Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 77,888 bioRxiv papers from 337,542 authors.
Most downloaded bioRxiv papers, all time
in category biochemistry
2,372 results found. For more information, click each entry to expand.
8,960 downloads biochemistry
The recent emergence of a novel coronavirus associated with an ongoing outbreak of pneumonia (Covid-2019) resulted in infections of more than 72,000 people and claimed over 1,800 lives. Coronavirus spike (S) glycoprotein trimers promote entry into cells and are the main target of the humoral immune response. We show here that SARS-CoV-2 S mediates entry in VeroE6 cells and in BHK cells transiently transfected with human ACE2, establishing ACE2 as a functional receptor for this novel coronavirus. We further demonstrate that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, which correlates with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and other SARS-related CoVs. We determined a cryo-electron microscopy structure of the SARS-CoV-2 S ectodomain trimer, demonstrating spontaneous opening of the receptor-binding domain, and providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal sera potently inhibited SARS-CoV-2 S-mediated entry into target cells, thereby indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
8,500 downloads biochemistry
Angiotensin-converting enzyme 2 (ACE2) is the surface receptor for SARS coronavirus (SARS-CoV) through interaction with its spike glycoprotein (S protein). ACE2 is also suggested to be the receptor for the new coronavirus (2019-nCoV), which is causing a serious epidemic in China manifested with severe respiratory syndrome. BAT1 (SLC6A19) is a neutral amino acid transporter whose surface expression in intestinal cells requires ACE2. Here we present the 2.9 Å resolution cryo-EM structure of full-length human ACE2 in complex with BAT1. The complex, assembled as a dimer of ACE2-BAT1 heterodimers, exhibits open and closed conformations due to the shifts of the peptidase domains of ACE2. A newly resolved Collectrin-like domain (CLD) on ACE2 mediates homo-dimerization. The extended TM7 in each BAT1 clamps CLD of ACE2. Structural analysis suggests that the ACE2-BAT1 complex can bind two S proteins simultaneously, providing important clues to the molecular basis for coronavirus recognition and infection.
7,930 downloads biochemistry
Zhenming Jin, Xiaoyu Du, Yechun Xu, Yongqiang Deng, Meiqin Liu, Yao Zhao, Bing Zhang, Xiaofeng Li, Leike Zhang, Chao Peng, Yinkai Duan, Jing Yu, Lin Wang, Kailin Yang, Fengjiang Liu, Rendi Jiang, Xinglou Yang, Tian You, Xiaoce Liu, Xiuna Yang, Fang Bai, Hong Liu, Xiang Liu, Luke W. Guddat, Wenqing Xu, Gengfu Xiao, Chengfeng Qin, Zhengli Shi, Hualiang Jiang, Zihe Rao, Haitao Yang
A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.
7,287 downloads biochemistry
In December 2019, the first cases of a novel coronavirus infection were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for COVID-19, therefore research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 Mpro protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 proteases, using natural and a large panel of unnatural amino acids. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.
6,746 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.
6,016 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.
5,796 downloads biochemistry
Angiotensin-converting enzyme 2 (ACE2) has been suggested to be the cellular receptor for the new coronavirus (2019-nCoV) that is causing the coronavirus disease 2019 (COVID-19). Like other coronaviruses such as the SARS-CoV, the 2019-nCoV uses the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) to engage ACE2. We most recently determined the structure of the full-length human ACE2 in complex with a neutral amino acid transporter BAT1. Here we report the cryo-EM structure of the full-length human ACE2 bound to the RBD of the 2019-nCoV at an overall resolution of 2.9 Å in the presence of BAT1. The local resolution at the ACE2-RBD interface is 3.5 Å, allowing analysis of the detailed interactions between the RBD and the receptor. Similar to that for the SARS-CoV, the RBD of the 2019-nCoV is recognized by the extracellular peptidase domain (PD) of ACE2 mainly through polar residues. Pairwise comparison reveals a number of variations that may determine the different affinities between ACE2 and the RBDs from these two related viruses.
5,317 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.
4,980 downloads biochemistry
Using mRNA-Seq and de novo transcriptome assembly, we identified, cloned and characterized nine previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from avGFP. Among these FPs are the brightest GFP homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including two that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.
4,784 downloads biochemistry
Three anti-HIV drugs, ritonavir, lopinavir and darunavir, might have therapeutic effect on coronavirus disease 2019 (COVID-19). In this study, the structure models of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteases, coronavirus endopeptidase C30 (CEP\_C30) and papain like viral protease (PLVP), were built by homology modeling. Ritonavir, lopinavir and darunavir were then docked to the models, respectively, followed by energy minimization of the protease-drug complexes. In the simulations, ritonavir can bind to CEP\_C30 most suitably, and induce significant conformation changes of CEP\_C30; lopinavir can also bind to CEP\_C30 suitably, and induce significant conformation changes of CEP\_C30; darunavir can bind to PLVP suitably with slight conformation changes of PLVP. It is suggested that the therapeutic effect of ritonavir and lopinavir on COVID-19 may be mainly due to their inhibitory effect on CEP\_C30, while ritonavir may have stronger efficacy; the inhibitory effect of darunavir on SARS-CoV-2 and its potential therapeutic effect may be mainly due to its inhibitory effect on PLVP.
4,695 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.
4,641 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.
4,522 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.
4,494 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].
4,489 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.
4,437 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.
4,132 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.
4,018 downloads biochemistry
Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis.
3,974 downloads biochemistry
2’3’-cyclic GMP-AMP (cGAMP) is characterized as an intracellular second messenger that is synthesized in response to cytosolic dsDNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, using mass spectrometry, we detected that cGAMP is continuously exported as a soluble factor by an engineered cell line but then efficiently cleared by ENPP1, explaining why it has escaped detection until now. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we detected that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In tumors, depletion of extracellular cGAMP using a neutralizing protein decreased tumor-associated immune cell infiltration in a tumor cGAS and host STING dependent manner. Depletion of extracellular cGAMP also abolished the curative effect of IR. Boosting extracellular cGAMP by ENPP1 inhibitors synergizes with IR to shrink tumors in mice. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be stimulated and harnessed to treat less immunogenic cancers.
3,860 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.
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