The 50 most-cited articles in Biochemistry, 2022

Targeted protein degradation is a rapidly exploding drug discovery strategy that uses small molecules to recruit disease-causing proteins for rapid destruction mainly via the ubiquitin-proteasome pathway. It shows great potential for treating diseases such as cancer and infectious, inflammatory, and neurodegenerative diseases, especially for those with "undruggable" pathogenic protein targets. With the recent rise of the "molecular glue" type of protein degraders, which tighten and simplify the connection of an E3 ligase with a disease-causing protein for ubiquitination and subsequent degradation, new therapies for unmet medical needs are being designed and developed. Here we use data from the CAS Content Collection and the publication landscape of recent research on targeted protein degraders to provide insights into these molecules, with a special focus on molecular glues. We also outline the advantages of the molecular glues and summarize the advances in drug discovery practices for molecular glue degraders. We further provide a thorough review of drug candidates in targeted…

Cysteine side chains can exist in distinct oxidation states depending on the pH and redox potential of the environment, and cysteine oxidation plays important yet complex regulatory roles. Compared with the effects of post-translational modifications such as phosphorylation, the effects of oxidation of cysteine to sulfenic, sulfinic, and sulfonic acid on protein structure and function remain relatively poorly characterized. We present an analysis of the role of cysteine reactivity as a regulatory factor in proteins, emphasizing the interplay between electrostatics and redox potential as key determinants of the resulting oxidation state. A review of current computational approaches suggests underdeveloped areas of research for studying cysteine reactivity through molecular simulations.

In addition to producing profound subjective effects following acute administration, psychedelic compounds can induce beneficial behavioral changes relevant to the treatment of neuropsychiatric disorders that last long after the compounds have been cleared from the body. One hypothesis with the potential to explain the remarkable enduring effects of psychedelics is related to their abilities to promote structural and functional neuroplasticity in the prefrontal cortex (PFC). A hallmark of many stress-related neuropsychiatric diseases, including depression, post-traumatic stress disorder (PTSD), and addiction, is the atrophy of neurons in the PFC. Psychedelics appear to be particularly effective catalysts for the growth of these key neurons, ultimately leading to restoration of synaptic connectivity in this critical brain region. Furthermore, evidence suggests that the hallucinogenic effects of psychedelics are not directly linked to their ability to promote structural and functional neuroplasticity. If we are to develop improved alternatives to psychedelics for treating…

In the scope of targeted protein degradation (TPD), proteolysis-targeting chimeras (PROTACs), leveraging the ubiquitin-proteasome system, have been extensively studied. However, they are limited to the degradation of soluble and membrane proteins, excluding the aggregated and extracellular proteins and dysfunctional organelles. As an alternative protein degradation pathway, lysosomes serve as a feasible tool for accessing these untouched proteins and/or organelles by proteosomes. Here, we focus on reviewing the emerging lysosome-mediated TPD, such as AUTAC, ATTEC, AUTOTAC, LYTAC, and MoDE-A. Intracellular targets, such as soluble and aggregated proteins and organelles, can be degraded via the autophagy-lysosome pathway. Extracellular targets, such as membrane proteins, and secreted extracellular proteins can be degraded via the endosome-lysosome pathway. In addition, we summarize the mechanism and regulation of autophagy, available methods and assays for monitoring the autophagy process, and the recently developed chemical probes for perturbing the autophagy pathways.

A variety of membraneless organelles, often termed "biological condensates", play an important role in the regulation of cellular processes such as gene transcription, translation, and protein quality control. On the basis of experimental and theoretical investigations, liquid-liquid phase separation (LLPS) has been proposed as a possible mechanism for the origin of biological condensates. LLPS requires multivalent macromolecules that template the formation of long-range, intermolecular interaction networks and results in the formation of condensates with defined composition and material properties. Multivalent interactions driving LLPS exhibit a wide range of modes from highly stereospecific to nonspecific and involve both folded and disordered regions. Multidomain proteins serve as suitable macromolecules for promoting phase separation and achieving disparate functions due to their potential for multivalent interactions and regulation. Here, we aim to highlight the influence of the domain architecture and interdomain interactions on the phase separation of multidomain protein…

More than 55 distinct classes of riboswitches that respond to small metabolites or elemental ions have been experimentally validated to date. The ligands sensed by these riboswitches are biased in favor of fundamental compounds or ions that are likely to have been relevant to ancient forms of life, including those that might have populated the "RNA World", which is a proposed biochemical era that predates the evolutionary emergence of DNA and proteins. In the following text, I discuss the various types of ligands sensed by some of the most common riboswitches present in modern bacterial cells and consider implications for ancient biological processes centered on the proven capabilities of these RNA-based sensors. Although most major biochemical aspects of metabolism are represented by known riboswitch classes, there are striking sensory gaps in some key areas. These gaps could reveal weaknesses in the performance capabilities of RNA that might have hampered RNA World evolution, or these could highlight opportunities to discover additional riboswitch classes that sense essential…

Fe(II)/α-ketoglutarate-dependent dioxygenases ( α- KGDs) are widespread enzymes in aerobic biology and serve a remarkable array of biological functions, including roles in collagen biosynthesis, plant and animal development, transcriptional regulation, nucleic acid modification, and secondary metabolite biosynthesis. This functional diversity is reflected in the enzymes’ catalytic flexibility as α- KGDs can catalyze an intriguing set of synthetically valuable reactions, such as hydroxylations, halogenations, and desaturations, capturing the interest of scientists across disciplines. Mechanistically, all α- KGDs are understood to follow a similar activation pathway to generate a substrate radical, yet how individual members of the enzyme family direct this key intermediate toward the different reaction outcomes remains elusive, triggering structural, computational, spectroscopic, kinetic, and enzyme engineering studies. In this Perspective, we will highlight how first enzyme and substrate engineering examples suggest that the chemical reaction pathway within α- KGDs can be…

The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations…

Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate…

Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibril-catalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein exhibit a lower β-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and…

CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained…

Promiscuous enzymes can be recruited to serve new functions when a genetic or environmental change makes catalysis of a novel reaction important for fitness or even survival. Subsequently, gene duplication and divergence can lead to evolution of an efficient and specialized new enzyme. Every organism likely has thousands of promiscuous enzyme activities that provide a vast reservoir of catalytic potential. However, much of this potential may not be accessible. We compiled kinetic parameters for promiscuous reactions catalyzed by 108 enzymes. The median value of k cat / K M is a very modest 31 M –1 s –1 . Based upon the fluxes through metabolic pathways in E. coli, we estimate that many, if not most, promiscuous activities are too inefficient to impact fitness. However, mutations can elevate the level of an insufficient promiscuous activity by increasing enzyme expression, improving k cat / K M, or altering concentrations of the promiscuous and native substrates and allosteric regulators. Particularly in large bacterial populations, stochastic mutations may provide a viable pathway…

Abstract The main protease (M pro ) of SARS-CoV-2 is essential for viral replication and has been the focus of many drug discovery efforts since the start of the COVID-19 pandemic. Nirmatrelvir (NTV) is an inhibitor of SARS-CoV-2 M pro that is used in the combination drug Paxlovid for the treatment of mild to moderate COVID-19. However, with increased use of NTV across the globe, there is a possibility that future SARS-CoV-2 lineages will evolve resistance to NTV. Early prediction and monitoring of resistance mutations could allow for measures to slow the spread of resistance and for the development of new compounds with activity against resistant strains. In this work, we have used in silico mutational scanning and inhibitor docking of M pro to identify potential resistance mutations. Subsequent in vitro experiments revealed five mutations (N142L, E166M, Q189E, Q189I, and Q192T) that reduce the potency of NTV and of a previously identified non-covalent cyclic peptide inhibitor of M pro . The E166M mutation reduced the half-maximal inhibitory concentration (IC 50 ) of NTV 24-fold,…

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.

F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide-peptide and peptide-lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

ABSTRACT The enzyme BesC from the β - e thynyl-L- s erine biosynthetic pathway in Streptomyces cattleya fragments 4-chloro-L-lysine (produced from L-Lysine by BesD) to ammonia, formaldehyde, and 4-chloro-L-allylglycine and can analogously fragment L-Lys itself. BesC belongs to the emerging family of O 2 -activating non-heme-diiron enzymes with the “heme-oxygenase-like” protein fold (HDOs). Here we show that binding of L-Lys or an analog triggers capture of O 2 by the protein’s diiron(II) cofactor to form a blue µ-peroxodiiron(III) intermediate analogous to those previously characterized in two other HDOs, the olefin-installing fatty acid decarboxylase, UndA, and the guanidino- N -oxygenase domain of SznF. The ∼ 5- and ∼ 30-fold faster decay of the intermediate in reactions with 4-thia-L-Lys and (4 RS )-chloro-DL-lysine than in the reaction with L-Lys itself, and the primary deuterium kinetic isotope effects (D-KIEs) on decay of the intermediate and production of L-allylglycine in the reaction with 4,4,5,5-[ 2 H]-L-Lys, imply that the peroxide intermediate or a successor complex with…

Over the years, protein engineers have studied nature and borrowed its tricks to accelerate protein evolution in the test tube. While there have been considerable advances, our ability to generate new proteins in the laboratory is seemingly limited. One explanation for these shortcomings may be that insertions and deletions (indels), which frequently arise in nature, are largely overlooked during protein engineering campaigns. The profound effect of indels on protein structures, by way of drastic backbone alterations, could be perceived as "saltation" events that bring about significant phenotypic changes in a single mutational step. Should we leverage these effects to accelerate protein engineering and gain access to unexplored regions of adaptive landscapes? In this Perspective, we describe the role played by indels in the functional diversification of proteins in nature and discuss their untapped potential for protein engineering, despite their often-destabilizing nature. We hope to spark a renewed interest in indels, emphasizing that their wider study and use may prove…

High Resolution Image Download MS PowerPoint Slide Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent…

Ribosomally synthesized and post-translationally modified peptides (RiPPs) make up a rapidly growing superfamily of natural products. RiPPs exhibit an extraordinary range of structures, but they all begin as gene-encoded precursor peptides that are linear chains of amino acids produced by ribosomes. Given the gene-encoded nature of RiPP precursor peptides, the toolbox of protein engineering can be directly applied to these precursors. This Perspective will discuss examples of site-directed mutagenesis, noncanonical amino acid mutagenesis, and the construction and screening of combinatorial libraries as applied to RiPPs. These studies have led to important insights into the biosynthesis and bioactivity of RiPPs and the reengineering of RiPPs for entirely new functions.

Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key…

High Resolution Image Download MS PowerPoint Slide Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether “staples” into a protein via cysteine alkylation with the noncanonical amino acid O -2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p -chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the…

The class I histone deacetylase (HDAC) enzymes;HDAC1,2 and 3 form the catalytic engine of at least seven structurally distinct multiprotein complexes in cells. These molecular machines play a vital role in the regulation of chromatin accessibility and gene activity via the removal of acetyl moieties from lysine residues within histone tails. Their inhibition via small molecule inhibitors has beneficial effects in a number of disease types, including the clinical treatment of hematological cancers. We have previously reported a library of proteolysis targeting chimeras (PROTACs) incorporating a benzamide-based HDAC ligand (from CI-994), with an alkyl linker and ligand for the von Hippel-Lindau (VHL) E3 ubiquitin ligase that degrade HDAC1-3 at submicromolar concentrations. Here we report the addition of two novel PROTACs (JPS026 and JPS027), which utilize a ligand for the cellular inhibitor of apoptosis (IAP) family of E3 ligases. We found that both VHL (JPS004)- and IAP (JPS026)-based PROTACs degrade HDAC1-3 and induce histone acetylation to a similar degree. However, JPS026 is…

The P450 superfamily comprises some of the most powerful and versatile enzymes for the site-selective oxidation of small molecules. One of the main drawbacks for the applications of the P450s in biotechnology is that the majority of these enzymes is multicomponent in nature and requires the presence of suitable redox partners to support their functions. Nevertheless, the discovery of several self-sufficient P450s, namely those from Classes VII and VIII, has served as an inspiration for fusion approaches to generate chimeric P450 systems that are self-sufficient. In this Perspective, we highlight the domain organizations of the Class VII and Class VIII P450 systems, summarize recent case studies in the engineering of catalytically self-sufficient P450s based on these systems, and outline outstanding challenges in the field, along with several emerging technologies as potential solutions.

High Resolution Image Download MS PowerPoint Slide In photosystem II (PSII) and photosynthetic reaction centers from purple bacteria (PbRC), the electron released from the electronically excited chlorophyll is transferred to the terminal electron acceptor quinone, Q B . Q B accepts two electrons and two protons before leaving the protein. We investigated the molecular mechanism of quinone exchange in PSII, conducting molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations. MD simulations suggest that the release of Q B leads to the transformation of the short helix (D1-Phe260 to D1-Ser264), which is adjacent to the stromal helix de (D1-Asn247 to D1-Ile259), into a loop and to the formation of a water-intake channel. Water molecules enter the Q B binding pocket via the channel and form an H-bond network. QM/MM calculations indicate that the H-bond network serves as a proton-transfer pathway for the reprotonation of D1-His215, the proton donor during Q B H – /Q B H 2 conversion. Together with the absence of the corresponding short helix but…

High Resolution Image Download MS PowerPoint Slide Ni–Fe–S-dependent carbon monoxide dehydrogenases (CODHs) are enzymes that interconvert CO and CO 2 by using their catalytic Ni–Fe–S C-cluster and their Fe–S B- and D-clusters for electron transfer. CODHs are important in the microbiota of animals such as humans, ruminants, and termites because they can facilitate the use of CO and CO 2 as carbon sources and serve to maintain redox homeostasis. The bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is responsible for acetate production via the Wood–Ljungdahl pathway, where acetyl-CoA is assembled from two CO 2 -derived one-carbon units. A Ni–Fe–S A-cluster is key to this chemistry. Whereas acetogens use the A- and C-clusters of CODH/ACS to produce acetate from CO 2, methanogens use A- and C-clusters of an acetyl-CoA decarbonylase/synthase complex (ACDS) to break down acetate en route to CO 2 and methane production. Here we review some of the recent advances in understanding the structure and mechanism of CODHs, CODH/ACSs, and ACDSs, their unusual…

by reversing methanogenesis, a theoretically reversible process. The pathway would start with alkane activation, an endergonic step catalyzed by methyl-coenzyme M reductase (MCR) homologues that would generate alkyl-thiols carried by coenzyme M. While the methane-generating MCR found in methanogens has been well characterized, the enzymatic activity of the putative alkane-fixing counterparts has not been validated so far. Such an absence of biochemical investigations contrasts with the current explosion of metagenomics data, which draws new potential alkane-oxidizing pathways in various archaeal phyla. Therefore, validating the physiological function of these putative alkane-fixing machines and investigating how their structures, catalytic mechanisms, and cofactors vary depending on the targeted alkane have become urgent needs. The first structural insights into the methane- and ethane-capturing MCRs highlighted unsuspected differences and proposed some explanations for their substrate specificity. This Perspective reviews the current physiological, biochemical, and structural…

Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid-liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Here, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over…

High Resolution Image Download MS PowerPoint Slide CRISPR gene editing and control systems continue to emerge and inspire novel research and clinical applications. Advances in CRISPR performance such as optimizing the duration of activity in cells, tissues, and organisms, as well as limiting off-target activities, have been extremely important for expanding the utility of CRISPR-based systems. By investigating the effects of various chemical modifications in guide RNAs (gRNAs) at defined positions and combinations, we find that 2′- O -methyl-3′-phosphonoacetate (MP) modifications can be substantially more effective than 2′- O -methyl-3′-phosphorothioate (MS) modifications at the 3′ ends of single-guide RNAs (sgRNAs) to promote high editing yields, in some instances showing an order of magnitude higher editing yield in human cells. MP-modified 3′ ends are especially effective at promoting the activity of guide RNAs cotransfected with Cas messenger RNA (mRNA), as the gRNA must persist in cells until the Cas protein is expressed. We demonstrate such an MP enhancement for sgRNAs…

Structured RNAs bind ligands and are attractive targets for small-molecule drugs. A wide variety of analytical methods have been used to characterize RNA-ligand interactions, but our experience is that most have significant limitations in terms of material requirements and applicability to complex RNAs. Surface plasmon resonance (SPR) potentially overcomes these limitations, but we find that the standard experimental framework measures notable nonspecific electrostatic-mediated interactions, frustrating analysis of weak RNA binders. SPR measurements are typically quantified relative to a non-target reference channel. Here, we show that referencing to a channel containing a non-binding control RNA enables subtraction of nonspecific binding contributions, allowing measurements of accurate and specific binding affinities. We validated this approach for small-molecule binders of two riboswitch RNAs with affinities ranging from nanomolar to millimolar, including low-molecular-mass fragment ligands. SPR implemented with reference subtraction reliably discriminates specific from…

and CGP37157 (termed paraptosis II herein) proceeds via a signaling pathway different from that of the previously known paraptosis induced by celastrol, a process that negligibly involves membrane fusion between mitochondria and the ER (termed paraptosis I herein).

The phytocannabinoid cannabigerol (CBG) is the central biosynthetic precursor to many cannabinoids, including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Though the use of CBG has recently witnessed a widespread surge because of its beneficial health effects and lack of psychoactivity, its metabolism by human cytochrome P450s is largely unknown. Herein, we describe comprehensive in vitro and in vivo cytochrome P450 (CYP)-mediated metabolic studies of CBG, ranging from LC-MS/MS-based primary metabolic site determination, synthetic validation, and kinetic behavior using targeted mass spectrometry. These investigations revealed that cyclo-CBG, a recently isolated phytocannabinoid, is the major metabolite that is rapidly formed by selected human cytochrome P450s (CYP2J2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9). Additionally, in vivo studies with mice administered with CBG, supported these studies, where cyclo-CBG is the major metabolite as well. Spectroscopic binding studies along with docking and modeling of CBG molecule near the heme in the active site of P450s confirmed these…

ADVERTISEMENT RETURN TO ISSUEPREVViewpointNEXTTargeting Scaffolding Functions of Enzymes Using PROTAC ApproachesChiho KimChiho KimDepartment of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York 10032, United StatesMore by Chiho Kim, Xu-Dong WangXu-Dong WangDepartment of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York 10032, United StatesMore by Xu-Dong Wang, Zhengshuai LiuZhengshuai LiuDepartment of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York 10032, United StatesMore by Zhengshuai Liu, Shan ZhaShan ZhaInstitute for Cancer Genetics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York 10032, United StatesDepartment of Pediatrics, Vagelos College of Physicians and Surgeons, Columbia University Irving Medical Center, New York, New York…

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments;…

The gut microbiota produce specialized metabolites that are important for maintaining host health homeostasis. Hence, unstable production of these metabolites can contribute to diseases such as inflammatory bowel disease and colon cancer. While fecal transplantation or dietary modification approaches can be used to correct the gut microbial community's metabolic output, this Perspective focuses on the use of engineered bacteria. We highlight recent advances in bacterial synthetic biology approaches for the treatment of colorectal cancer and systemic tumors and discuss the functionality and biochemical properties of novel containment approaches using hydrogel-based and electronic devices. Synthetic circuitry refinement and incorporation of novel functional modules have enabled more targeted detection of colonic tumors and delivery of anticancer compounds inside the gastrointestinal (GI) tract, as well as the design of tumor-homing bacteria capable of recruiting infiltrating T cells. Engineering challenges in these applications include the stability of the genetic circuits, long-term…

lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or…

Abstract The opportunistic pathogen Pseudomonas aeruginosa causes antibiotic resistant, nosocomial infections in immuno-compromised individuals, and is a high priority for antimicrobial development. Key to pathogenicity in P. aeruginosa are biofilm formation and virulence factor production. Both traits are controlled by the cell-to-cell communication process called quorum sensing (QS). QS involves the synthesis, release, and population-wide detection of signal molecules called autoinducers. We previously reported that activity of the RhlR QS transcription factor depends on a protein-protein interaction with the hydrolase, PqsE, and PqsE catalytic activity is dispensable for this interaction. Nonetheless, the PqsE-RhlR interaction could be disrupted by substitution of an active site glutamate residue with tryptophan (PqsE(E182W)). Here, we show that disruption of the PqsE-RhlR interaction via either the E182W change or alteration of PqsE surface residues that are essential for the interaction with RhlR, attenuates P. aeruginosa infection in a murine host. We use crystallography to…

Type II diabetes is characterized by the loss of pancreatic β-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, that is, the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP…

ABSTRACT Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo -inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologs display phosphohydrolase activity with a high specificity for the 5-β-phosphate of inositol pyrophosphates. We…

High Resolution Image Download MS PowerPoint Slide The structural origin of enzyme cold-adaptation has been the subject of considerable research efforts in recent years. Comparative studies of orthologous mesophilic–psychrophilic enzyme pairs found in nature are an obvious strategy for solving this problem, but they often suffer from relatively low sequence identity of the enzyme pairs. Small bacterial lipases adapted to distinctly different temperatures appear to provide an excellent model system for these types of studies, as they may show a very high degree of sequence conservation. Here, we report the first crystal structures of lipase A from the psychrophilic bacterium Bacillus pumilus, which confirm the high structural similarity to the mesophilic Bacillus subtilis enzyme, as indicated by their 81% sequence identity. We further employ extensive QM/MM calculations to delineate the catalytic reaction path and its energetics. The computational prediction of a rate-limiting deacylation step of the enzymatic ester hydrolysis reaction is verified by stopped-flow experiments, and…

Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with…

High Resolution Image Download MS PowerPoint Slide Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S- adenosylmethionine (SAM)-dependent m 6 A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3′ triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6…

CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposon-associated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile…

Mo-nitrogenase catalyzes the challenging N 2 -to-NH 3 reduction. This complex reaction proceeds through a series of intermediate states (E n ) of its active site FeMo-cofactor. An understanding of the kinetics of the conversion between E n states is central to defining the mechanism of nitrogenase. Here, rate constants of key steps have been determined through a steady-state kinetic model with fits to experimental data. The model reveals that the rate for H 2 formation from the early electron populated state E 2 (2H) is much slower than that from the more reduced E 4 (4H) state. Further, it is found that the competing reactions of H 2 formation and N 2 binding at the E 4 (4H) state occur with equal rate constants. The H 2 -dependent reverse reaction of the N 2 binding step is found to have a rate constant of 5.5 ± 0.2 (atm H 2 ) −1 s –1 (7.2 ± 0.3 (mM H 2 ) −1 s –1 ). Importantly, the reduction of N 2 bound to FeMo-cofactor proceeds with a rate constant of 1 ± 0.1 s –1, revealing a previously unrecognized slow step in the Mo-nitrogenase catalytic cycle associated with the chemical…

The rapid growth of sequence databases over the past two decades means that protein engineers faced with optimizing a protein for any given task will often have immediate access to a vast number of related protein sequences. These sequences encode information about the evolutionary history of the protein and the underlying sequence requirements to produce folded, stable, and functional protein variants. Methods that can take advantage of this information are an increasingly important part of the protein engineering tool kit. In this Perspective, we discuss the utility of sequence data in protein engineering and design, focusing on recent advances in three main areas: the use of ancestral sequence reconstruction as an engineering tool to generate thermostable and multifunctional proteins, the use of sequence data to guide engineering of multipoint mutants by structure-based computational protein design, and the use of unlabeled sequence data for unsupervised and semisupervised machine learning, allowing the generation of diverse and functional protein sequences in unexplored regions…

CRISPR diagnostics have recently emerged as powerful diagnostic tools for the rapid detection of infections. The ultimate goal is to develop these diagnostics for the point of care, where patients quickly receive and easily interpret results. Although they are in their infancy, the COVID-19 pandemic has accelerated innovation of CRISPR diagnostics and led to an explosion of improvements to these systems. Challenges that have impeded the implementation at the point of care have been addressed, and CRISPR diagnostics have been dramatically simplified. Here we outline recent developments and advancements in CRISPR diagnostics that have pushed these technologies to the point of care.

to gain insights into their biochemical and physiological differences in the brain. Using Fourier transform infrared and nuclear magnetic resonance spectroscopy, we measure the relative propensities of Aβ40-Dutch and Aβ40-Iowa to form antiparallel structure and compare these propensities to those of the wild-type Aβ40 and Aβ42 isoforms. We find that both Aβ40-Dutch and Aβ40-Iowa have strong propensities to form antiparallel β-hairpins in the first step of the fibrillization process. However, there is a marked difference in the ability of these peptides to form elongated antiparallel structures. Importantly, we find marked differences in the stability of the protofibril or fibril states formed by the four Aβ peptides. We discuss these differences with respect to the mechanisms of Aβ fibril formation in CAA.

The ABC efflux pump P-glycoprotein (P-gp) transports a wide variety of drugs and is inhibited by others. Some drugs stimulate ATP hydrolysis at the nucleotide binding domains (NBDs) and are transported, others uncouple ATP hydrolysis and transport, and others inhibit ATP hydrolysis. The molecular basis for the different behavior of these drugs is not well understood despite the availability of several structural models of P-gp complexes with ligands bound. Hypothetically, ligands differentially alter the conformational dynamics of peptide segments that mediate the coupling between the drug binding sites and the NBDs. Here, we explore by hydrogen-deuterium exchange mass spectrometry the dynamic consequences of a classic substrate and inhibitor, vinblastine and zosuquidar, binding to mouse P-gp (mdr1a) in lipid nanodiscs. The dynamics of P-gp in nucleotide-free, pre-hydrolysis, and post-hydrolysis states in the presence of each drug reveal distinct mechanisms of ATPase stimulation and implications for transport. For both drugs, there are common regions affected in a similar manner,…

High Resolution Image Download MS PowerPoint Slide The linker histone H1 is a highly prevalent protein that compacts chromatin and regulates DNA accessibility and transcription. However, the mechanisms behind H1 regulation of transcription factor (TF) binding within nucleosomes are not well understood. Using in vitro fluorescence assays, we positioned fluorophores throughout human H1 and the nucleosome, then monitored the distance changes between H1 and the histone octamer, H1 and nucleosomal DNA, or nucleosomal DNA and the histone octamer to monitor the H1 movement during TF binding. We found that H1 remains bound to the nucleosome dyad, while the C terminal domain (CTD) releases the linker DNA during nucleosome partial unwrapping and TF binding. In addition, mutational studies revealed that a small 16 amino acid region at the beginning of the H1 CTD is largely responsible for altering nucleosome wrapping and regulating TF binding within nucleosomes. We then investigated physiologically relevant post-translational modifications (PTMs) in human H1 by preparing fully synthetic H1…

Proteins and protein assemblies often tether interaction partners to strengthen interactions, to regulate activity through auto-inhibition or -activation, or to boost enzyme catalysis. Tethered reactions are regulated by the architecture of the tether, which defines an effective concentration of the interactor. Effective concentrations can be estimated theoretically for simple linkers via polymer models, but there is currently no general method for estimating effective concentrations for complex linker architectures consisting of both flexible and folded domains. We describe how effective concentrations can be estimated computationally for any protein linker architecture by defining a realistic conformational ensemble. We benchmark against prediction from a worm-like chain and values measured by competition experiments and find minor differences likely due to excluded volume effects. Systematic variation of the properties of flexible and folded segments shows that the effective concentration is mainly determined by the combination of the total length of flexible segments and the…

Estrogen receptor alpha (ERα) is a ligand-responsive transcription factor critical for sex determination and development. Recent reports challenge the canonical view of ERα function by suggesting an activity beyond binding dsDNA at estrogen-responsive promotor elements: association with RNAs in vivo. Whether these interactions are direct or indirect remains unknown, which limits the ability to understand the extent, specificity, and biological role of ERα-RNA binding. Here we demonstrate that an extended DNA-binding domain of ERα directly binds a wide range of RNAs in vitro with structural specificity. ERα binds RNAs that adopt a range of hairpin-derived structures independent of sequence, while interacting poorly with single- and double-stranded RNA. RNA affinities are only 4-fold weaker than consensus dsDNA and significantly tighter than nonconsensus dsDNA sequences. Moreover, RNA binding is competitive with DNA binding. Together, these data show that ERα utilizes an extended DNA-binding domain to achieve a high-affinity/low-specificity mode for interacting with RNA.