Phosphinothricin-tripeptide (PTT, phosphinothricyl-alanyl-alanine) is a natural product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 217051 and Streptomyces viridochromogenes DSM 407362. PTT has attracted widespread interest due to its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of heretofore undescribed catalyses, including a highly unusual reaction for carbon bond cleavage. In sum, we define a more complex pathway for early PTT biosynthesis that includes biochemically unprecedented and chemically interesting steps.
Streptomyces viridochromogenes; phosphinothricin; biosynthesis; bialaphos; phosphonate metabolism
To identify the physiological targets of drugs and bioactive small molecules we have developed an approach, named DrugTargetSeqR, which combines high-throughput sequencing, computational mutation discovery and CRISPR/Cas9-based genome editing. We apply this approach to ispinesib and YM155, drugs that have undergone clinical trials as anti-cancer agents, and demonstrate target identification and uncover genetic and epigenetic mechanisms likely to cause drug resistance in human cancer cells.
The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during iPSC reprogramming. Here, we show that inhibition of Notch significantly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.
Altered glycosylation of cancer cells confers phenotypic changes that promote spread and evasion of immune responses. A novel method for engineering cell surface glycans is providing insights into these mechanisms.
Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux, without altering the hDAT physiological function of DA uptake. We evaluated, for the first time, the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.
dopamine; transporter; amphetamine; Drosophila melanogaster; phosphatidylinositol 4,5-bisphosphate
Despite the remarkable versatility displayed by flavin-dependent monooxygenases (FMOs) in natural product biosynthesis, one notably missing activity is the oxidative generation of carbonate functional groups. We describe a multifunctional Baeyer-Villiger monooxygenase CcsB, which catalyzes the formation of an in-line carbonate in the macrocyclic portion of cytochalasin E. This study expands the repertoire of activities of FMOs and provides a possible synthetic strategy for transformation of ketones into carbonates.
PTP1B, a validated therapeutic target for diabetes and obesity, plays a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We defined a novel mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small molecule inhibitor, MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.
Lytic polysaccharide mono-oxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They currently attract much attention due to their potential use in biomass conversion, notably in the production of biofuels. Past work has identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the 3-D structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active centre featuring the histidine brace active site, but is distinct in terms of its active site details and its EPR spectroscopy. The new AA11 family expands the LPMO clan with the potential to broaden both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.
Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. Of practical necessity, inferences are often drawn from in vitro assays on target fragments, but such inferences may belie actual mechanisms. One key example of such ambiguity concerns calcium-binding proteins (CaBPs) that tune signaling molecules regulated by calmodulin (CaM). Since CaBPs resemble CaM, CaBPs are believed to competitively replace CaM on targets. Yet, brain CaM expression far surpasses that of CaBPs, so how can CaBPs exert appreciable biological actions? Here, we devise a live-cell, holomolecule approach that reveals an allosteric mechanism for calcium channels, whose CaM-mediated inactivation is eliminated by CaBP4. Our strategy is to covalently link CaM and/or CaBP to holochannels, enabling live-cell FRET assays to resolve a cyclical allosteric binding scheme for CaM and CaBP4 to channels, thus explaining how trace CaBPs prevail. This approach may apply generally for discerning allostery in live cells.
Development of specific inhibitors of allergy has had limited success, in part, owing to a lack of experimental models that reflect the complexity of allergen-IgE interactions. We designed a heterotetravalent allergen (HtTA) system, which reflects epitope heterogeneity, polyclonal response and number of immunodominant epitopes observed in natural allergens, thereby providing a physiologically relevant experimental model to study mast cell degranulation. The HtTA design revealed the importance of weak-affinity epitopes in allergy, particularly when presented with high-affinity epitopes. The effect of selective inhibition of weak-affinity epitope-IgE interactions was investigated with heterobivalent inhibitors (HBIs) designed to simultaneously target the antigen- and nucleotide-binding sites on the IgE Fab. HBI demonstrated enhanced avidity for the target IgE and was a potent inhibitor of degranulation in vitro and in vivo. These results demonstrate that partial inhibition of allergen-IgE interactions was sufficient to prevent mast cell degranulation, thus establishing the therapeutic potential of the HBI design.
Major histocompatibility complex (MHC) class I and II are glycoproteins that can present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes. Here, the importance of the modification of protein antigens by glycans on cellular uptake, proteolytic processing, presentation by MHC and subsequent T-cell priming is reviewed. Antigen glycosylation is important for a number of diseases and vaccine design. All of the key proteins involved in antigen recognition and the orchestration of downstream effector functions are glycosylated. The influence of protein glycosylation on immune function and disease is covered.
Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester, and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is the first non-patent report of the enzyme currently used for the manufacture of simvastatin, as well as the intermediate evolved variants. Crystal structures and microsecond molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations dramatically altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF.
Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.
Uniquely among known ribozymes, the glmS ribozyme-riboswitch requires a small-molecule coenzyme, glucosamine-6-phosphate (GlcN6P). Although consistent with its gene-regulatory function, use of GlcN6P is unexpected because all other characterized self-cleaving ribozymes employ RNA functional groups or divalent cations for catalysis. To determine what active site features make this ribozyme reliant on GlcN6P, and to evaluate whether it might have evolved from a coenzyme-independent ancestor, we isolated a GlcN6P-independent variant through in vitro selection. Three active site mutations suffice to generate a highly reactive RNA that adopts the wildtype fold but employs divalent cations for catalysis and is insensitive to GlcN6P. Biochemical and crystallographic comparisons of wildtype and mutant ribozymes show that a handful of functional groups fine-tune the RNA to be either coenzyme- or cation-dependent. These results indicate that a few mutations can confer novel biochemical activities on structured RNAs. Thus, families of structurally related ribozymes with divergent function may exist.
Specific chemicals can prime the plant immune system for augmented defence. β-aminobutyric acid (BABA) is a priming agent that provides broad-spectrum disease protection. However, BABA also suppresses plant growth when applied in high doses, which has hampered its application as a crop defence activator. Here we describe a mutant of Arabidopsis thaliana that is impaired in BABA-induced disease immunity (ibi1) but hypersensitive to BABA-induced growth repression. IBI encodes an aspartyl-tRNA synthetase. Enantiomer-specific binding of R-BABA to IBI1 primed the protein for non-canonical defence signalling in the cytoplasm after pathogen attack. This priming was associated with aspartic acid accumulation and tRNA-induced phosphorylation of translation initiation factor eIF2α. However, mutation of eIF2α-phosphorylating GCN2 kinase did not affect BABA-induced immunity, but relieved BABA-induced growth repression. Hence, BABA-activated IBI1 controls plant immunity and growth via separate pathways. Our results open new opportunities to separate broad-spectrum disease resistance from the associated costs on plant growth.
Proteins that traffic through the eukaryotic secretory pathway are commonly modified with N-linked carbohydrates. These bulky amphipathic modifications at asparagines intrinsically enhance solubility and folding energetics through carbohydrate-protein interactions. N-linked glycans can also extrinsically enhance glycoprotein folding by utilizing the glycoprotein homeostasis or “glycoproteostasis” network, comprising numerous glycan binding and/or modification enzymes or proteins that synthesize, transfer, sculpt and utilize N-linked glycans to direct folding vs. degradation, and trafficking of nascent N-glycoproteins through the cellular secretory pathway. If protein maturation is perturbed by misfolding and/or aggregation, stress pathways are often activated that result in transcriptional remodeling of the secretory pathway, in an attempt to alleviate the insult(s). The inability to achieve glycoproteostasis is linked to several pathologies, including amyloidoses, cystic fibrosis, and lysosomal storage diseases. Recent progress on genetic and pharmacologic adaptation of the glycoproteostasis network provides hope that drugs can be developed for these maladies in the near future.
Protein degradation plays a central role in many cellular functions. Misfolded and damaged proteins are removed from the cell to avoid toxicity. The concentrations of regulatory proteins are adjusted by degradation at the appropriate time. Both foreign and native proteins are digested into small peptides as part of the adaptive immune response. In eukaryotic cells, an ATP-dependent protease called the proteasome is responsible for much of this proteolysis. Proteins are targeted for proteasomal degradation by a two-part degron, which consists of a proteasome binding signal and a degradation initiation site. Here we describe how both components contribute to the specificity of degradation.
Concatenation of engineered biocatalysts into multistep pathways dramatically increases their utility, but development of generalizable assembly methods remains a significant challenge. Herein we evaluate ‘bioretrosynthesis’, which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2,3-dideoxyinosine). Applying both directed evolution and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and 3-fold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase, but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.
Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.
The lasso peptide microcin J25 is known to hijack the siderophore receptor FhuA for initiating internalization. Here, we provide the first structural evidence on the recognition mechanism and our biochemical data show that another closely related lasso peptide cannot interact with FhuA. Our work provides an explanation on the narrow activity spectrum of lasso peptides and opens the path to the development of new antibacterials.
Amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans for over 50 years with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of the first derivatives of amphotericin that kill yeast but not human cells.