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Most downloaded bioRxiv papers, all time

in category biochemistry

1,600 results found. For more information, click each entry to expand.

1: Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells
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Posted to bioRxiv 16 Jan 2018

Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells
5,833 downloads biochemistry

Philip JR Roche, Heidi Gytz, Faiz Hussain, Christopher JF Cameron, Denis Paquette, Mathieu Blanchette, Josée Dostie, Bhushan Nagar, Uri David Akavia

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.

2: CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells
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Posted to bioRxiv 04 Jul 2017

CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells
5,754 downloads biochemistry

Samuel A. Myers, Jason Wright, Feng Zhang, Steven A. Carr

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.

3: Platform for rapid nanobody discovery in vitro
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Posted to bioRxiv 16 Jun 2017

Platform for rapid nanobody discovery in vitro
4,835 downloads biochemistry

Conor McMahon, Alexander S Baier, Sanduo Zheng, Roberta Pascolutti, Janice X. Ong, Sarah C. Erlandson, Daniel Hilger, Aaron M. Ring, Aashish Manglik, Andrew C. Kruse

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: Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
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Posted to bioRxiv 06 Jul 2017

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
4,599 downloads biochemistry

Janice S Chen, Yavuz S. Dagdas, Benjamin P. Kleinstiver, Moira M. Welch, Lucas B Harrington, Samuel H. Sternberg, J. Keith Joung, Ahmet Yildiz, Jennifer A Doudna

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.

5: DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute
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Posted to bioRxiv 20 Jan 2017

DNA-dependent RNA cleavage by the Natronobacterium gregoryi Argonaute
4,356 downloads biochemistry

Ye Sunghyeok, Bae Taegeun, Kim Kyoungmi, Habib Omer, Lee Seung Hwan, Kim Yoon Young, Lee Kang-In, Kim Seokjoong, Kim Jin-Soo

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.

6: CRISPR-Cas12a target binding unleashes single-stranded DNase activity
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Posted to bioRxiv 29 Nov 2017

CRISPR-Cas12a target binding unleashes single-stranded DNase activity
4,198 downloads biochemistry

Janice S Chen, Enbo Ma, Lucas B Harrington, Xinran Tian, Jennifer A Doudna

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.

7: A chemical biology view of bioactive small molecules and a binder-based approach to connect biology to precision medicines
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Posted to bioRxiv 07 Aug 2018

A chemical biology view of bioactive small molecules and a binder-based approach to connect biology to precision medicines
4,194 downloads biochemistry

Stuart L Schreiber

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.

8: Doubling healthy lifespan using drug synergies
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Posted to bioRxiv 21 Jun 2017

Doubling healthy lifespan using drug synergies
3,762 downloads biochemistry

Tesfahun Dessale, Krishna Chaithanya Batchu, Diogo Barardo, Ng Li Fang, Vanessa Yuk Man Lam, Linfan Xiao, Markus R. Wenk, Nicholas S. Tolwinski, Jan Gruber

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.

9: Thermal Decomposition Of The Amino Acids Glycine, Cysteine, Aspartic Acid, Asparagine, Glutamic Acid, Glutamine, Arginine And Histidine
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Posted to bioRxiv 22 Mar 2017

Thermal Decomposition Of The Amino Acids Glycine, Cysteine, Aspartic Acid, Asparagine, Glutamic Acid, Glutamine, Arginine And Histidine
3,551 downloads biochemistry

Ingrid M Weiss, Christina Muth, Robert Drumm, Helmut O.K. Kirchner

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.

10: REASSESSING THE REVOLUTIONS RESOLUTIONS
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Posted to bioRxiv 24 Nov 2017

REASSESSING THE REVOLUTIONS RESOLUTIONS
3,446 downloads biochemistry

Marin van Heel, Michael Schatz

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].

11: Aequorea victoria’s secrets
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Posted to bioRxiv 19 Jun 2019

Aequorea victoria’s secrets
3,345 downloads biochemistry

Talley Lambert, Hadrien Depernet, Guillaume Gotthard, Darrin T Schultz, lsabelle Navizet

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.

12: T cell co-stimulatory receptor CD28 is a primary target for PD-1-mediated inhibition
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Posted to bioRxiv 09 Nov 2016

T cell co-stimulatory receptor CD28 is a primary target for PD-1-mediated inhibition
3,047 downloads biochemistry

Enfu Hui, Jeanne Cheung, Jing Zhu, Xiaolei Su, Marcus J Taylor, Heidi A. Wallweber, Dibyendu K Sasmal, Jun Huang, Jeong M. Kim, Ira Mellman, Ronald D Vale

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.

13: Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d
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Posted to bioRxiv 04 May 2018

Structural basis for the RNA-guided ribonuclease activity of CRISPR-Cas13d
2,938 downloads biochemistry

Cheng Zhang, Silvana Konermann, Nicholas J Brideau, Peter Lotfy, Scott J Novick, Timothy Strutzenberg, Patrick R Griffin, Patrick D Hsu, Dmitry Lyumkis

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.

14: Cannabinoid glycosides: In vitro production of a new class of cannabinoids with improved physicochemical properties
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Posted to bioRxiv 30 Jan 2017

Cannabinoid glycosides: In vitro production of a new class of cannabinoids with improved physicochemical properties
2,887 downloads biochemistry

Janee' M. Hardman, Robert T. Brooke, Brandon J. Zipp

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.

15: A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins
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Posted to bioRxiv 17 May 2019

A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins
2,692 downloads biochemistry

Hansjoerg Goetzke, Markus Kilisch, Markel Martinez-Carranza, Shama Sograte-Idrissi, Abirami Rajavel, Thomas Schlichthaerle, Niklas Engels, Ralf Jungmann, Paul Stenmark, Felipe Opazo, Steffen Frey

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We developed a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in-vivo detection of proteins. By solving the crystal structure of the complex we were able to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

16: Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking
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Posted to bioRxiv 21 Jun 2017

Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking
2,680 downloads biochemistry

Pornchai Kaewsapsak, David M Shechner, William Mallard, John L. Rinn, Alice Y. Ting

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.

17: Modified TCA/acetone precipitation of plant proteins for proteomic analysis
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Posted to bioRxiv 01 Aug 2018

Modified TCA/acetone precipitation of plant proteins for proteomic analysis
2,448 downloads biochemistry

Liangjie Niu, Hang Zhang, Zhaokun Wu, Wei Wang, Hui Liu, Xiaolin Wu

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.

18: Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16
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Posted to bioRxiv 15 Oct 2018

Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16
2,342 downloads biochemistry

Xiaoyu Zhang, Vincent M Crowley, Thomas G Wucherpfennig, Melissa M Dix, Benjamin F Cravatt

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.

19: Atomic structure of Hsp90:Cdc37:Cdk4 reveals Hsp90 regulates kinase via dramatic unfolding
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Posted to bioRxiv 23 Feb 2016

Atomic structure of Hsp90:Cdc37:Cdk4 reveals Hsp90 regulates kinase via dramatic unfolding
2,265 downloads biochemistry

Kliment A Verba, Ray Yu-Ruei Wang, Akihiko Arakawa, Yanxin Liu, Mikako Shirouzu, Shigeyuki Yokoyama, David A Agard

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.

20: Architectures of a lipid transport systems for the bacterial outer membrane
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Posted to bioRxiv 18 Jul 2016

Architectures of a lipid transport systems for the bacterial outer membrane
2,195 downloads biochemistry

Damian C Ekiert, Gira Bhabha, Garrett Greenan, Sergey Ovchinnikov, Jeffery S Cox, Ronald D Vale

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.

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