Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 52,519 bioRxiv papers from 243,473 authors.
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in category biochemistry
1,426 results found. For more information, click each entry to expand.
5,680 downloads biochemistry
The activation or repression of a gene's expression is primarily controlled by changes in the proteins that occupy its regulatory elements. The most common method to identify proteins associated with genomic loci is chromatin immunoprecipitation (ChIP). While having greatly advanced our understanding of gene expression regulation, ChIP requires specific, high quality, IP-competent antibodies against nominated proteins, which can limit its utility and scope for discovery. Thus, a method able to discover and identify proteins associated with a particular genomic locus within the native cellular context would be extremely valuable. Here, we present a novel technology combining recent advances in chemical biology, genome targeting, and quantitative mass spectrometry to develop genomic locus proteomics, a method able to identify proteins which occupy a specific genomic locus.
5,390 downloads biochemistry
Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.
4,673 downloads biochemistry
Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single immunoglobulin VHH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available.
4,536 downloads biochemistry
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
4,329 downloads biochemistry
We show here that, unlike most other prokaryotic Argonaute (Ago) proteins, which are DNA-guided endonucleases, the Natronobacterium gregoryi-derived Ago (NgAgo) can function as a DNA-guided endoribonuclease, cleaving RNA, rather than DNA, in a targeted manner. The NgAgo protein, in complex with 5′-hydroxylated or 5′-phosphrylated oligodeoxyribonucleotides (ODNs) of variable lengths, split RNA targets into two or more fragments in vitro, suggesting its physiological role in bacteria and demonstrating a potential for degrading RNA molecules such as mRNA or lncRNA in eukaryotic cells for study of their functions.
4,072 downloads biochemistry
Evidence is provided that the discovery of small-molecule binders can yield compounds that alter interactomes, protein modifications, cellular lifetimes, and ultimately the specific functions of proteins relevant to human health. These novel mechanisms of action are needed to accelerate the translation of insights from human biology into medicines.
4,000 downloads biochemistry
CRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes.
3,572 downloads biochemistry
Pharmacological interventions that target human ageing would extend individual healthspan and result in dramatic economic benefits to rapidly ageing societies worldwide. For such interventions to be contemplated they need to comprise drugs that are efficacious when given to adults and for which extensive human safety data are available. Here we show that dramatic lifespan extension can be achieved in C.elegans by targeting multiple, evolutionarily conserved ageing pathways using drugs that are already in human use. By targeting multiple synergistic ageing pathways, we are able to slow ageing rate, double lifespan and improves healthspan while minimize developmental and fitness trade-offs. Moreover, we established that there is no synergistic benefit in a daf-2 or daf-7 background, implying the involvement of the TGFβ and IGF pathways in this synergy. Employing lipidomics and transcriptomics analysis we found lipid metabolism to be affected resulting in increased monounsaturated fatty acids (MUFA) and decrease membrane peroxidation index. Our best drug combination showed a conserved lifespan extension in fruit flies. To the best of our knowledge, this is the largest lifespan effect ever reported for any adult-onset drug treatment in C. elegans. This drug-repurposing approach, using drugs already approved for humans to target multiple conserved aging pathways simultaneously, could lead to interventions that prevent age-related diseases and overall frailty in a rapidly ageing population.
3,346 downloads biochemistry
We are currently facing an avalanche of cryoEM (cryogenic Electron Microscopy) publications presenting beautiful structures at resolution levels of ~3 Angstrom: a true resolution revolution [Kuehlbrandt, Science 343(2014)1443,1444]. Impressive as these results may be, a fundamental statistical error has persisted in the literature that affects the numerical resolution values for practically all published structures. The error goes back to a misinterpretation of basic statistics and pervades virtually all popular cryo EM quality metrics. The resolution in cryo EM is typically assessed by the Fourier Shell Correlation FSC [Harauz & van Heel: Optik 73(1986)146,156] using a fixed threshold value of 0.143 (FSC 0.143) [Rosenthal, Henderson, J.Mol.Biol. 333(2003)721,745]. Using a simple model experiment we illustrate why this fixed threshold is flawed and we pinpoint the source of the resolution confusion. When two vectors are uncorrelated the expectation value of their inner-product is zero. That, however, does not imply that each individual inner-product of the vectors is zero (the vectors are not orthogonal). This error was introduced to electron microscopy in [Frank & Al Ali, Nature 256(1975)376,379] and has since proliferated into virtually all quality and resolution related metrics in EM. One criterion not affected by this error is the information-based half bit FSC threshold [van Heel & Schatz: J.Struct.Biol. 151(2005)250-262].
3,338 downloads biochemistry
Calorimetry, thermogravimetry and mass spectrometry were used to follow the thermal decomposition of the eight amino acids G, C, D, N, E, Q, R and H between 185 °C and 280 °C. Endothermic heats of decomposition between 72 and 151 kJ/mol are needed to form 12 to 70 % volatile products. This process is neither melting nor sublimation. With exception of cysteine they emit mainly H2O, some NH3 and no CO2. Cysteine produces CO2 and little else. The reactions are described by polynomials, AA → a (NH3) + b (H2O) + c (CO2) + d (H2S) + e (residue), with integer or half integer coefficients. The solid monomolecular residues are rich in peptide bonds.
2,989 downloads biochemistry
Programmed death-1 (PD-1) is a co-inhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). Here, by titrating the strength of PD-1 signaling in both biochemical reconstitution systems and in T cells, we demonstrate that the coreceptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1- recruited Shp2 phosphatase. We also show that PD-1 colocalizes with the costimulatory receptor CD28 in plasma membrane microclusters but partially segregates from the TCR. These results reveal that PD-1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting that costimulatory pathways may play unexpected roles in regulating effector T cell function and therapeutic responses to anti-PD-L1/PD-1.
2,716 downloads biochemistry
CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, explain the compact molecular architecture of Cas13d and provide insights into the structural transitions required for enzyme activation. Our comprehensive analysis of Cas13d in diverse enzymatic states facilitated site-specific truncations for minimal size and delineates a blueprint for improving biomolecular applications of RNA targeting.
2,647 downloads biochemistry
The spatial organization of RNA within cells is a crucial factor in a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER), with higher specificity and coverage than do conventional approaches. We furthermore identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.
2,599 downloads biochemistry
The cannabinoid signaling system has recently garnered attention as a therapeutic target for numerous indications, and cannabinoids are now being pursued as new treatment options in diverse medical fields such as neurology, gastroenterology, pain management, and oncology. Cannabinoids are extremely hydrophobic and relatively unstable compounds, and as a result, formulation and delivery options are severely limited. Enzymatic glycosylation is a strategy to alter the physicochemical properties of small molecules, often improving their stability and aqueous solubility, as well as enabling site-specific drug targeting strategies. To determine if cannabinoids are a candidate for glycosylation, a library of glucosyltransferase (UGT) enzymes was screened for glycosylation activity towards various cannabinoids. The UGT76G1 enzyme from Stevia rebaudiana has been identified as having glucosyltransferase activity towards a broad range of cannabinoids. Compounds that were successfully glycosylated by UGT76G1 include the phytocannabinoids cannabidiol (CBD), Δ9-tetrahydrocannabinol (Δ9-THC), cannabidivarin (CBDV), and cannabinol (CBN), and the human endocannabinoids anandamide (AEA), 2-arachidonoyl-glycerol (2AG), 1-arachidonoyl-glycerol (1AG), and synaptamide (DHEA). Interestingly, UGT76G1 is able to transfer primary, secondary, and tertiary glycosylations at each acceptor of most of the cannabinoids tested. Additionally, Os03g0702000p, a glycosyltransferase from Oryza sativa, was able to transfer secondary glucose residues onto cannabinoid monoglycosides previously established by UGT76G1. This new class of cannabinoid-glycosides has been termed cannabosides. The compounds have greatly improved solubility in aqueous solutions. This increased aqueous solubility may enable new oral pharmaceutical delivery options for cannabinoids, as well as targeted delivery and release of cannabinoids within the intestines through glycoside prodrug metabolism.
2,222 downloads biochemistry
The Hsp90 molecular chaperone and its Cdc37 co-chaperone help stabilize and activate over half of the human kinome. However, neither the mechanism by which these chaperones assist their client kinases nor why some kinases are addicted to Hsp90 while closely related family members are independent is known. Missing has been any structural understanding of these interactions, with no full-length structures of human Hsp90, Cdc37 or either of these proteins with a kinase. Here we report a 3.9A cryoEM structure of the Hsp90:Cdc37:Cdk4 kinase complex. Cdk4 is in a novel conformation, with its two lobes completely separated. Cdc37 mimics part of the kinase N-lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, safely trapping the kinase in an unfolded state. Based on this novel structure and extensive previous data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.
2,144 downloads biochemistry
How phospholipids are trafficked between the bacterial inner and outer membranes through the intervening hydrophilic space of the periplasm is not known. Here we report that members of the mammalian cell entry (MCE) protein family form structurally diverse hexameric rings and barrels with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring as part of a larger ABC transporter complex in the inner membrane, and employs a soluble lipid-binding protein to ferry lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the entire periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the inner and outer membranes of bacteria and some eukaryotic organelles.
2,100 downloads biochemistry
Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a gene's synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized ~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.
2,052 downloads biochemistry
CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to proteolytic degradation and unsuitable for applications requiring function at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage over a wide temperature range and has an enhanced protein lifetime in human plasma. GeoCas9 is active at temperatures up to 70°C, compared to 45°C for Streptococcus pyogenes Cas9 (SpyCas9), which greatly expands the temperature range for CRISPR-Cas9 applications. By comparing features of two closely related Geobacillus homologs, we created a variant of GeoCas9 that doubles the DNA target sequences that can be recognized by this system. We also found that GeoCas9 is an effective tool for editing mammalian genomes when delivered as a ribonucleoprotein (RNP) complex. Together with an increased lifetime in human plasma, the thermostable GeoCas9 provides the foundation for improved RNP delivery in vivo and expands the temperature range of CRISPR-Cas9.
2,042 downloads biochemistry
Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy to discover that DCAF16 - a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases - promotes nuclear-restricted protein degradation upon modification by cysteine-directed heterobifunctional electrophilic compounds.
1,977 downloads biochemistry
Chemicals such as drugs, hormones, and odorants can have many potential interactions with endogenous targets, and uncovering these relationships is critical for understanding and modulating function. Mammalian olfactory receptors (ORs), a large family of G protein-coupled receptors, mediate olfaction through activation by small molecules. Each OR can respond to many odorants, and vice versa, making exploring this space one interaction at a time difficult. We developed a high-throughput receptor screening platform in human cell lines to screen libraries of chemicals against a multiplexed library of receptors using next-generation sequencing of barcoded genetic reporters. We screened three concentrations of 181 odorants, where in each well we record the activity of 39 ORs simultaneously, and identified 79 novel associations, including ligands for 15 orphan receptors. This platform allows the cost-effective mapping of large chemical libraries to receptor repertoires at scale.
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