Enzymes that contain metal ions--that is, metalloenzymes--possess the reactivity of a transition metal centre and the potential of molecular evolution to modulate the reactivity and ...substrate-selectivity of the system. By exploiting substrate promiscuity and protein engineering, the scope of reactions catalysed by native metalloenzymes has been expanded recently to include abiological transformations. However, this strategy is limited by the inherent reactivity of metal centres in native metalloenzymes. To overcome this limitation, artificial metalloproteins have been created by incorporating complete, noble-metal complexes within proteins lacking native metal sites. The interactions of the substrate with the protein in these systems are, however, distinct from those with the native protein because the metal complex occupies the substrate binding site. At the intersection of these approaches lies a third strategy, in which the native metal of a metalloenzyme is replaced with an abiological metal with reactivity different from that of the metal in a native protein. This strategy could create artificial enzymes for abiological catalysis within the natural substrate binding site of an enzyme that can be subjected to directed evolution. Here we report the formal replacement of iron in Fe-porphyrin IX (Fe-PIX) proteins with abiological, noble metals to create enzymes that catalyse reactions not catalysed by native Fe-enzymes or other metalloenzymes. In particular, we prepared modified myoglobins containing an Ir(Me) site that catalyse the functionalization of C-H bonds to form C-C bonds by carbene insertion and add carbenes to both β-substituted vinylarenes and unactivated aliphatic α-olefins. We conducted directed evolution of the Ir(Me)-myoglobin and generated mutants that form either enantiomer of the products of C-H insertion and catalyse the enantio- and diastereoselective cyclopropanation of unactivated olefins. The presented method of preparing artificial haem proteins containing abiological metal porphyrins sets the stage for the generation of artificial enzymes from innumerable combinations of PIX-protein scaffolds and unnatural metal cofactors to catalyse a wide range of abiological transformations.
Cytochrome P450 enzymes have been engineered to catalyze abiological C–H bond amination reactions, but the yields of these reactions have been limited by low chemoselectivity for the amination of C–H ...bonds over competing reduction of the azide substrate to a sulfonamide. Here we report that P450s derived from a thermophilic organism and containing an iridium porphyrin cofactor (Ir(Me)-PIX) in place of the heme catalyze enantioselective intramolecular C−H bond amination reactions of sulfonyl azides. These reactions occur with chemoselectivity for insertion of the nitrene units into C−H bonds over reduction of the azides to the sulfonamides that is higher and with substrate scope that is broader than those of enzymes containing iron porphyrins. The products from C−H amination are formed in up to 98% yield and ∼300 TON. In one case, the enantiomeric excess reaches 95:5 er, and the reactions can occur with divergent site selectivity. The chemoselectivity for C–H bond amination is greater than 20:1 in all cases. Variants of the Ir(Me)-PIX CYP119 displaying these properties were identified rapidly by evaluating CYP119 mutants containing Ir(Me)-PIX in cell lysates, rather than as purified enzymes. This study sets the stage to discover suitable enzymes to catalyze challenging C–H amination reactions.
Medicine, emotience, and reason Clark, John F
Philosophy, ethics, and humanities in medicine,
04/2024, Letnik:
19, Številka:
1
Journal Article
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Medicine is faced with a number of intractable modern challenges that can be understood in terms of hyper-intellectualization; a compassion crisis, burnout, dehumanization, and lost meaning. These ...challenges have roots in medical philosophy and indeed general Western philosophy by way of the historic exclusion of human emotion from human reason. The resolution of these medical challenges first requires a novel philosophic schema of human knowledge and reason that incorporates the balanced interaction of human intellect and human emotion. This schema of necessity requires a novel extension of dual-process theory into epistemology in terms of both intellect and emotion each generating a distinct natural kind of knowledge independent of the other as well as how these two forms of mental process together construct human reason. Such a novel philosophic schema is here proposed. This scheme is then applied to the practice of medicine with examples of practical applications with the goal of reformulating medical practice in a more knowledgable, balanced, and healthy way. This schema's expanded epistemology becomes the philosophic foundation for more fully incorporating the humanities in medicine.
The selective functionalization of one C−H bond over others in nearly identical steric and electronic environments can facilitate the construction of complex molecules. We report site‐selective ...functionalizations of C−H bonds, differentiated solely by remote substituents, catalyzed by artificial metalloenzymes (ArMs) that are generated from the combination of an evolvable P450 scaffold and an iridium‐porphyrin cofactor. The generated systems catalyze the insertion of carbenes into the C−H bonds of a range of phthalan derivatives containing substituents that render the two methylene positions in each phthalan inequivalent. These reactions occur with site‐selectivity ratios of up to 17.8:1 and, in most cases, with pairs of enzyme mutants that preferentially form each of the two constitutional isomers. This study demonstrates the potential of abiotic reactions catalyzed by metalloenzymes to functionalize C−H bonds with site selectivity that is difficult to achieve with small‐molecule catalysts.
Ir‐replaceable: Site‐selective functionalizations of C−H bonds differentiated solely by remote substituents were achieved with nearly 10:1 ratios between constitutional isomers by using two distinct mutants of an artificial metalloenzyme generated from an evolvable P450 scaffold and an Ir‐porphyrin cofactor. This site selectivity is difficult to achieve with small‐molecule catalysts.
Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are an emerging class of catalyst for non‐natural reactions. Although many classes of ArMs are ...known, in vitro reconstitution of cofactors and proteins has been a limiting step in the high‐throughput screening and directed evolution of ArMs because purification of individual host proteins is time‐consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from α‐diazopropanoate esters (Me‐EDA) into the N−H bonds of N‐alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me‐EDA with N‐methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo.
Whole‐cell E. coli platform enables directed evolution campaigns with ArMs containing an abiotic Ir‐porphyrin cofactor involving the generation of more than 4000 mutants to develop an enantioselective insertion of a simple acyclic carbene from a diazo ester into the N−H bond of N‐methyl aniline that has formed product with poor enantioselectivity when catalyzed by prior enzyme systems.
Synthetic biology enables microbial hosts to produce complex molecules from organisms that are rare or difficult to cultivate, but the structures of these molecules are limited to those formed by ...reactions of natural enzymes. The integration of artificial metalloenzymes (ArMs) that catalyse unnatural reactions into metabolic networks could broaden the cache of molecules produced biosynthetically. Here we report an engineered microbial cell expressing a heterologous biosynthetic pathway, containing both natural enzymes and ArMs, that produces an unnatural product with high diastereoselectivity. We engineered Escherichia coli with a heterologous terpene biosynthetic pathway and an ArM containing an iridium-porphyrin complex that was transported into the cell with a heterologous transport system. We improved the diastereoselectivity and product titre of the unnatural product by evolving the ArM and selecting the appropriate gene induction and cultivation conditions. This work shows that synthetic biology and synthetic chemistry can produce, by combining natural and artificial enzymes in whole cells, molecules that were previously inaccessible to nature.
Beliefs that individuals hold about whether emotions are malleable or fixed, also referred to as emotion malleability beliefs, may play a crucial role in individuals' emotional experiences and their ...engagement in changing their emotions. The current review integrates affective science and clinical science perspectives to provide a comprehensive review of how emotion malleability beliefs relate to emotionality, emotion regulation, and specific clinical disorders and treatment. Specifically, we discuss how holding more malleable views of emotion could be associated with more active emotion regulation efforts, greater motivation to engage in active regulatory efforts, more effort expended regulating emotions, and lower levels of pathological distress. In addition, we explain how extending emotion malleability beliefs into the clinical domain can complement and extend current conceptualizations of major depressive disorder, social anxiety disorder, and generalized anxiety disorder. This may prove important given the increasingly central role emotion dysregulation has been given in conceptualization and intervention for these psychiatric conditions. Additionally, discussion focuses on how emotion beliefs could be more explicitly addressed in existing cognitive therapies. Promising future directions for research are identified throughout the review.
•Emotion malleability beliefs can be incorporated into depression and anxiety conceptualizations.•Emotion malleability beliefs provide the implicit premise for cognitive behavioral therapy.•Emotion malleability beliefs could represent shape emotion regulatory behavior.
Human pluripotent and trophoblast stem cells have been essential alternatives to blastocysts for understanding early human development
. However, these simple culture systems lack the complexity to ...adequately model the spatiotemporal cellular and molecular dynamics that occur during early embryonic development. Here we describe the reprogramming of fibroblasts into in vitro three-dimensional models of the human blastocyst, termed iBlastoids. Characterization of iBlastoids shows that they model the overall architecture of blastocysts, presenting an inner cell mass-like structure, with epiblast- and primitive endoderm-like cells, a blastocoel-like cavity and a trophectoderm-like outer layer of cells. Single-cell transcriptomics further confirmed the presence of epiblast-, primitive endoderm-, and trophectoderm-like cells. Moreover, iBlastoids can give rise to pluripotent and trophoblast stem cells and are capable of modelling, in vitro, several aspects of the early stage of implantation. In summary, we have developed a scalable and tractable system to model human blastocyst biology; we envision that this will facilitate the study of early human development and the effects of gene mutations and toxins during early embryogenesis, as well as aiding in the development of new therapies associated with in vitro fertilization.
In this Perspective, we present progress, outstanding challenges, and opportunities for the incorporation of artificial metalloenzymes (ArMs) into biosynthetic pathways. We first explain discoveries ...within the field of ArMs that led to the potential inclusion of these enzymes in biosynthesis. We then describe the specific barriers that our laboratory, in collaboration with the laboratories of Keasling and Mukhopadhyay, addressed to establish a biosynthetic pathway containing an ArM. This biosynthesis produced an unnatural cyclopropyl terpenoid by combining heterologous production of the terpene with modification of its terminal alkene by an ArM built from a cytochrome P450. Finally, we describe the remaining challenges and opportunities related to the application of ArMs in synthetic biology.
Tau is a highly abundant and multifunctional brain protein that accumulates in neurofibrillary tangles (NFTs), most commonly in Alzheimer's disease (AD) and primary age-related tauopathy. Recently, ...microRNAs (miRNAs) have been linked to neurodegeneration; however, it is not clear whether miRNA dysregulation contributes to tau neurotoxicity. Here, we determined that the highly conserved brain miRNA miR-219 is downregulated in brain tissue taken at autopsy from patients with AD and from those with severe primary age-related tauopathy. In a Drosophila model that produces human tau, reduction of miR-219 exacerbated tau toxicity, while overexpression of miR-219 partially abrogated toxic effects. Moreover, we observed a bidirectional modulation of tau levels in the Drosophila model that was dependent on miR-219 expression or neutralization, demonstrating that miR-219 regulates tau in vivo. In mammalian cellular models, we found that miR-219 binds directly to the 3'-UTR of the tau mRNA and represses tau synthesis at the post-transcriptional level. Together, our data indicate that silencing of tau by miR-219 is an ancient regulatory mechanism that may become perturbed during neurofibrillary degeneration and suggest that this regulatory pathway may be useful for developing therapeutics for tauopathies.
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