodies (bsTCEs), can potently redirect the immune system to attack cancer cells but are also associated with unmanageable toxicities that have been observed in early clinical trials (4, 7, 9). First-generation bsTCEs had short half-lives (<5 h) that allowed for rapid termination of treatment in response to toxicities but necessitated the use of burdensome continuous infusion pumps. Extended half-life bsTCEs were explored as a way to achieve more convenient dosing, but physicians lack a mechanism to quickly terminate activity in response to adverse events. Thus, efforts to increase the half-life of this powerful modality must be paired with approaches for managing toxicity. An ideal solution to address these challenges would combine the long half-life advantages of biologic drugs with the precise temporal control of activity associated with small molecules. An antibody-based drug with these features would allow for convenient dosing but also enable a clinician to quickly respond to a patient''s needs by increasing drug activity or reversing toxicity.Here, we demonstrate a general approach for ligand-induced transient engagement (LITE) of multiple antibody domains, whereby chemically induced dimerization is applied to enable switchable antibody activity by modulating the dose of an Food and Drug Administration (FDA)-approved small molecule (Fig. 1B). We provide three examples demonstrating the broad utility of this approach. First, we show the induced association of a tumor-targeting domain to reversibly control the biodistribution and tumor localization of an antibody in vivo. Second, we demonstrate that the inducible transient engagement of a therapeutically relevant cytokine to an Fc domain dramatically increases its half-life in vivo. Finally, we show small-molecule–regulated formation of a functional, bispecific T cell engager complex capable of redirecting T cells to kill tumor cells in vitro and in vivo. In summary, the LITE platform enables a new class of biologic drugs with functions that can be precisely switched on and off after intravenous (i.v.) administration.     

Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.

The radical S-adenosylmethionine (rSAM) enzyme superfamily is the largest known in nature, with over 570,000 annotated and predominantly uncharacterized members spanning all domains of life (1–4). The uniting feature of rSAM enzymes is a [4Fe-4S] cluster, usually bound by a CX₃CX₂C motif that catalyzes reductive cleavage of SAM to form L-Met and a strongly oxidizing 5′-deoxyadenosyl radical (5′-dA•) (5–7). Recent studies on a suite of rSAM enzymes have revealed the presence of a previously unknown organometallic intermediate in this process, termed Ω, in which the 5′-C of 5′-dA• is bound to the unique iron of the [4Fe-4S] cluster (Fig. 1A) (8, 9). Homolysis of the Fe–C bond ultimately liberates 5′-dA•, which abstracts a hydrogen atom from substrate to initiate a profoundly diverse set of chemical reactions in both primary and secondary metabolism, including DNA, cofactor, vitamin, and antibiotic biosynthesis (5, 10–13).Open in a separate windowFig. 1.(A) Accepted scheme for radical initiation in rSAM enzymes. (B) X-ray crystal structure of SuiB (PDB ID: 5V1T). The RS domain, SPASM domain, and RiPP recognition element are rendered blue, green, and pink, respectively. [4Fe-4S] clusters are shown as spheres with the distances separating them indicated. (C) Lys–Trp cross-link formation (20) catalyzed by SuiB. The carbon–carbon bond installed by SuiB is shown in red. (D and E) Previously proposed EAS (D) and rEAS (E) mechanisms for SuiB-catalyzed Lys–Trp cross-link formation.Of the 570,000 rSAM enzyme superfamily members, over a quarter (∼165,000 genes from the Enzyme Function Initiative-Enzyme Similarity Tool) possess C-terminal extensions, called SPASM and twitch domains, which bind auxiliary Fe-S clusters (4, 14–19). The SPASM domain typically binds two auxiliary Fe-S clusters and is named after the rSAM enzymes involved in the synthesis of subtilosin, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin. The twitch domain is a truncated SPASM domain and only binds one auxiliary cluster (15). Despite the wide prevalence of these domains and the characterization of several different SPASM/twitch rSAM enzymes by spectroscopic and structural studies, direct evidence for their catalytic function(s) has remained elusive.We previously performed functional and structural characterization on the SPASM rSAM enzyme SuiB (Fig. 1B), which is involved in the biosynthesis of a ribosomal peptide natural product in human and mammalian microbiome streptococci (14, 20–22). SuiB introduces an unusual carbon–carbon bond onto its substrate peptide, SuiA, between the sidechains of Lys2 and Trp6 (Fig. 1C). The mechanism for this transformation is of broader relevance, as a number of enzymes, such as RrrB, PqqE, and MqnC (2, 23, 24), are known to join unactivated aliphatic and aromatic carbons to generate sp³-sp² cross-links. A general mechanistic paradigm for this class of transformations is not yet available. For SuiB, two pathways have been proposed (20), one through a typical electrophilic aromatic substitution (EAS) mechanism, which is involved in other enzyme-catalyzed indole modifications, such as indole prenylation or flavin adenine dinucleotide (FAD)-enzyme-dependent indole chlorination (25–27). In this pathway, the 5′-dA• generates an alkyl radical, which upon a second one-electron oxidation, creates an α,β-unsaturated amide electrophile with which the indole sidechain reacts via Michael addition (Fig. 1D). Lanthionine cross-links observed in diverse lanthipeptides are built by this general scheme, though via heterolytic chemistry, with Cys acting as the nucleophile (28, 29). Alternatively, a radical electrophilic aromatic substitution (rEAS) reaction has been proposed, wherein the alkyl radical, formed by 5′-dA•, would react with the indole sidechain to generate a radical σ complex, a cross-linked Lys–Trp radical (Lys–Trp•), which upon oxidation and rearomatization would yield product (Fig. 1E). In both mechanisms, AuxI is proposed as an oxidant. Although this role for an rSAM auxiliary cluster has been previously suggested (30, 31), it has yet to be directly demonstrated experimentally. Mechanistic studies have favored the rEAS pathway (20); however, intermediates in the reaction of SuiB and enzymes that catalyze similar reactions have not yet been detected (15).In the current work, we sought to differentiate between the proposed mechanisms by trapping intermediates in the catalytic cycle of SuiB and characterizing them using electron paramagnetic resonance (EPR) spectroscopy. We report observation of three transient reaction intermediates, most importantly the sought-after Lys–Trp•, which is fundamentally different from previously characterized Trp radicals, as it is cross-linked and carries an indole tetrahedral center. We also provide evidence for AuxI as the oxidant of the Lys–Trp• intermediate as well as insights into redox potential changes of Fe-S clusters in SuiB that accompany SuiA binding. Together, our findings support the rEAS pathway for formation of the sp³-sp² cross-link and carry important implications for other enzymes that catalyze related transformations.     

Structural properties of Abeta protofibrils stabilized by a small molecule

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of Abeta protofibrils. Library screening yielded several molecules that stimulate Abeta aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts Abeta(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37 degrees C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-Abeta aggregates exhibit a low thioflavin T response. Like Abeta fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-Abeta aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous Abeta fibril formation reaction: approximately 12 of the 39 Abeta(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the Abeta molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in Abeta conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester Abeta in a form and/or location that cannot engage the toxic pathway.     

PEP005, a selective small-molecule activator of protein kinase C, has potent antileukemic activity mediated via the delta isoform of PKC

Ingenol 3-angelate (PEP005) is a selective small molecule activator of protein kinase C (PKC) extracted from the plant Euphorbia peplus, whose sap has been used as a traditional medicine for the treatment of skin conditions including warts and cancer. We report here that PEP005 also has potent antileukemic effects, inducing apoptosis in myeloid leukemia cell lines and primary acute myeloid leukemia (AML) cells at nanomolar concentrations. Of importance, PEP005 did not induce apoptosis in normal CD34+ cord blood myeloblasts at up to 2-log concentrations higher than those required to induce cell death in primary AML cells. The effects of PEP005 were PKC dependent, and PEP005 efficacy correlated with expression of PKC-delta. The delta isoform of PKC plays a key role in apoptosis and is therefore a rational potential target for antileukemic therapies. Transfection of KG1a leukemia cells, which did not express PKC-delta or respond to PEP005, with enhanced green fluorescent protein (EGFP)-PKC-delta restored sensitivity to induction of apoptosis by PEP005. Our data therefore suggest that activation of PKC-delta provides a novel approach for treatment of acute myeloid leukemia and that screening for PKC-delta expression may identify patients for potential responsiveness to PEP005.     

Calculation of Cement Composition Using a New Model Compared to the Bogue Model

The major cement composition ratios of alite, belite, aluminate, and ferrite have been calculated with the Bogue models until now. However, a recent comprehensive analysis based on various experimental data has revealed that the chemical composition of alite, belite, aluminate, and ferrite implemented by the Bogue models are slightly different than the experimental data, where small amounts of Al₂O₃ and Fe₂O₃ existing in alite and belite can change the prediction of cement composition. Since the amounts of cement compound are very important factors in determining the properties of concrete, improvement in the calculation would give more precise prediction for application usages such as climate change adaptable cement and high durable concrete manufacturing. For this purpose, 20 new models are proposed by modifying chemical compositions of the cement compounds and verified with the 50 experimental data sets. From the verification, the most accurate models are identified. The calculation using new models exhibit an accuracy improvement of approximately 5% compared to the Bogue models. Their applicable range is also presented. The study results are discussed in detail in the paper.     

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation.Bioluminescence in insects is almost exclusively confined to a small subset of beetles, including click beetles (1), railroad worm beetle larvae (2), and perhaps the best known example, the firefly Photinus pyralis (3). However, all insects express long-chain fatty acyl-CoA synthetases (ACSLs) that share high homology to beetle luciferases and are hypothesized to be their evolutionary antecedents (4–6). These two classes of enzymes are both members of the adenylate-forming superfamily (7) and share the ability to make AMP esters of fatty acids as well as the ability to displace the AMP ester with CoASH (Fig. 1) (8). Beetle luciferases differ from other insect ACSLs in their ability to chemically generate light by adenylating and oxidizing d-luciferin, a small molecule naturally found in bioluminescent beetles. How this additional activity developed is unknown, although weak bioluminescence has been reported by treating a beetle ACSL with d-luciferin (6, 9).Open in a separate windowFig. 1.Firefly luciferase and long-chain fatty acyl-CoA synthetases catalyze similar two-step mechanisms. (A) Firefly luciferase catalyzes the formation of an activated AMP ester of its native substrate, d-luciferin. Subsequent oxidation within the luciferase binding pocket generates an excited-state oxyluciferin molecule that is responsible for light emission. (B) Long-chain fatty acyl-CoA synthetases catalyze the formation of activated AMP esters from long-chain fatty acids such as arachidonic acid. AMP is then displaced by CoASH to form the fatty acyl-CoA product.We have previously found that mutation of firefly luciferase can improve light emission from synthetic luciferin substrates while concurrently reducing light emission from d-luciferin (10). This suggested that the requirements for d-luciferin bioluminescence and for bioluminescence with synthetic luciferin substrates are not the same in mutant luciferases and, by extension, in luciferase homologs. Consequently, we reasoned that even though insect ACSLs from nonbioluminescent organisms outside the order of beetles fail to emit light with d-luciferin, this does not necessarily mean that they are incapable of luciferase activity. Clearly, the catalytic machinery needed to form AMP esters from carboxylic acids is present in ACSLs (Fig. 1), potentially allowing access to an adenylate of a luciferin analog. Furthermore, oxygen has ready access to ligand binding sites in proteins (11), and luciferin active esters in basic DMSO are known to be readily oxidized to generate an excited-state molecule that can emit light (12, 13). We therefore speculated that ACSLs lack luciferase activity with d-luciferin because d-luciferin is a poor ligand for ACSLs and/or binds in a geometry that is not conducive to adenylation. If this hypothesis were true, treatment with a suitable synthetic luciferin substrate that possessed higher affinity and/or conformational rigidity could potentially reveal latent luciferase activity in an ACSL.     

From the Cover: Metabolomics and proteomics reveal impacts of chemically mediated competition on marine plankton

Competition is a major force structuring marine planktonic communities. The release of compounds that inhibit competitors, a process known as allelopathy, may play a role in the maintenance of large blooms of the red-tide dinoflagellate Karenia brevis, which produces potent neurotoxins that negatively impact coastal marine ecosystems. K. brevis is variably allelopathic to multiple competitors, typically causing sublethal suppression of growth. We used metabolomic and proteomic analyses to investigate the role of chemically mediated ecological interactions between K. brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. The impact of K. brevis allelopathy on competitor physiology was reflected in the metabolomes and expressed proteomes of both diatoms, although the diatom that co-occurs with K. brevis blooms (A. glacialis) exhibited more robust metabolism in response to K. brevis. The observed partial resistance of A. glacialis to allelopathy may be a result of its frequent exposure to K. brevis blooms in the Gulf of Mexico. For the more sensitive diatom, T. pseudonana, which may not have had opportunity to evolve resistance to K. brevis, allelopathy disrupted energy metabolism and impeded cellular protection mechanisms including altered cell membrane components, inhibited osmoregulation, and increased oxidative stress. Allelopathic compounds appear to target multiple physiological pathways in sensitive competitors, demonstrating that chemical cues in the plankton have the potential to alter large-scale ecosystem processes including primary production and nutrient cycling.Marine phytoplankton are responsible for ∼50% of global net primary production (1), which ultimately drives the global carbon cycle (2). This primary production is vital for higher trophic levels with interactions among species playing a critical role in controlling the flux of biomass and nutrients in the water column (3). Chemical cues and signals mediate many interactions among planktonic organisms, including competition (4, 5), defense against grazers (6, 7), predator detection (8), prey capture (9), and signaling between neighbor cells during bloom events (10). Therefore, chemical cues that affect ecological interactions between microalgae are hypothesized to alter these large-scale ecosystem processes.Allelopathy, the production and release of chemical compounds to inhibit or kill competitor species, is a form of interference competition that alters community composition in terrestrial (11) and benthic aquatic communities (12, 13). In the plankton, allelopathy is noted as a strong structuring force that alters species succession (14, 15) and community composition (16, 17) by influencing a variety of species-specific physiological targets in competitors. Some allelopathic interactions result in massive mortality of competitors (18) or initiation of programmed cell death pathways (15). However, allelopathy is not always lethal as it can cause sublethal physiological responses in competitors, such as cyst formation (19), altered cell swimming behavior (5, 6), or reduced growth rate (e.g., refs. 20 and 21).The red-tide dinoflagellate Karenia brevis is known for a suite of potent neurotoxins, brevetoxins, responsible for neurotoxic shellfish poisoning in humans as well as fish and marine mammal mortalities during bloom events in the Gulf of Mexico (22). Although brevetoxins have been found to lack allelopathic potency in most studies thus far (4, 23–25), K. brevis produces an additional group of unstable, polar compounds (24) that negatively influence several competing phytoplankton species (4, 25). As a relatively weak exploitation competitor (26), K. brevis may use allelopathy to maintain monospecific blooms in near-shore waters (4). Competitor susceptibility to K. brevis allelopathy is at least partly mediated by ecological context and not all competitors are equally affected (4). The presence of particular competitor species modulates allelopathic potency (27), and competitors in earliest growth stages are most susceptible to K. brevis allelopathy (23). Allelopathic compounds produced by K. brevis cause sublethal reductions in growth and photosynthetic efficiency as well as increased cell membrane permeability (25), although the exact cellular metabolic targets of K. brevis allelopathy are unknown.Metabolomics and proteomics provide systems-level snapshots of the metabolism of a cell or organism at the time of harvest (28–30). Proteins are responsible for cellular signals, structural integrity, and catalysis of most biochemical reactions including the production and conversion of the vast array of metabolites required for cellular survival. Although metabolomics and proteomics have been used in the past to examine diatoms adapting to various stressors (31–34), the current work represents, to our knowledge, the first instance of metabolites and proteins measured simultaneously to understand the effects of allelopathy or any form of competition. To identify potential cellular targets of K. brevis allelopathy and better understand effects of sublethal chemical cues in marine ecosystems, we examined two diatom species exposed to K. brevis allelopathic compounds using whole-cell proteomics and metabolomics. This integrated systems biology approach provided multiple lines of evidence from which we distinguished metabolic responses of these two competitors to K. brevis allelopathy.     

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.Although the ubiquitin proteasome pathway plays essential roles in eukaryotes (reviewed in refs. 1 and 2), most bacterial species do not have proteasome systems and instead degrade proteins using ATP-dependent proteases like ClpP, Lon, and HslUV (reviewed in refs. 3 and 4). However, bacteria of the orders Actinomycetales and Nitrospirales also encode proteasomes that are structurally highly similar to eukaryotic and archaeal proteasomes (reviewed in refs. 5 and 6). Importantly, the human pathogen Mycobacterium tuberculosis (Mtb), an Actinomycete, requires proteasomal function to cause lethal infections in mice (7). Ablation of proteasomal degradation sensitizes bacteria to nitric oxide, an antimicrobial free radical made by macrophages and other cell types, and attenuates bacterial growth in mice (7–9). The potential to target persistent or latent bacteria has made the Mtb proteasome system a prioritized target for the development of antituberculosis drugs (10, 11). Indeed, Mtb-specific proteasome inhibitors have been identified that may provide a promising lead for new drugs to treat tuberculosis (12, 13).There are numerous similarities and differences between eukaryotic and bacterial proteasomes. The 20S proteasome core particle (20S CP), which consists of two seven-membered β-rings between two seven-membered α-rings, is highly conserved structurally between prokaryotes and eukaryotes (14–16). However, the accessory factors that associate with the 20S CPs quickly diverge among the domains of life. Both bacteria and eukaryotes use a covalent small protein modification to mark substrate proteins for degradation; however, the eukaryotic ubiquitin tag is a well-folded protein whereas the Mtb Pup (prokaryotic ubiquitin-like protein) tag is intrinsically disordered (17, 18). Furthermore, degradation of ubiquitylated proteins by eukaryotic 20S CPs largely relies on a complex regulatory particle that caps one or both ends of the 20S CP and includes a heterohexameric ring of adenosine triphosphatases (ATPases) for substrate recognition and unfolding (reviewed in refs. 19 and 20). In contrast, the mycobacterial 20S CP uses a homohexameric ATPase ring called Mpa (mycobacterial proteasome ATPase) for both the recognition and unfolding of pupylated proteins (18, 21, 22).In addition to the ATPase activators, proteolysis by eukaryotic proteasomes can also be stimulated by several ATP-independent factors, such as the 11S activators PA26 and PA28, as well as Blm10 (23–28). We and another group recently discovered that Mtb has an analogous factor encoded by Rv3780 that we call PafE (proteasome accessory factor E; also known as Bpa for bacterial proteasome activator), which stimulates the degradation of small peptides and β-casein in vitro (29, 30). Both studies also showed that a carboxyl (C)-terminal glycine-glutamine-tyrosine-leucine (GQYL) motif is essential for interacting with and activating 20S CPs, and the penultimate tyrosine residue contributes to activation similarly to tyrosines observed in the “HbYX” (hydrophobic-tyrosine-any amino acid) motif in other characterized proteasome activators (reviewed in ref. 28). Our work further showed that PafE promotes the degradation of at least one native Mtb protein substrate, heat-shock protein repressor (HspR), and that an Mtb pafE mutant is sensitive to heat shock and is attenuated for growth in mice (30). Importantly, PafE-mediated degradation does not require pupylation. Thus, there appear to be at least two independent paths for targeting proteins to the mycobacterial proteasome for degradation.Like the eukaryotic 11S proteasome activators, PafE does not require ATP to stimulate proteolysis. However, it was unknown if PafE formed heptameric complexes like PA26 or PA28. In this work, we show that PafE monomers assume a four-helix bundle structure that is similar to that found in 11S activators, but assemble differently into an unprecedented dodecameric ring structure with 12-fold symmetry. We used isothermal titration calorimetry, cryo-electron microscopy (cryo-EM), and X-ray crystallography to analyze interactions between PafE and 20S core particles, and found that PafE binding induces a larger gate-opening change than has been described for other organisms. We also found that PafE has an extended C terminus that limits the ability of PafE to activate proteasomal degradation in vitro and in vivo.     

Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K_d = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC₅₀ = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC₅₀ = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC₅₀ value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.     

Accurate prediction of autologous stem cell apheresis yields using a double variable-dependent method assures systematic efficiency control of continuous flow collection procedures

BACKGROUND AND OBJECTIVES: Stem cell collection is a standard procedure for the procurement of autologous grafts to rescue myelosuppression induced by high-dose treatments. Accurate prediction of collection yields may contribute to optimize planning and quality control of collection. MATERIALS AND METHODS: Data of 313 autologous haematopoietic stem cell (AHSC) evaluable collections performed in 208 patients with haematologic and non-haematologic neoplasms from seven centres were prospectively analysed to test the accuracy of yield predictions generated by a formula that required the input of peripheral blood (PB) CD34+ cell precount and desired PB volume to be processed. Data were matched in a standard linear regression, in a zero-point regression analysis and tested for prediction accuracy. Further 165 AHSC collections were analysed on a single-centre basis, using yield predictions as reference standards. RESULTS: Analysis showed high levels of correlation between measured collection yields (my) and predictions (py) (R = 0.85; P = 0.000000) as well as high degree of prediction accuracy (my vs. py at paired t-test: P = 0.114781; median my/py ratio = 1.23). Analysis of additional 165 AHSC collections on a single-centre basis showed that the analysed centres had 70% or more measured yields comprising the 0.6-1.8 interval of the my/py ratio. The observance of the 'efficiency' my/py interval assured collection quality control in these centres confirming the reliability of the method. CONCLUSIONS: This prediction method generates accurate and immediate yield predictions allowing collection planning and rapid efficiency control. As a consequence of our study, four centres out of seven use the described method to plan both leukapheresis number and single-procedure blood processing volume while the remaining three centres plan leukapheresis number on the basis of our predictions, maintaining a fixed single-procedure 200 ml/kg blood volume processing, according to their centre AHSC collection policy.     

Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3^CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)–bound forms. AtCESA3^CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3^CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3^CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Cellulose, a linear homopolymer of d-glucopyranose linked by β-1,4-glycosidic bonds, is the major structural component of the cell walls of plants, oomycetes, and algae and constitute the most abundant biopolymer on Earth (1). Cellulose is synthesized by cellulose synthases (CESAs) that belongs to the glycosyltransferase GT-2 superfamily (1, 2). In land plants, cellulose is produced at the plasma membrane by six-lobed rosette-shaped CESA complexes (CSCs) where each CESA is thought to synthesize one cellulose chain (3). The precise number of CESAs per CSC is unresolved but estimated to range between 18 and 36 (4–6).Plants contain multiple cesa genes, with 10 found in the Arabidopsis genome (7). Of these, CESA1, CESA3, CESA6, and the CESA6-like CESAs (i.e., CESA2, CESA5, and CESA9) are involved in primary cell wall formation, whereas CESA4, CESA7, and CESA8 participate in secondary cell wall formation (8–12). These two types of CSCs form heterotrimeric complexes with a ratio of 1:1:1 (13, 14). The Arabidopsis CESAs share an overall sequence identity of ∼60% and have seven transmembrane helices (15). In plants, the catalytic domain (CatD) of the CESAs is located between the second and third transmembrane helices and contains a canonical D, D, D, QxxRW motif (1). While there are similarities between the plant CatD and its counterpart in bacterial cellulose synthases, the CatD is flanked by two plant-specific domains, the so-called plant-conserved region (P-CR) and class-specific region (C-SR) (16). These domains are proposed to have important functions in cellulose synthesis and CESA oligomerization (17).The oligomerization of plant CESAs is thought to be important for the final CSC assembly, and multiple oligomeric states of CESAs, including homodimers, have been reported (18, 19). For example, immunoprecipitation assays using CESA7 fused to a dual His/STRP-tag demonstrated that CESA4, CESA7, and CESA8 could form independent homodimers, and it was hypothesized that the CESA homodimerization may contribute to early stages of CSC assembly. These homodimers might then be converted into CSC heterotrimeric configurations (19). This feature poses a marked difference from the bacterial cellulose synthase complex. However, how CESA homodimers are formed and how they function in cellulose synthesis are unknown.To comprehend the mechanisms behind plant cellulose synthesis, it is essential to acquire structural information about plant CESAs. Indeed, the BcsA–BcsB complex structure from Rhodobacter greatly aided our understanding of the cellulose synthesis in bacteria (20). Nevertheless, there are many differences between bacterial and plant CESAs and the corresponding protein complexes. Extensive efforts have been undertaken to acquire plant CESA structural information, including homology modeling and small-angle X-ray scattering analyses (5, 6, 16, 21, 22). While these efforts have been important to form new hypotheses, they did not reveal significant insights into substrate coordination, cellulose chain extrusion, and complex assembly. Recently, a homotrimeric CESA8 structure from Populus tremula × tremuloides was resolved by cryogenic electron microscopy (cryo-EM), which offered significant new molecular understanding of cellulose microfibril biosynthesis and CESA coordination within the CSC (15). Here we report the crystal structures of Arabidopsis CESA3 CatD (AtCESA3^CatD) in apo and uridine diphosphate (UDP)-glucose (UDP-Glc) bound forms and outline how the CatD might contribute to CESA homodimerization and substrate coordination.     

In vivo translation rates can substantially delay the cotranslational folding of the Escherichia coli cytosolic proteome

A question of fundamental importance concerning protein folding in vivo is whether the kinetics of translation or the thermodynamics of the ribosome nascent chain (RNC) complex is the major determinant of cotranslational folding behavior. This is because translation rates can reduce the probability of cotranslational folding below that associated with arrested ribosomes, whose behavior is determined by the equilibrium thermodynamics of the RNC complex. Here, we combine a chemical kinetic equation with genomic and proteomic data to predict domain folding probabilities as a function of nascent chain length for Escherichia coli cytosolic proteins synthesized on both arrested and continuously translating ribosomes. Our results indicate that, at in vivo translation rates, about one-third of the Escherichia coli cytosolic proteins exhibit cotranslational folding, with at least one domain in each of these proteins folding into its stable native structure before the full-length protein is released from the ribosome. The majority of these cotranslational folding domains are influenced by translation kinetics which reduces their probability of cotranslational folding and consequently increases the nascent chain length at which they fold into their native structures. For about 20% of all cytosolic proteins this delay in folding can exceed the length of the completely synthesized protein, causing one or more of their domains to switch from co- to posttranslational folding solely as a result of the in vivo translation rates. These kinetic effects arise from the difference in time scales of folding and amino-acid addition, and they represent a source of metastability in Escherichia coli''s proteome.     

Seriniquinone,a selective anticancer agent,induces cell death by autophagocytosis,targeting the cancer-protective protein dermcidin

Natural products continue to provide vital treatment options for cancer. Although their translation into chemotherapeutics is complex, collaborative programs continue to deliver productive pipelines for cancer chemotherapy. A new natural product, seriniquinone, isolated from a marine bacterium of the genus Serinicoccus, demonstrated potent activity over a select set of tumor cell lines with particular selectivity toward melanoma cell lines. Upon entering the cell, its journey began by localization into the endoplasmic reticulum. Within 3 h, cells treated with seriniquinone underwent cell death marked by activation of autophagocytosis and gradually terminated through a caspase-9 apoptotic pathway. Using an immunoaffinity approach followed by multipoint validation, we identified the target of seriniquinone as the small protein, dermcidin. Combined, these findings revealed a small molecule motif in parallel with its therapeutic target, whose potential in cancer therapy may be significant. This discovery defines a new pharmacophore that displayed selective activity toward a distinct set of cell lines, predominantly melanoma, within the NCI 60 panel. This selectivity, along with the ease in medicinal chemical modification, provides a key opportunity to design and evaluate new treatments for those cancers that rely on dermcidin activity. Further, the use of dermcidin as a patient preselection biomarker may accelerate the development of more effective personalized treatments.Over the last decade, we have found that integrating a streamlined transition between marine natural products discovery (1), detailed cell and molecular biological studies (2), and medicinal chemical optimization offers a rich forum to advance leads that escape industrialized high-throughput practices (3–5). One place where drug discovery continues to be challenged lies in the discovery of new agents, which are highly selective for specific cancers (6–8).Malignant melanoma, classified by genetic defects within pigment-producing melanocytes, is attributed to the largest number of skin-related cancer deaths (9). Although protective measures can be taken, the aggressive and rapid metastatic properties of melanoma-based tumors continue to challenge clinical practices. The recent introduction of dabrafenib (Tafinlar), trametinib (Mekinist), and ipilimumab (Yervoy) promises to provide improved melanoma treatment (9); however, selective melanoma drugs are still of great interest. In addition, the unique epidemiological concerns for the generation of melanoma (10) and the lack of curative treatment options (11) places melanoma as one of the most dangerous of cancers.     

A multiply convergent platform for the synthesis of trioxacarcins

Many first-line cancer drugs are natural products or are derived from them by chemical modification. The trioxacarcins are an emerging class of molecules of microbial origin with potent antiproliferative effects, which may derive from their ability to covalently modify duplex DNA. All trioxacarcins appear to be derivatives of a nonglycosylated natural product known as DC-45-A2. To explore the potential of the trioxacarcins for the development of small-molecule drugs and probes, we have designed a synthetic strategy toward the trioxacarcin scaffold that enables access to both the natural trioxacarcins and nonnatural structural variants. Here, we report a synthetic route to DC-45-A2 from a differentially protected precursor, which in turn is assembled in just six steps from three components of similar structural complexity. The brevity of the sequence arises from strict adherence to a plan in which strategic bond-pair constructions are staged at or near the end of the synthetic route.