Showing papers in "ACS Chemical Biology in 2018"
TL;DR: This work outlines for the first time all necessary considerations for the appropriate use of PAINS filters.
Abstract: Pan-Assay Interference Compounds (PAINS) are very familiar to medicinal chemists who have spent time fruitlessly trying to optimize these nonprogressible compounds. Electronic filters formulated to recognize PAINS can process hundreds and thousands of compounds in seconds and are in widespread current use to identify PAINS in order to exclude them from further analysis. However, this practice is fraught with danger because such black box treatment is simplistic. Here, we outline for the first time all necessary considerations for the appropriate use of PAINS filters.
384 citations
TL;DR: One example in which the genetic code itself has been expanded with new building blocks that allow chemists to probe and manipulate the structures and functions of proteins with unprecedented precision is reviewed.
Abstract: Our understanding of the complex molecular processes of living organisms at the molecular level is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of macromolecules, is allowing chemists to to harness and reprogram the cellular machinery in ways previously unimaged. Here we review one example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins with unprecedented precision.
233 citations
TL;DR: The underlying technology and different types of CRISPRi/a screens, including those based on cell survival/proliferation, sensitivity to drugs or toxins, fluorescent reporters, and single-cell transcriptomes, are introduced and compared.
Abstract: Next-generation DNA sequencing technologies have led to a massive accumulation of genomic and transcriptomic data from patients and healthy individuals. The major challenge ahead is to understand the functional significance of the elements of the human genome and transcriptome, and implications for diagnosis and treatment. Genetic screens in mammalian cells are a powerful approach to systematically elucidating gene function in health and disease states. In particular, recently developed CRISPR/Cas9-based screening approaches have enormous potential to uncover mechanisms and therapeutic strategies for human diseases. The focus of this review is the use of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for genetic screens in mammalian cells. We introduce the underlying technology and present different types of CRISPRi/a screens, including those based on cell survival/proliferation, sensitivity to drugs or toxins, fluorescent reporters, and single-cell transcriptomes. Combinatorial screens, in...
224 citations
TL;DR: The ability to efficiently tag endogenous proteins with a small luminescent peptide is demonstrated, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.
Abstract: Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Witho...
202 citations
TL;DR: This work employed stimulated Raman scattering (SRS) microscopy coupled with small vibrational tags to image the distribution of ferrostatins in cells and found that they accumulate in lysosomes, mitochondria, and the endoplasmic reticulum.
Abstract: Ferroptosis is a form of nonapoptotic cell death characterized by the unchecked accumulation of lipid peroxides. Ferrostatin-1 and its analogs (ferrostatins) specifically prevent ferroptosis in multiple contexts, but many aspects of their molecular mechanism of action remain poorly described. Here, we employed stimulated Raman scattering (SRS) microscopy coupled with small vibrational tags to image the distribution of ferrostatins in cells and found that they accumulate in lysosomes, mitochondria, and the endoplasmic reticulum. We then evaluated the functional relevance of lysosomes and mitochondria to ferroptosis suppression by ferrostatins and found that neither is required for effective ferroptosis suppression.
190 citations
TL;DR: An innovative, modular live-cell platform utilizing endogenous tagging technologies is presented and applied to monitoring PROTAC-mediated degradation of the bromodomain and extra-terminal family members, showing comprehensive real-time degradation and recovery profiles for each target.
Abstract: A new generation of heterobifunctional small molecules, termed proteolysis targeting chimeras (PROTACs), targets proteins for degradation through recruitment to E3 ligases and holds significant therapeutic potential. Despite numerous successful examples, PROTAC small molecule development remains laborious and unpredictable, involving testing compounds for end-point degradation activity at fixed times and concentrations without resolving or optimizing for the important biological steps required for the process. Given the complexity of the ubiquitin proteasomal pathway, technologies that enable real-time characterization of PROTAC efficacy and mechanism of action are critical for accelerating compound development, profiling, and improving guidance of chemical structure–activity relationship. Here, we present an innovative, modular live-cell platform utilizing endogenous tagging technologies and apply it to monitoring PROTAC-mediated degradation of the bromodomain and extra-terminal family members. We show c...
168 citations
TL;DR: This research synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752 and identified the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target, resulting from inadvertent E3 ligase modulation.
Abstract: Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. While both series failed to induce degradation of their consensus targets, individual molecules displayed pronounced efficacy in leukemia cell lines. Orthogonal target identification supported by molecular docking led us to identify the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target, resulting from inadvertent E3 ligase modulation. This research highlights the importance of monitoring degradation events that are independent of t...
109 citations
TL;DR: A survey by enzyme classification is organized, with emphasis on fluorophore masking strategies, modes of enzymatic activation, and the breadth of current and future applications of enzyme-activated fluorogenic probes for biological imaging.
Abstract: Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure to specific stimuli, are powerful tools for chemical biology. Those probes that respond to enzymatic catalysis illuminate the complex dynamics of biological processes at a level of spatiotemporal detail and sensitivity unmatched by other techniques. Here, we review recent advances in enzyme-activated fluorogenic probes for biological imaging. We organize our survey by enzyme classification, with emphasis on fluorophore masking strategies, modes of enzymatic activation, and the breadth of current and future applications. Key challenges such as probe selectivity and spectroscopic requirements are described alongside therapeutic, diagnostic, and theranostic opportunities.
108 citations
TL;DR: This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identifyAllosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.
Abstract: SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.
103 citations
TL;DR: In this article, a proteolysis-targeting chimera (PROTAC) was developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines.
Abstract: B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen w...
102 citations
TL;DR: Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the molecular mechanism of thalidomide analogues.
Abstract: The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degradation of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degradation. By linking two pomalidomide molecules, we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degradation. The homodimerized compound 15a was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of 15a for CRBN degradation was confirmed at the proteome level by quantitative mass spectrometry. Inactivation by compound 15a did not affect proliferation of different cell lines, prevented pomalidomide-induced degradation of IKZF1 and IKZF3, an...
TL;DR: Advances toward modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering are reviewed.
Abstract: Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either nonhomologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site. However, genetic recombination in mammalian systems through the HDR pathway is an inefficient process and requires cumbersome laboratory methods to identify the desired accurate insertion events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repairs the majority of nuclease induced DNA DSBs and also is responsible for mutagenic insertion and deletion events at off-target locations throughout the genome. Various methodologies have been developed to increase the efficiency of designer nuclease-based HDR mediated gene editing. Here, we review these advances toward modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering.
TL;DR: The data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate, making HDAC11 the most proficient fatty-acid deacylase of the HDAC family.
Abstract: Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here, we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8–18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides were 77 700 and 149 000 M–1 s–1, respectively, making HDAC11 the most proficient fatty-acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic, and stearic acids with inhibition constants of 6.5, 0.9, and 1.6 μM, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by...
Barcelona Supercomputing Center1, University of Hamburg2, University of Toronto3, Bangor University4, University of Düsseldorf5, University of Oviedo6, University of Bergen7, Argonne National Laboratory8, University of Stuttgart9, University of Oxford10, Max Planck Society11, Jacobs University Bremen12, Immanuel Kant Baltic Federal University13, Forschungszentrum Jülich14, Catalan Institution for Research and Advanced Studies15
TL;DR: An extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters, finds a structural parameter that helps rank (classify) the promiscuity level of esterase from sequence data at 94% accuracy.
Abstract: Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases’ substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein–ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, ...
TL;DR: The biochemical and cellular activities of ClpP are discussed along with the mechanisms by whichClpP affects bacterial pathogenesis and various human diseases.
Abstract: In prokaryotic cells and eukaryotic organelles, the ClpP protease plays an important role in proteostasis. The disruption of the ClpP function has been shown to influence the infectivity and virulence of a number of bacterial pathogens. More recently, ClpP has been found to be involved in various forms of carcinomas and in Perrault syndrome, which is an inherited condition characterized by hearing loss in males and females and by ovarian abnormalities in females. Hence, targeting ClpP is a potentially viable, attractive option for the treatment of different ailments. Herein, the biochemical and cellular activities of ClpP are discussed along with the mechanisms by which ClpP affects bacterial pathogenesis and various human diseases. In addition, a comprehensive overview is given of the new classes of compounds in development that target ClpP. Many of these compounds are currently primarily aimed at treating bacterial infections. Some of these compounds inhibit ClpP activity, while others activate the prot...
TL;DR: This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells.
Abstract: Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitc...
TL;DR: By generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), small molecules able to degrade and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells are identified.
Abstract: P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAF-deficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multidomain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.
TL;DR: Alternative, mechanistically motivated strategies to perform chemistry on the genome of unmodified cells without introducing DSBs are described and a brief analysis of future prospects for genome and transcriptome editing without double-stranded DNA cleavage is analyzed.
Abstract: Genome editing methods have commonly relied on the initial introduction of double-stranded DNA breaks (DSBs), resulting in stochastic insertions, deletions, and translocations at the target genomic locus To achieve gene correction, these methods typically require the introduction of exogenous DNA repair templates and low-efficiency homologous recombination processes In this review, we describe alternative, mechanistically motivated strategies to perform chemistry on the genome of unmodified cells without introducing DSBs One such strategy, base editing, uses chemical and biological insights to directly and permanently convert one target base pair to another Despite its recent introduction, base editing has already enabled a number of new capabilities and applications in the genome editing community We summarize these advances here and discuss the new possibilities that this method has unveiled, concluding with a brief analysis of future prospects for genome and transcriptome editing without double-st
TL;DR: The NCI Program for Natural Product Discovery (NPNPD), a newly launched, national program to advance natural product discovery technologies and facilitate the discovery of structurally defined, validated lead molecules ready for translation will create a prefractionated library from over 125,000 natural product extracts with the aim of producing a publicly-accessible, HTS-amenable library of >1,000,000 fractions.
Abstract: The US National Cancer Institute’s (NCI) Natural Product Repository is one of the world’s largest, most diverse collections of natural products containing over 230,000 unique extracts derived from plant, marine, and microbial organisms that have been collected from biodiverse regions throughout the world. Importantly, this national resource is available to the research community for the screening of extracts and the isolation of bioactive natural products. However, despite the success of natural products in drug discovery, compatibility issues that make extracts challenging for liquid handling systems, extended timelines that complicate natural product-based drug discovery efforts and the presence of pan-assay interfering compounds have reduced enthusiasm for the high-throughput screening (HTS) of crude natural product extract libraries in targeted assay systems. To address these limitations, the NCI Program for Natural Product Discovery (NPNPD), a newly launched, national program to advance natural produ...
TL;DR: It is found that Hsp70 blocks the early stages of t Tau aggregation by suppressing the formation of tau nuclei, and sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation.
Abstract: As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remain unknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature. Here, we used ensemble and single-molecule fluorescence measurements to dissect how Hsp70 counteracts the self-assembly process of the K18 ΔK280 tau variant. We found that Hsp70 blocks the early stages of tau aggregation by suppressing the formation of tau nuclei. Additionally, Hsp70 sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation. Our results provide novel insights into the molecular mechanisms by which the chaperone Hsp70 counteracts the formation, propagation, and toxicity of tau aggregates.
TL;DR: This work is a successful attempt to construct PROTACs based on cell-permeable stabilized peptides, which significantly broadens the chemical space ofprotACs and stabilized peptided.
Abstract: Peptide modulators targeting protein–protein interactions (PPIs) exhibit greater potential than small-molecule drugs in several important aspects including facile modification and relative large contact surface area. Stabilized peptides constructed by variable chemistry methods exhibit improved peptide stability and cell permeability compared to that of the linears. Herein, we designed a stabilized peptide-based proteolysis-targeting chimera (PROTAC) targeting estrogen receptor α (ERα) by tethering an N-terminal aspartic acid cross-linked stabilized peptide ERα modulator (TD-PERM) with a pentapeptide that binds the Von Hippel–Lindau (VHL) E3 ubiquitin ligase complex. The resulting heterobifunctional peptide (TD-PROTAC) selectively recruits ERα to the VHL E3 ligase complex, leading to the degradation of ERα in a proteasome-dependent manner. Compared with the control peptides, TD-PROTAC shows significantly enhanced activities in reducing the transcription of the ERα-downstream genes and inhibiting the proli...
TL;DR: It is shown that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses.
Abstract: The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders.
TL;DR: A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4-20 natural and non-natural amino acids and some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.
Abstract: A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 1012 members composed of 4–20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.
TL;DR: The biology, mechanism, and applications of the type II-C CRISPR systems are reviewed with particular emphasis on their Cas9s, which are central players in multiple steps of theCRISPR pathway, including adaptation and interference.
Abstract: Genome editing technologies have been revolutionized by the discovery of prokaryotic RNA-guided defense system called CRISPR-Cas. Cas9, a single effector protein found in type II CRISPR systems, has been at the heart of this genome editing revolution. Nearly half of the Cas9s discovered so far belong to the type II-C subtype but have not been explored extensively. Type II-C CRISPR-Cas systems are the simplest of the type II systems, employing only three Cas proteins. Cas9s are central players in type II-C systems since they function in multiple steps of the CRISPR pathway, including adaptation and interference. Type II-C CRISPR systems are found in bacteria and archaea from very diverse environments, resulting in Cas9s with unique and potentially useful properties. Certain type II-C Cas9s possess unusually long PAMs, function in unique conditions (e.g., elevated temperature), and tend to be smaller in size. Here, we review the biology, mechanism, and applications of the type II-C CRISPR systems with parti...
TL;DR: This work discusses noncanonical protein folding patterns, with an emphasis on metamorphic proteins, and discusses research areas surrounding metamorphIC proteins that are primed for future exploration, including evolution, drug discovery, and the quest for previously unrecognized metamorphs.
Abstract: Since the proposal of Anfinsen’s thermodynamic hypothesis in 1963, our understanding of protein folding and dynamics has gained significant appreciation of its nuance and complexity. Intrinsically disordered proteins, chameleonic sequences, morpheeins, and metamorphic proteins have broadened the protein folding paradigm. Here, we discuss noncanonical protein folding patterns, with an emphasis on metamorphic proteins, and we review known metamorphic proteins that occur naturally and that have been engineered in the laboratory. Finally, we discuss research areas surrounding metamorphic proteins that are primed for future exploration, including evolution, drug discovery, and the quest for previously unrecognized metamorphs. As we enter an age where we are capable of complex bioinformatic searches and de novo protein design, we are primed to search for previously unrecognized metamorphic proteins and to design our own metamorphs to act as targeted, switchable drugs; biosensors; and more.
TL;DR: The discovery and taxonomy, secondary metabolites, characteristics and application, gene function, and molecular research of B. velezensis are reviewed to provide more clear and comprehensive understanding of this strain for researchers.
Abstract: Bacillus velezensis has been investigated and applied more and more widely recently because it can inhibit fungi and bacteria and become a potential biocontrol agent. In order to provide more clear and comprehensive understanding of B. velezensis for researchers, we collected the recent relevant articles systematically and reviewed the discovery and taxonomy, secondary metabolites, characteristics and application, gene function, and molecular research of B. velezensis. This review will give some direction to the research and application of this strain for the future.
TL;DR: It is demonstrated for the first time that heme can mediate the activation and death of human platelets through ferroptosis, which is an iron-dependent form of nonapoptotic cell death.
Abstract: Hemolysis, a process by which the destruction of red blood cells leads to the release of hemoglobin, is a critical event observed during hemolytic disorders. Under oxidative stress conditions, hemoglobin can release its heme prosthetic group, which is highly cytotoxic and can catalyze the generation of reactive oxygen species (ROS), leading to several undesired redox reactions in the cells. Herein, we demonstrate for the first time that heme can mediate the activation and death of human platelets through ferroptosis, which is an iron-dependent form of nonapoptotic cell death. This study also suggests that the heme-mediated lipid peroxidation and ferroptosis in platelets may play an important role in hemolytic disorders.
TL;DR: The first application of the Automated Ligand Detection System (ALIS) is reported, an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds that target the FMN riboswitch.
Abstract: Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule–ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, l
TL;DR: It is demonstrated that reduction of ferric Cyt C by H2S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cytochrome c reduced/mol of H 2S oxidized.
Abstract: Hydrogen sulfide (H2S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome c (Cyt C) by H2S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H2S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H2S oxidized. H2S increases O2 consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H2S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling.
TL;DR: The fact that gene drive can lead to the spread of fitness-reducing traits makes it an attractive process to consider exploiting to control disease vectors and other pests.
Abstract: Drive is a process of accelerated inheritance from one generation to the next that allows some genes to spread rapidly through populations even if they do not contribute to—or indeed even if they detract from—organismal survival and reproduction Genetic elements that can spread by drive include gametic and zygotic killers, meiotic drivers, homing endonuclease genes, B chromosomes, and transposable elements The fact that gene drive can lead to the spread of fitness-reducing traits (including lethality and sterility) makes it an attractive process to consider exploiting to control disease vectors and other pests There are a number of efforts to develop synthetic gene drive systems, particularly focused on the mosquito-borne diseases that continue to plague us