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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 199 records · Page 11Linked to original sources

bioRxiv2016

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the ATPase that powers DNA translocation and an endonuclease that cleaves the concatemeric genome both at initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage is still mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nucleolysis. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of nucleolysis suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid shell during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the procapsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

biochemistry↗

bioRxiv2016

A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed \"gel shifting\" appears to be common for histidine-rich proteins but not yet studied in detail. We investigated \"gel shifting\" in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining \"gel shifting\" of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3-4 kDa) than apo-Hpn, phenomenon termed \"metal gel-shift\" demonstrating an intimate link between Ni2+ binding and \"gel shifting\". To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66{+/-}0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact \"metal-gel shift\" band on SDS-PAGE. In view of presented data metal-binding and \"metal-gel shift\" models are discussed.

biochemistry↗

bioRxiv2016

Extremely fast and incredibly close: co-transcriptional splicing in budding yeast

RNA splicing, an essential part of eukaryotic pre-messenger RNA processing, can be simultaneous with transcription by RNA polymerase II. Here, we compare and review independent next-generation sequencing methods that quantify co-transcriptional splicing in budding yeast. Splicing in yeast is fast, taking place within seconds of intron transcription, while polymerase is within a few dozens of nucleotides of the 3 splice site. Ribosomal protein mRNAs are spliced particularly fast and co-transcriptionally. Intron-mediated regulation of some genes is also likely to be co-transcriptional. We suggest that intermediates of the splicing reaction, missing from current datasets, may hold key information about splicing kinetics.\n\nTrendsO_LIIndependent next-generation sequencing methods quantify co-transcriptional splicing in budding yeast\nC_LIO_LIRibosomal protein mRNAs are spliced particularly fast and co-transcriptionally\nC_LIO_LIIntron-mediated regulation of DBP2 and RPS9A is likely co-transcriptional\nC_LIO_LISplicing intermediates, missing from current datasets, may hold key information\nC_LI

biochemistry↗

bioRxiv2016

Experimental and Computational Investigation of the Structure of Peptide Monolayers on Gold Nanoparticles

The self-assembly and self-organization of small molecules at the surface of nanoparticles constitute a potential route towards the preparation of advanced protein-like nanosystems. However, their structural characterization, critical to the design of bio-nanomaterials with well-defined biophysical and biochemical properties, remains highly challenging. Here, a computational model for peptide-capped gold nanoparticles is developed using experimentally characterized CALNN-and CFGAILSS-capped gold nanoparticles as a benchmark. The structure of CALNN and CFGAILSS monolayers is investigated by both structural biology techniques and molecular dynamics simulations. The calculations reproduce the experimentally observed dependence of the monolayer secondary structure on peptide capping density and on nanoparticle size, thus giving us confidence in the model. Furthermore, the computational results reveal a number of new features of peptide-capped monolayers, including the importance of sulfur movement for the formation of secondary structure motifs, the presence of water close to the gold surface even in tightly packed peptide monolayers, and the existence of extended 2D parallel {beta}-sheet domains in CFGAILSS monolayers. The model developed here provides a predictive tool that may assist in the design of further bio-nanomaterials.

biochemistry↗

bioRxiv2016

Structural and Functional analysis of the GABARAP Interaction Motif (GIM).

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence (W/F-V-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed eleven-fold more specific for GABARAP than LC3B. Selective mutation of the X1 and X2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of the canonical p62/SQSTM1-LIR into our newly defined GIM, by introducing two valine residues, enhances p62/SQSTM1 interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the continually expanding array of autophagy receptor and adaptor proteins and their in vivo functions.

biochemistry↗

bioRxiv2016

The best formula for estimating the low density lipoprotein cholesterol on a North African population

We often use the estimation of low density lipoprotein cholesterol by using the Friedwald Formula, however limitation and uncertainties make calculation its limits use. Otherwise, simple and inexpensive formulas exists, rarely used in clinical laboratories: Hatorri, Puavilai, Anandaraja, Ahmadi, Vujovic, Saidullah and Cordova. These formulas were studied on small geographically and ethnically populations and require validation in a larger population.\n\nWe intended by this work, highlight the formula that best estimates the low density lipoprotein cholesterol more accurately than the Friedewald formula on a North African population.\n\nIt seems that the Puivalai formula is the most suitable to be applied on this population.

biochemistry↗

bioRxiv2016

A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry

To facilitate high-throughput proteomic analyses we have developed a modified FASP protocol which improves the rate at which protein samples can be processed prior to mass spectrometry. Adapting the original FASP protocol to a 96-well format necessitates extended spin times for buffer exchange due to the low centrifugation speeds tolerated by these devices. However, by using 96-well plates with a more robust polyethersulfone molecular weight cutoff membrane, instead of the cellulose membranes typically used in these devices, we could use isopropanol as a wetting agent, decreasing spin times required for buffer exchange from an hour to 30 minutes. In a typical work flow used in our laboratory this equates to a reduction of 3 hours per plate. To test whether our modified protocol produced similar results to FASP and other FASP-like protocols we compared the performance of our modified protocol to the original FASP and the more recently described eFASP and MStern-blot. We show that all FASP-like methods, including our modified protocol, display similar performance in terms of proteins identified and reproducibility. Our results show that our modified FASP protocol is an efficient method for the high-throughput processing of protein samples for mass spectral analysis.

biochemistry↗

bioRxiv2016

Quod erat demonstrandum? No restriction endonuclease fold in MIF

It was claimed in a recently published article that MIF functions as an exo/endo-DNase mediating cell-death upon being induced by DNA damage and PARP1. MIF, for which tautomerase activity has been previously reported, is a member of the tautomerase superfamily which does not feature nucleases. The central premise of the authors to suggest that MIF functions as a DNase is the supposed structural relationship to nucleases of the Restriction endonuclease (REase) fold, which frequently but not always contain a motif of the form PD-(D/E)XK. However, we present evidence to show that this claim is entirely unsupported.

biochemistry↗

bioRxiv2016

Microbes use encapsulin protein organelles to sequester toxic reactions

Cells organize and regulate their metabolism via membrane-or protein-bound organelles. In this way, incompatible processes can be spatially separated and controlled. In prokaryotes, protein-based compartments are used to sequester harmful reactions and store useful compounds. These protein compartments play key roles in various metabolic and ecological processes ranging from iron homeostasis to carbon fixation. We identified more than 900 proteinaceous encapsulin nanocompartment systems in bacterial and archaeal genomes. Encapsulins can be found in 15 bacterial and 2 archaeal phyla. Our analysis reveals 1 new capsid type and 9 previously unknown cargo proteins targeted to the interior of encapsulins. We experimentally characterize 3 newly identified encapsulin systems and illustrate their involvement in iron mineralization, oxidative and nitrosative stress resistance and anaerobic ammonium oxidation, a process responsible for 30% of the N lost from the oceans. We propose that encapsulins represent a widespread strategy for toxic reaction sequestration in prokaryotes.

biochemistry↗

bioRxiv2016

Odorant cues linked to social immunity induce lateralized antenna stimulation in honey bees (Apis mellifera L.)

Hygienic behaviour (HB) is a social immunity trait in honey bees (Apis mellifera L.) whereby workers detect, uncap and remove unhealthy brood, improving disease resistance in the colony. This is clearly economically valuable; however, the molecular mechanism behind it is not well understood. The freeze-killed brood (FKB) assay is the conventional method of HB selection, so we compared odour profiles of FKB and live brood. Surprisingly, we found that significantly more brood pheromone ({beta}-ocimene) was released from FKB. {beta}-ocimene abundance also positively correlated with HB, suggesting there could be a brood effect contributing to overall hygiene. We found that β ocimene stimulated worker antennae in a dose-dependent manner, with the left antennae responding significantly stronger than right antennae in hygienic bees, but not in non-hygienic bees. This suggests that HB depends not only on odour detection, but also lateralization of sensitivity. We also compared odour profiles of Varroa-infested brood to healthy brood and found an overall interactive effect between developmental stage and infestation, but specific odours did not drive these differences. Overall, the data we present here is an important foundation on which to build our understanding the molecular mechanism behind this complex behaviour.

biochemistry↗

bioRxiv2016

T cell co-stimulatory receptor CD28 is a primary target for PD-1-mediated inhibition

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.

biochemistry↗

bioRxiv2016

Interpreting deep mutational scanning data resulting from selections on solid media

Deep mutational scanning is now used in directed evolution experiments to quantify the change in frequency of a cellular variant in a mixed population. A key concern is the extent to which the enrichment of a variant in a population corresponds to a fitness metric like relative growth rate or survival percentage. We present here analytical equations converting the enrichment of a variant to fitness metrics for plate-based selections. Using isogenic and mixed cultures we show that growth rates and survival percentages correlate for antibiotic plate-based selections. These results are important for proper interpretation of data resulting from deep sequencing.

biochemistry↗

bioRxiv2016

Structure-based alignment of human caspase recruitment domains provides a framework for understanding their function

Intracellular signalling is driven by protein-protein interactions. Members of the Death Domain superfamily mediate protein-protein interactions in both cell death and innate immune signalling pathways. They drive the formation of macromolecular complexes that act as a scaffold for protein recruitment and downstream signal transduction. Death Domain family members have low sequence identity, complicating their identification and predictions of their structure and function. We have taken all known human caspase recruitment domains (CARDs), a subfamily of the Death Domain superfamily, and generated a structure-guided sequence alignment. This alignment has enabled the identification of 14 positions that define the hydrophobic core and present a template for the identification of novel CARD sequences. We identify a conserved salt bridge in over half of all human CARDs and find a subset of CARDs likely to be regulated by tyrosine phosphorylation in their type I interface. Our alignment highlights that the CARDs of NLRC3 and NLRC5 are likely to be pseudodomains that have lost some of their original functionality. Together these studies demonstrate the benefits of structure-guided sequence alignments in understanding protein functionality.

biochemistry↗

bioRxiv2016

Simulations of CYP51A from Aspergillus fumigatus in a model bilayer provide insights into triazole drug resistance

1Azole antifungal drugs target CYP51A in Aspergillus fumigatus by binding with the active site of the protein, blocking ergosterol biosynthesis. Resistance to azole anti-fungal drugs is now common, with a leucine to histidine amino acid substitution at position 98 the most frequent, conferring resistance to itraconazole. In this study, we create a homology model of CYP51A using a recently published crystal structure of the paralog protein CYP51B. The derived structures, wild-type and L98H mutant, are positioned within a lipid membrane bilayer and subjected to molecular dynamics simulations in order improve the accuracy of both models. The structural analysis from our simulations suggests a decrease in active site surface from the formation of hydrogen bonds between the histidine substitution and neighbouring polar side chains, potentially preventing the binding of azole drugs. This study yields a biologically relevant structure and set dynamics of the A. fumigatus Lanosterol 14 alpha-demethylase enzyme and provides further insight into azole antifungal drug resistance.

biochemistry↗

bioRxiv2016

Conformational landscape of the p28-bound human proteasome regulatory particle

The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9 and S5b. We determined a cryo-EM structure of the human RP in complex with its assembly chaperone p28 at 4.5-[A] resolution. The Rpn1-p28-AAA subcomplex in the p28-bound RP is highly dynamic and was resolved to subnanometer resolution in seven states, which recapitulate the conformational landscape of the complex. Surprisingly, the p28-bound AAA ring does not form a channel in the free RP. Instead, it spontaneously samples multiple open and closed topologies. Our analysis suggests that p28 guides the proteolytic core particle to select certain conformation of the ATPase ring for RP engagement in the last step of the chaperone-mediated proteasome assembly.

biochemistry↗

bioRxiv2016

Gluten-Free Beer via Hydrodynamic Cavitation Assisted Brewing ofBarley Malts

We provide evidence that novel brewing technology based on controlled hydrodynamic cavitation greatly reduces gluten concentration in wort and finished beer. We advance the hypothesis that the degradation of proline, the most recalcitrant among gluten constituents, leads to gluten concentration reduction in the unfermented as well as in the fermenting wort and later during maturation. These findings are significant as the new cavitation-assisted technology could provide coeliac patients and gluten-intolerant people with gluten-free beer of high quality, offering an alternative to existing methods to lower the gluten concentration, which are detrimental to flavor and taste.

biochemistry↗

bioRxiv2016

Towards new cholera prophylactics and treatment: Crystal structures of bacterial enterotoxins in complex with GM1 mimics

Cholera is a life-threatening disease in many countries, and new drugs are clearly needed. C-glycosidic antagonists may serve such a purpose. Atomic resolution crystal structures of these compounds in complexes with the cholera toxin give unprecedented atomic details of the molecular interactions, and show how the inhibitors efficiently block the GM1 binding site. These molecules are well suited for development into low-cost prophylactic drugs, due to their relatively easy syntheses and their resistance to glycolytic enzymes. One of the compounds links two toxin B-pentamers in the crystal structure, which may yield improved inhibition through the formation of toxin aggregates. These structures can spark the improved design of GM1 mimics, either alone or as multivalent inhibitors connecting multiple GM1-binding sites. Future developments may further include compounds that link the primary and secondary binding sites. Serving as decoys, receptor mimics may lessen symptoms while avoiding the use of antibiotics.

biochemistry↗

bioRxiv2016

PolyCut: a computer programme for assignments of MSn signals of metallo-biomolecules by considering the entire isotope patterns

A computer programme PolyCut has been written to assist mass spectrometry-based bioinorganic studies via top-down and bottom-up approaches in positive and negative ion modes. PolyCut is designed to cope with metalloproteins, metallo-polymers and biomolecules with heavy element(s) as one (or more) of the charging agents at defined but different oxidation states. It does automatic assignment by considering the entire experimental and simulated isotopic patterns of an ion rather than reading only the mono-isotopic and average m/z values (and/or mass values). The calculation of chemical formula and the simulation of isotopic pattern for an ion at various charge states require PolyCut to be informed of the user-defined elements, the user-defined abundance of isotopes for each element, the user-defined oxidation state of an element, the user-defined residues and sequence of a polymer, and the user-defined fragmentation (or digestion). The programme successfully assisted MSn studies of two copper proteins coordinated to platinum(II) centres (a functional group possessing an intrinsic positive charge rather than a neutral organic modification), published as J. Biol. Inorg. Chem. 2009, 14(2), 163-165; Metallomics 2013, 5(8), 946-954; and [more]. Besides polypeptides, polynucleotides and polysaccharides, PolyCut can be extended to other polymers using user-defined monomer residues. For the metallo-community.\n\nAbbreviations

biochemistry↗