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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 67,095 bioRxiv papers from 295,233 authors.

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in category biochemistry

1,938 results found. For more information, click each entry to expand.

1841: HOPS recognizes each SNARE, assembling ternary trans-complexes for sudden fusion upon engagement with the 4th SNARE
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Posted to bioRxiv 29 Aug 2019

HOPS recognizes each SNARE, assembling ternary trans-complexes for sudden fusion upon engagement with the 4th SNARE
88 downloads biochemistry

Hongki Song, Amy Orr, Max Harner, William T Wickner

Vacuole fusion requires SNAREs, Sec17/18, a Rab, and HOPS. We find that co-incubation of HOPS, proteoliposomes bearing the Rab and R-SNARE, and proteoliposomes with the Rab and any two Q-SNAREs yields a trans complex which includes these 3 SNAREs. The missing Q-SNARE then triggers a burst of fusion, indicating that each HOPS, R-, and QxQy-SNARE trans -complex is an activated intermediate for functional Qz-SNARE incorporation. HOPS can assemble activated fusion intermediates because it recognizes each of the four SNAREs, binding them independently. HOPS-dependent fusion is saturable for each Q-SNARE, indicating saturable functional sites on HOPS. Though a nonspecific tether allows fusion with pre-assembled Q-SNAREs, only HOPS catalyzes fusion when the Q-SNAREs are not pre-assembled by ushering each Q-SNARE into a functional complex. In contrast, there is little spontaneous functional assembly of the 3 Q-SNAREs. HOPS thus recognizes each of the 4 SNAREs to assemble a versatile set of activated fusion intermediates.

1842: Characterization of xyloglucan-specific fucosyltransferase activity in Golgi-enriched microsomal preparations from wheat seedlings
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Posted to bioRxiv 15 Oct 2019

Characterization of xyloglucan-specific fucosyltransferase activity in Golgi-enriched microsomal preparations from wheat seedlings
88 downloads biochemistry

Richard E Wiemels, Wei Zeng, Nan Jiang, Ahmed Faik

Xyloglucan (XyG) is a major hemicellulosic polymer in primary cell walls of dicotyledonous plants but represents only a minor constituent of cell walls from graminaceous monocotyledons (Poaceae). Our current information on XyG biosynthesis in vitro comes exclusively from studies on dicotyledonous plants. While XyG has been reported in grass cell walls, there are no studies of XyG biosynthesis in vitro in grasses. In this report, we investigated XyG structure and biosynthesis in etiolated wheat seedlings and showed that their walls contain small amounts (4-14%) of XyG. Furthermore, structural analysis using electrospray ionization mass spectrometry (ESI-MS) and high pH anion exchange chromatography (HPAEC) revealed that wheat XyG may be of XXGGG-type. Interestingly, detergent extracts from root microsomes were able to fucosylate tamarind XyG in vitro in a similar way as fucosyltransferase activity from Arabidopsis thaliana (AtFUT1) and pea (PsFUT1). Endoglucanase digestion of the [14C]fucosylated-tamarind XyG formed by the wheat fucosyltransferase activity released radiolabeled oligosaccharides that co-eluted with authentic fucoslyated XyG oligosaccharides (XXFG and XLFG). Although wheat fucosyltransferase activity was low, it appeared to be specific to XyG and required divalent ions (Mg2+ or Mn2+) for full activity. Together, these results suggest that the XyG fucosylation mechanism is conserved between monocots and dicots.

1843: A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry
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Posted to bioRxiv 21 Nov 2019

A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry
88 downloads biochemistry

Janelle A. Hayes, Brendan J. Hilbert, Christl Gaubitz, Nicholas P. Stone, Brian A. Kelch

Tailed bacteriophage use a DNA packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component acts as a molecular matchmaker by recognizing the viral genome as well as the main motor component, the large terminase (TerL). How TerS binds DNA and the TerL protein remains unclear. Here, we identify the TerS protein of the thermophilic bacteriophage P74-26. TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. Our cryo-EM structure shows that TerSP76-26 forms a ring with a wide central pore and radially arrayed helix-turn-helix (HTH) domains. These HTH domains, which are thought to bind DNA by wrapping the helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA binding domain imposes strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacks the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL, suggesting that a well-ordered C-terminal β-barrel domain is not necessary for TerS to carry out its function as a matchmaker.

1844: Modular protein-oligonucleotide signal exchange
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Posted to bioRxiv 31 Oct 2019

Modular protein-oligonucleotide signal exchange
88 downloads biochemistry

Deepak Kumar Agrawal

The ability to detect a protein selectively and produce a predicted signal in real time is a long-lasting engineering challenge in the field of biochemistry. Such a mechanism typically requires a sensing module to recognize the input protein and a translation module to produce a programmable output signal that reflects the concentration of the input. Here we present a generic biomolecular reaction process that exchanges the concentration of an input protein with a DNA oligonucleotide. This approach uses the unique characteristic of DNA oligonucleotide aptamer that can either bind to a specific protein or to a complementary DNA oligonucleotide reversibly. We then pass the information of the protein concentration to the output signal through DNA strand displacement reactions. Using this strategy, we design and characterize four different exchange processes that can produce modular DNA oligonucleotides in response to different proteins such as clinically important human α-thrombin and vascular endothelial growth factor (VEGF). These exchange processes are capable of real time sensing and are modular such that they can be used for concurrent detection of different proteins with well-defined input-output characteristics. The novelty and simplicity of our approach encourage to develop advanced biochemical systems for point-of-care testing of infectious diseases and treatments.

1845: A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity
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Posted to bioRxiv 07 Oct 2019

A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity
88 downloads biochemistry

Christina R Bourne, Eswar Reddem, Tamiko Murphy, Jessica R Ames, Julia McGillick

Toxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB can perform both ribosome dependent and independent RNA cleavage. We have also characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence of the ribosome. The toxin concentration inside bacterial cells is unknown and according to current models, should increase upon activation of YoeB through degradation of the YefM antitoxin. The discovery of general nuclease activity by AtYoeB, and perhaps other YoeB toxins, offers an opportunity to explore the plasticity of this protein fold and its potential role in the evolution of nucleases.

1846: Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor
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Posted to bioRxiv 21 Nov 2019

Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor
87 downloads biochemistry

Oliver Wilkinson, Carolina Carrasco, Clara Aicart-Ramos, Fernando Moreno-Herrero, Mark S Dillingham

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5prime to 3prime ssDNA translocase and 5prime to 3prime helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of ~200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

1847: Proteins That Interact with the Mucin-Type Glycoprotein Msb2p Include Regulators of the Actin Cytoskeleton
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Posted to bioRxiv 29 Sep 2019

Proteins That Interact with the Mucin-Type Glycoprotein Msb2p Include Regulators of the Actin Cytoskeleton
87 downloads biochemistry

Aditi Prabhakar, Nadia Vadaie, Thomas Krzystek, Paul J Cullen

Transmembrane mucin-type glycoproteins can regulate signal transduction pathways. In yeast, signaling mucins regulate mitogen-activated protein kinase (MAPK) pathways that induce cell differentiation to filamentous growth (fMAPK pathway) and the response to osmotic stress (HOG pathway). To explore regulatory aspects of signaling mucin function, protein microarrays were used to identify proteins that interact with the cytoplasmic domain of the mucin-like glycoprotein, Msb2p. Eighteen proteins were identified that comprised functional categories of metabolism, actin filament capping and depolymerization, aerobic and anaerobic growth, chromatin organization and bud growth, sporulation, ribosome biogenesis, protein modification by iron-sulfur clusters, RNA catabolism, and DNA replication and DNA repair. A subunit of actin capping protein, Cap2p, interacted with the cytoplasmic domain of Msb2p. Cells lacking Cap2p showed altered localization of Msb2p and increased shedding of Msb2p N-terminal glycosylated domain. Consistent with its role in regulating the actin cytoskeleton, Cap2p, and another Msb2p-interacting protein, Aip1p, were required for the enhanced cell polarization during filamentous growth. Our study identifies proteins that connect a signaling mucin to diverse cellular processes and may provide insight into new aspects of mucin function.

1848: Hepcidin and conventional markers to detect iron deficiency in severely anaemic HIV-infected patients in Malawi.
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Posted to bioRxiv 10 Jun 2019

Hepcidin and conventional markers to detect iron deficiency in severely anaemic HIV-infected patients in Malawi.
87 downloads biochemistry

Minke Nl Hendrina Willemina Huibers, Job C Calis, Theresa J Allain, Sarah E. Coupland, Chimota Phiri, Kamija S. Phiri, Dorien W Swinkels, Michael Boele van Hensbroek, Imelda Bates

Introduction: Iron deficiency is a treatable cause of severe anaemia in low-and-middle-income-countries (LMIC). Diagnosing it remains challenging as peripheral blood markers poorly reflect bone-marrow iron deficiency (BM-ID), especially in the context of HIV-infection. Methods: Severe anaemic (haemoglobin ≤70g/l) HIV-infected adults were recruited at Queen Elizabeth Central Hospital, Blantyre, Malawi. BM-ID was evaluated. Accuracy of blood markers including hepcidin alongside mean corpuscular volume, mean cellular haemoglobin concentration, serum iron, serum ferritin, soluble transferrin receptor (sTfR), sTfR -index, sTfR –ratio to detect BM-ID was valued by ROC area under the curve (AUCROC ). Results: Seventy-three patients were enrolled and 35 (48.0%) had BM-ID. Hepcidin and MCV performed best; AUCROC of 0.593 and 0.545. Other markers performed poorly (ROC<0.5). The AUCROC of hepcidin in males was 0.767 (sensitivity 80%, specificity 78%) and in women 0.490 (sensitivity 60%, specificity 61%). Conclusion: BM-ID deficiency was common in severely anaemic HIV-infected patients and is an important and potential treatable contributor to severe anaemia. Hepcidin was the best, though still suboptimal, marker of BM-ID. Hepcidin, which is directly linked to iron absorption, is a very promising marker to guide curative iron supplementation policies in severely anaemic HIV-infected patients.

1849: An experimentally-derived measure of inter-replicate variation in reference samples: the same-same permutation methodology
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Posted to bioRxiv 08 Oct 2019

An experimentally-derived measure of inter-replicate variation in reference samples: the same-same permutation methodology
86 downloads biochemistry

David C.L. Handler, Paul A. Haynes

The multiple testing problem is a well-known statistical stumbling block in high-throughput data analysis, where large scale repetition of statistical methods introduces unwanted noise into the results. While approaches exist to overcome the multiple testing problem, these methods focus on theoretical statistical clarification rather than incorporating experimentally-derived measures to ensure appropriately tailored analysis parameters. Here, we introduce a method for estimating inter-replicate variability in reference samples for a quantitative proteomics experiment using permutation analysis. This can function as a modulator to multiple testing corrections such as the Benjamini-Hochberg ordered Q value test. We refer to this as a same-same analysis, since this method incorporates the use of six biological replicates of the reference sample and determines, through non-redundant triplet pairwise comparisons, the level of quantitative noise inherent within the system. The method can be used to produce an experiment-specific Q value cut-off that achieves a specified false discovery rate at the quantitation level, such as 1%. The same-same method is applicable to any experimental set that incorporates six replicates of a reference sample. To facilitate access to this approach, we have developed a same-same analysis R module that is freely available and ready to use via the internet.

1850: A microplate screen for metal-binding activity based on a nickel-binding protein from Clostridium carboxidivorans
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Posted to bioRxiv 28 Oct 2019

A microplate screen for metal-binding activity based on a nickel-binding protein from Clostridium carboxidivorans
86 downloads biochemistry

Patrick Diep, Radhakrishnan Mahadevan, Alexander Yakunin

Metal ion specificity of the solute binding protein (SBP) component of ATP-binding cassette (ABC) transporters is determined through binding studies. Clostridium carboxidivorans P7T possesses an operon that encodes for an ABC transporter with two SBPs ( Cc SBPI and Cc SBPII) of unknown specificity. Bioinformatics analysis of the operon reveals that the ABC transporter may have a potential role in nickel acquisition based on sequence similarity between the two SBPs and the previously characterized NikZ from Campylobacter jejuni . To screen for the uncharacterized ABC transporter substrate specificity, Cc SBPII was purified with minimal metal contamination and affinity tag removal. A ligand titration assay in microplate format was then developed and optimized to detect metal-binding activity using intrinsic protein fluorescence quenching. Cc SBPII was found to bind Cu2+ > Ni2+ > Co2+ (Kd 12.2, 32.6, and 142.6 μM, respectively). However, the presence of L-histidine greatly increased Cc SBPII affinity to Ni2+ allowing it to bind Ni2+ with nanomolar affinity (Kd 0.19 μM). This is the first demonstration of a microplate-based screen for metal-binding activity with SBPs, which is amenable to automation for high-throughput applications.

1851: Lipidomics analysis of juveniles’ blue mussels (Mytilus edulis L. 1758), a key economic and ecological species.
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Posted to bioRxiv 13 Sep 2019

Lipidomics analysis of juveniles’ blue mussels (Mytilus edulis L. 1758), a key economic and ecological species.
85 downloads biochemistry

Vincenzo Alessandro Laudicella, Christine Beveridge, Stefano Carboni, Sofia Cota Franco, Mary K. Doherty, Nina Long, Elaine Mitchell, Michele S. Stanley, Philip D. Whitfield, Adam D. Hughes

Blue mussels (Mytilus edulis L.) are important components of coastal ecosystems functioning through benthopelagic coupling and ecosystem engineering. At the same time, mussel production is central in the economy of coastal areas. Therefore, understanding their nutritional, physiological and metabolic processes at key life stages is important for their management, both within food production systems and in wild populations. Lipids are crucial molecules for bivalve growth, but their diversity and roles have been considered from fatty acid (FA) perspective. In this paper, we applied lipidomics to bivalve nutrition. Lipidomics provides a holistic perspective on lipid patterns; by examining the lipidome, important physiological information can be acquired. Here, we use controlled laboratory experiments to elucidate the responses to changes in the diet of newly settled mussels juveniles, one of the most critical life stages. The diets considered in this study are single strains diet of Cylindrotheca fusiformis CCAP 1017/2 – CYL, Isochrysis galbana CCAP 927/1– ISO, Monodopsis subterranean CCAP 848/1 – MONO, Nannochloropsis oceanica CCAP 849/10– NANNO and a commercial algae paste –SP. The diets had a significant effect on spat GR and WI, and according to their efficacy resulted ranked as follows: ISO>NANNO/CYL>SP>MONO. Spat FA composition and neutral lipid content (principally triacylglycerols - TG), were influenced by the diets. Furthermore, untargeted lipidomics also showed shifts in several phospholipid species, with changes related to the essential PUFA available from the diet. TG content, neutral lipids and several TG and FA species were correlated (Spearman R2>0.8 FDR p<0.05) with spat WI, suggesting their possible application as markers of mussel juvenile condition. The availability of dietary essential PUFA deeply modified the spat lipidome both for neutral and for polar lipids. This change in the lipidome could have major impacts on their ecology and their production for food.

1852: Revealing acquired resistance mechanisms of kinase-targeted drugs using an on-the-fly, function-site interaction fingerprint approach
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Posted to bioRxiv 13 Nov 2019

Revealing acquired resistance mechanisms of kinase-targeted drugs using an on-the-fly, function-site interaction fingerprint approach
85 downloads biochemistry

Zheng Zhao, Philip E. Bourne

Although kinase-targeted drugs have achieved significant clinical success, they are frequently subject to the limitations of drug resistance, which has become a primary vulnerability to targeted drug therapy. Therefore, deciphering resistance mechanisms is an important step in designing more efficacious, anti-resistant, drugs. Here we studied two FDA-approved kinase drugs: Crizotinib and Ceritinib, which are first- and second-generation anaplastic lymphoma kinase (ALK) targeted inhibitors, to unravel drug-resistance mechanisms. We used an on-the-fly, function-site interaction fingerprint (on-the-fly Fs-IFP) approach by combining binding free energy surface calculations with the Fs-IFPs. Establishing the potentials of mean force and monitoring the atomic-scale protein-ligand interactions, before and after the L1196M-induced drug resistance, revealed insights into drug-resistance/anti-resistant mechanisms. Crizotinib prefers to bind the wild type ALK kinase domain, whereas Ceritinib binds more favorably to the mutated ALK kinase domain, in agreement with experimental results. We determined that ALK kinase-drug interactions in the region of the front pocket are associated with drug resistance. Additionally, we find that the L1196M mutation does not simply alter the binding modes of inhibitors, but also affects the flexibility of the entire ALK kinase domain. Our work provides an understanding of the mechanisms of ALK drug resistance, confirms the usefulness of the on-the-fly Fs-IFP approach and provides a practical paradigm to study drug-resistance mechanisms in prospective drug discovery.

1853: Suramin potently inhibits binding of the mammalian high mobility group protein AT-hook 2 to DNA
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Posted to bioRxiv 12 Nov 2019

Suramin potently inhibits binding of the mammalian high mobility group protein AT-hook 2 to DNA
85 downloads biochemistry

Linjia Su, Nadezda Bryan, Sabrina Battista, Juliano Freitas, Alyssa Garabedian, Federica DAlessio, Miriam Romano, Fabiana Falanga, Alfredo Fusco, Lidia Kos, Jeremy Chambers, Francisco Fernandez-Lima, Prem P Chapagain, Stefan Vasile, Layton Smith, Fenfei Leng

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein which plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen high throughput screening (HTS) assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we discovered that suramin, a negatively charged antiparasitic drug potently inhibits the HMGA2-DNA interaction. Our results also show that the inhibition is through suramin binding to the AT-hooks of HMGA2, therefore blocking its DNA binding capacity. Furthermore, we demonstrate that suramin can induce brain tumor stem cells differentiation into cells with neurite-like structures, a process triggered by disrupting HMGA2-DNA interactions. Since suramin has strong antitumor and anti-metastasis activities, our discovery suggests that HMGA2 and HMGA2-like proteins may be the cellular target of this century-old drug.

1854: Amyloid damage to islet β-cells in type 2 diabetes: hypoxia or pseudo-hypoxia?
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Posted to bioRxiv 24 Oct 2019

Amyloid damage to islet β-cells in type 2 diabetes: hypoxia or pseudo-hypoxia?
85 downloads biochemistry

Vittorio Bellotti, Alessandra Corazza, Beatrice Foglia, Erica Novo, J. Paul Simons, P. Patrizia Mangione, Guglielmo Verona, Diana Canetti, Paola Nocerino, Laura Obici, Alessandro Vanoli, Marco Paulli, Sofia Giorgetti, Sara Raimondi, Raya Al-Shawi, Glenys A. Tennent, Graham W. Taylor, Julian D. Gillmore, Mark B. Pepys, Maurizio Parola

Aggregation of islet amyloid polypeptide (IAPP) and amyloid deposition in the islets of Langerhans may significantly contribute to the multifactorial pathogenic mechanisms leading to type 2 diabetes. A direct toxic effect on β-cells of oligomeric IAAP has been demonstrated in in vitro models, but the mechanism operating in vivo is still unclear. Mice models presenting amyloid deposition and glucose intolerance represent a good tool for exploring in vivo a putative mechanism of toxicity directly related to the physical expansion of the extracellular matrix by the amyloid fibrillar aggregates. Based on our hypothesis that deposition of amyloid may influence the oxygen perfusion, we have calculated that the mean distribution of oxygen partial pressure would drop by more than 50% in the presence of amyloid deposits in the islet. This condition of hypoxia caused by the remodelling of the extracellular space may explain the metabolic abnormalities in the Langerhans islets, otherwise interpreted as pseudo-hypoxic response to IAPP oligomers.

1855: Benchmarking Computational Methods for Estimating the Pathogenicity of Wilson's Disease Mutations
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Posted to bioRxiv 25 Sep 2019

Benchmarking Computational Methods for Estimating the Pathogenicity of Wilson's Disease Mutations
84 downloads biochemistry

Ning Tang, Thomas Sandahl, Peter Ott, Kasper Planeta Kepp

Genetic variations in the gene encoding the copper-transport protein ATP7B are the primary cause of Wilson's disease. Controversially, clinical prevalence seems much smaller than prevalence estimated by genetic screening tools, causing fear that many people are undiagnosed although early diagnosis and treatment is essential. To address this issue, we benchmarked 16 state-of-the-art computational disease-prediction methods against established data of missense ATP7B mutations. Our results show that the quality of the methods vary widely. We show the importance of optimizing the threshold of the methods used to distinguish pathogenic from non-pathogenic mutations against data of clinically confirmed pathogenic and non-pathogenic mutations. We find that most methods use thresholds that predict too many ATP7B mutations to be pathogenic. Thus, our findings explain the current controversy on Wilson's disease prevalence, because meta analysis and text search methods include many computational estimates that lead to higher disease prevalence than clinically observed. Since proteins differ widely, a one-size-fits-all threshold for all proteins cannot distinguish efficiently pathogenic and non-pathogenic mutations, as shown here. We also show that amino acid changes with small evolutionary substitution probability, mainly due to amino acid volume, are more associated with disease, implying a pathological effect on the conformational state of the protein, which could affect copper transport or ATP recognition and hydrolysis. These findings may be a first step towards a more quantitative genotype-phenotype relationship of Wilson's disease.

1856: Development of a novel HPTLC-based method for the simultaneous quantification of clinically relevant lipids from cells and tissue extracts
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Posted to bioRxiv 23 Sep 2019

Development of a novel HPTLC-based method for the simultaneous quantification of clinically relevant lipids from cells and tissue extracts
84 downloads biochemistry

Michelle Pinault, Cyrille Guimaraes, Céline Ben Hassen, Jorge L Gutierrez-Pajares, Stéphan Chevalier, Caroline Goupille, Pierre Bernard-Savary, Philippe G Frank

Lipids such as cholesterol, triglycerides, and fatty acids play important roles in the regulation of cellular metabolism and cellular signaling pathways and, as a consequence, in the development of various diseases. It is therefore important to understand how their metabolism is regulated to better define the components involved in the development of various human diseases. In the present work, we described the development and validation of an HPTLC method allowing the separation and quantification of free cholesterol, cholesteryl esters, non-esterified fatty acids, and triglycerides. This method will be of interest as the quantification of these lipids in one single assay is difficult to perform.

1857: Characterization of OTUB1 activation and inhibition by different E2 enzymes
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Posted to bioRxiv 20 Nov 2019

Characterization of OTUB1 activation and inhibition by different E2 enzymes
84 downloads biochemistry

Lauren T Que, Marie E Morrow, Cynthia Wolberger

OTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subsset of E2 ubiquitin conjugating enzymes, forming complexes that regulate one another's activity. OTUB1 can non-catalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2~Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. In order to assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the autoubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3.

1858: Impact of substrate-template stability, temperature, phosphate location, and nick-site base pairs on non-enzymatic DNA ligation: Defining parameters for optimization of ligation rates and yields with carbodiimide activation
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Posted to bioRxiv 28 Oct 2019

Impact of substrate-template stability, temperature, phosphate location, and nick-site base pairs on non-enzymatic DNA ligation: Defining parameters for optimization of ligation rates and yields with carbodiimide activation
84 downloads biochemistry

Chiamaka Obianyor, Gary Newnam, Bryce Clifton, Martha A. Grover, Nicholas V Hud

Non-enzymatic, chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields range from 30% to 95% for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation using a well-defined bimolecular test system and water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results reveal interplay between template-substrate stability and the rates of the chemical steps of ligation, which can cause yields to increase or decrease with increasing temperature. Phosphate location at the nick site also exhibits a strong influence on ligation rates and yields, with a 3' phosphate providing yields near 100% after 24 hours for particularly favourable reaction conditions, while comparable reactions with the phosphate on the 5' position of the nick site only reach 40% ligation even after 48 hours. Ligation rates are also shown to be sensitive to the identity of base pairs flanking a nick site, with some varying by more than three-fold. Finally, DNA substrate modification by EDC can, in some cases, make long reaction times and repeated addition of EDC an ineffective strategy for increasing ligation yields.

1859: Study of the AMP-activated protein kinase role in energy metabolism changes during the postmortem aging of yak longissimus lumborum
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Posted to bioRxiv 13 Jun 2019

Study of the AMP-activated protein kinase role in energy metabolism changes during the postmortem aging of yak longissimus lumborum
83 downloads biochemistry

Yayuan Yang, Ling Han, Qunli Yu, Yongfang Gao, Rende Song

To explore the postmortem physiological mechanism of muscle, activity of adenosine monophosphate activated protein kinase (AMPK) as well as its role in energy metabolism of postmortem yaks were studied. In this experiment, we injected 5-amino-1-beta-d-furanonyl imidazole-4-formamide (AICAR), a specific activator of AMPK, and the specific AMPK inhibitor STO-609, to observe the changes in glycolysis, energy metabolism, AMPK activity and AMPK gene expression (PRKA1 and PRKA2) in postmortem yaks during maturation. The results showed that AICAR could increase the expression of the PRKKA1 and PRKAA2 genes, activate AMPK and increase its activity. The effects of AICAR include a lower concentration of ATP, an increase in AMP production, an acceleration of glycolysis, an increase in the lactic acid concentration, and a decrease in the pH value. In contrast, STO-609 had the opposite effect. Under hypoxic adaptation, the activity of the meat AMPK increased, which accelerated glycolysis and metabolism, and more effectively regulated energy production.

1860: High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the "inserted" state of the ATP synthase ϵ-subunit
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Posted to bioRxiv 28 Aug 2019

High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the "inserted" state of the ATP synthase ϵ-subunit
83 downloads biochemistry

Yang Liu, Jiayu Yu, Mengyuan Wang, Qingfang Zeng, Xinmiao Fu, Zengyi Chang

ATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of α3β3γδϵab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (α3β3γδϵ) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the ϵ-subunit could adopt either an "inserted" or "non-inserted" state (with or without interacting with the α/β-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an "inserted" state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the ϵ-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the "inserted" and "non-inserted" states of the ϵ-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

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