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Results for “Biochemistry”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,459 records · Page 81Linked to original sources

bioRxiv2019

In vitro evaluation of factor IX as novel treatment for factor XI deficiency

Factor XI deficiency is associated with mild to moderate bleeding upon injury. Treatment of bleeding in patients can be a challenge due to the limited availability of factor XI concentrates that may also have thrombotic side effects, and the volume overload as a result of plasma transfusion. In our in vitro study, we established that recombinant factor IX concentrate Benefix(R) (Pfizer) was able to potently enhance thrombin generation in factor XI depleted plasma when coagulation was initiated via the extrinsic pathway. This was due to the contamination of Benefix(R) with very low amounts of factor IXa that compensated for the lack of factor XI in plasma. Our data suggest that bleeding due to factor XI deficiency or antithrombotic therapy targeting factor XI may be treated with certain factor IX concentrates, which should be investigated in future clinical studies.

biochemistry↗

bioRxiv2019

A Genetically Encoded Fluorescent Sensor Enables Real-time Detection of the Intracellular GTP:GDP Ratio

The interconversion of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) is integral to a wide variety of biological cellular activities. However, analytical methods which directly detect the ratio of intracellular GTP and GDP concentrations have not been available. Herein, we report GNEPS, a genetically encoded fluorescent sensor that enables real-time monitoring of the GTP:GDP ratio, which is a fusion protein comprising a eukaryotic G-protein and a circularly permuted yellow fluorescent protein. GNEPS has distinct fluorescence spectra between its GTP-bound and GDP-bound states. Its apparent fluorescence signal therefore depends upon the competitive binding of GTP and GDP. Live cell imaging experiments demonstrated that GNEPS can be used to monitor spatiotemporal changes in the intracellular GTP:GDP ratio in various cell types and organelles in response to metabolic perturbations. We anticipate that GNEPS will become a valuable tool for understanding the metabolic and regulatory contributions of guanosine nucleotides.

biochemistry↗

bioRxiv2019

Small molecule inhibitors of a human recombination-associated ATPase, RAD54

Homologous recombination (HR) is a principal support pathway for DNA replication and for recovery from DNA breaks and interstrand crosslinks, making it a rational target for inhibition in cancer therapy. The ATPase RAD54 functions in molecular events that promote DNA sequence-preservation during HR-mediated damage repair, including homology search, DNA strand exchange, and transition to DNA repair synthesis within a displacement loop intermediate. We developed a high-throughput biochemical screen to identify small-molecule inhibitors of human RAD54, using a phosphate detection assay to monitor RAD54 ATPase activity in the presence of double-stranded DNA (dsDNA). After filtering potential DNA intercalators and frequent hitters, we identified two chemotypes that reproducibly inhibited RAD54 ATPase in vitro. We evaluated these chemotypes for inhibition of RAD54-dsDNA binding and cancer cell survival. A halogenated carbazole/dihydroacridine scaffold inhibited a panel of SWI2/SNF2-related ATPases but not VCP/p97, an unrelated ATPase. Small molecules that interfere with key steps in HR-- such as inhibitors of RAD54--may expose DNA repair-dependent vulnerabilities in cancer cells.

biochemistry↗

bioRxiv2019

Normal development and fertility of Fut1, Fut2, and Sec1 triple knockout mice

The fucose alpha(1,2) galactose structure (H antigen) is synthesized by 1,2 fucosyltransferases Fut1, Fut2 and Sec1. H antigen has been reported to be involved in cancer progression, neurite migration, synaptic plasticity, host-microbe interaction, blastocyst implantation, and the maintenance of gut microbiome. Genetic depletion of Fut1 or Fut2 only cause defects of alpha1,2 fucosylation in limited tissues because of enzyme redundancy. In this study, we generated mice with deficiencies in Fut1, Fut2, and Sec1 genes to deplete H antigen through BAC Engineering for the generation of ES Cell-Targeting construct. The homogenous triple knockout mice showed no significant decrease of viability or development. Mass spectrometry and Western blot analysis confirmed the absence of H blood group antigen in multiple organs. These results indicate normal development and fertility of mice devoid of blood group H. The fine pathophysiological alterations in these mice remain to be carefully studied, and they may serve as valuable tools to study gut microbiome and host-microbe interactions.

biochemistry↗

bioRxiv2019

MicroED with the Falcon III direct electron detector

Microcrystal electron diffraction (MicroED) combines crystallography and electron cryomicroscopy (cryo-EM) into a method that can be used for high-resolution structure determination. In MicroED nanosized crystals, often intractable by other techniques, are probed by high-energy electrons in a transmission electron microscope and the diffracted signal is recorded on an electron detector. Since only a small number of different detectors have been used for MicroED measurements in the past, their impact on data quality has not been investigated. Here we evaluate two different cameras using crystals of the well-characterized serine protease proteinase K. Compared to previously used equipment, the Falcon III direct electron detector and the CMOS-based CetaD camera can collect complete datasets both faster and using lower total exposure. As an effect of the lower dose, radiation damage is reduced, which is confirmed in both real and reciprocal space. The increased speed and lower exposure requirements have implications on model quality and the prospects for further automation of MicroED.

biochemistry↗

bioRxiv2019

A lipophilicity-based energy function for membrane-protein modelling and design

Membrane-protein design is an exciting and increasingly successful research area which has led to landmarks including the design of stable and accurate membrane-integral proteins based on coiled-coil motifs. Design of topologically more complex proteins, such as most receptors, channels, and transporters, however, demands an energy function that balances contributions from intra-protein contacts and protein-membrane interactions. Recent advances in water-soluble all-atom energy functions have increased the accuracy in structure-prediction benchmarks. The plasma membrane, however, imposes different physical constraints on protein solvation. To understand these constraints, we recently developed a high-throughput experimental screen, called dsT{beta}L, and inferred apparent insertion energies for each amino acid at dozens of positions across the bacterial plasma membrane. Here, we express these profiles as lipophilicity energy terms in Rosetta and demonstrate that the new energy function outperforms previous ones in modelling and design benchmarks. Rosetta ab initio simulations starting from an extended chain recapitulate two-thirds of the experimentally determined structures of membrane-spanning homo-oligomers with <2.5 [A] root-mean-square deviation within the top-predicted five models. Furthermore, in two sequence-design benchmarks, the energy function improves discrimination of stabilizing point mutations and recapitulates natural membrane-protein sequences of known structure, thereby recommending this new energy function for membrane-protein modelling and design.

biochemistry↗

bioRxiv2019

Revisiting Mitchell’s chemiosmotic theory in light of new stoichiometric reaction equations of ATPase II

Chemiosmotic theory has been reining the field of bioenergetics since its inception. As stoichiometric mechanisms of the underlying chemical reactions have now been elucidated, it calls for a revision in the relationships derived by Mitchell in his seminal review paper. Here in this work, new formulation of the relationship between the pH difference and transmembrane potential, as derived in light of modified ATPase II reaction stoichiometry, has been proposed for the first time. This formulation results in accurate estimation of dependence of transmembrane potential on the pH difference across the two sides of the mitochondrial membrane. Thus, this work is of potential interest and will enable researchers working in the field of bioenergetics involving chemiosmotic theory to come-up with more exact mechanistic explanations.

biochemistry↗

bioRxiv2019

Zinc restriction promotes β-cell hyper-hormonemia and endocrine pancreas degeneration in mice

Zinc is a key component of proteins, including interaction with varying pancreatic hormones, including insulin and amylin. Zinc is key in insulin crystallinity in ZnT8 knock-out mice models, although the dietary role of zinc restriction over both energetic metabolism and {beta}-pancreatic hormonemia and morphology remained unexplored. We aimed to test whether dietary zinc restriction on swiss male mice would impact over endocrine pancreas and metabolic phenotype. We evaluated the role of dietary zinc restriction on {beta}-pancreatic hormonemia on non-transgenic Swiss male mice weaned onto a control or low-zinc diet for 4 weeks. Growth, glycemia, insulinemia, amylinemia and pancreatic islet were smaller in intervention group despite insulin crystallinity in secretory granules. We have found overlabelling for insulin, amylin and toxic oligomers in apoptotic pancreatic islet. High production of {beta}-pancreatic hormones in zinc-restricted animals counteract the decreasing islet size due to their apoptotic cells. We conclude that zinc deficiency is sufficient to promote islet {beta}-cell hormonal disruption and degeneration.

biochemistry↗

bioRxiv2019

Rapid and highly sensitive detection of pyocyanin biomarker in different Pseudomonas aeruginosa infections using gold nanoparticles modified sensor

Successful antibiotic treatment of infections relies on accurate and rapid identification of the infectious agents. Pseudomonas aeruginosa is implicated in a wide range of human infections that almost complicated and become life threating especially in immunocompromised and critically ill patients. Conventional microbiological methods take more than 3 days to obtain accurate results. Pyocyanin is a distinctive electroactive biomarker for Pseudomonas aeruginosa. Here, we have developed a rapid diagnostic (polyaniline) PANI gold nanoparticles (Au NPs) modified indium tin oxide (ITO) electrode that showed 100% sensitivity for pyocyanin in culture of Pseudomonas aeruginosa clinical isolates and high selectivity for pyocyanin at low concentration when measured in the presence of other substances like ascorbic acid, uric acid, and glucose as interferences. The constructed electrode was characterized using scanning electron microscopy and cyclic voltammetry. The determined linear range for pyocyanin detection was from 238 {micro}M to 1.9 {micro}M with a detection limit of 500 nM. Compared to the screen-printed electrode used before, the constructed electrode showed a 4-fold enhanced performance.

biochemistry↗

bioRxiv2019

Mass Cytometry Study on the Heterogeneity in Cellular Association and Cytotoxicity of Silver Nanoparticles in Human Immune Cells

There have been many reports about the adverse effects of nanoparticles (NPs) on the environment and human health. Conventional toxicity assessments of NPs frequently assume uniform distribution of monodisperse NPs in homogeneous cell populations, and provide information on the relationships between the administered dose of NPs and cellular responses averaged for a large number of cells. They may have limitations in describing the wide heterogeneity of cell-NP interactions, caused by cell-to-cell and NP-to-NP variances. To achieve more detailed insight into the heterogeneity of cell-NP interactions, it is essential to understand the cellular association and adverse effects of NPs at single-cell level. In this study, we applied mass cytometry to investigate the interactions between silver nanoparticles (AgNPs) and primary human immune cells. High dimensionality of mass cytometry allowed us to identify various immune cell types and observe the cellular association and toxicity of AgNPs in each population. Our findings showed that AgNPs had higher affinity with phagocytic cells like monocytes and dendritic cells and caused more severe toxic effects than with T cells, B cells and NK cells. Multi-element detection capability of mass cytometry also enabled us to simultaneously monitor cellular AgNP dose and intracellular signaling of individual cells, and subsequently investigate the dose-response relationships of each immune population at single-cell level, which are often hidden in conventional toxicity assays at bulk-cell level. Our study will assist future development of single-cell dose-response models for various NPs and will provide key information for the safe use of nanomaterials for biomedical applications.

biochemistry↗

bioRxiv2019

Regulation of the 20S proteasome by a novel family of inhibitory proteins

The protein degradation machinery plays a critical role in the maintenance of cellular homeostasis, preventing the accumulation of damaged or misfolded proteins and controlling the levels of regulatory proteins. The 20S proteasome degradation machinery is able to cleave any protein with a partially unfolded region, however uncontrolled degradation of the myriad of potential substrates is improbable. Thus, there must exist a regulatory mechanism to control 20S proteasome mediated degradation. Here we have discovered a family of 20S proteasome regulators, named Catalytic Core Regulators (CCRs). They coordinate the function of the 20S proteasome and are involved in the oxidative stress response via Nrf2. The CCRs organize into a feed-forward loop regulatory circuit, with some members stabilizing Nrf2, others being induced by Nrf2, and all of them inhibiting the 20S proteasome. This provides a fine-tuned mechanism to carefully modulate the 20S proteasome, ensuring its proper functioning by controlling the degradative flux.

biochemistry↗

bioRxiv2019

Bactofilins form non-polar filaments that bind to membranes directly

Bactofilins are small beta-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions: filaments in Caulobacter crescentus are involved in cell stalk formation whereas Myxococcus xanthus filaments aid chromosome segregation and motility. The precise molecular architecture of bactofilin filaments has remained unclear. Here we revealed by sequence analyses and electron microscopy that in addition to wide distribution across bacteria and archaea, bactofilins are also present in a few eukaryotic cells such as oomycetes. The sole bactofilin from Thermus thermophilus was demonstrated to form constitutive filaments and cryo-EM analysis revealed that protofilaments formed through end-to-end association of the beta-helical domains. Using a nanobody against Thermus bactofilin we determined the near-atomic filament structure, showing that the filaments are non-polar, with subunits arranged head-to-head and tail-to-tail. A polymerisation-impaired mutant F105R, that disrupts one of the two protofilament interfaces, enabled crystallisation. The crystal structure also revealed non-polar protofilaments, and the dominance of the beta-stacking interface that formed despite the inhibiting mutation. To confirm the generality of the lack of polarity, we performed co-evolutionary analysis of a large set of sequences. Finally, using Thermus bactofilin, we determined that the N-terminal disordered tail of the protein is responsible for direct binding to lipid membranes both on liposomes and by electron cryotomography in E. coli cells. The tail is conserved, suggesting that membrane binding is likely a general feature of these very common but only recently discovered filaments of the prokaryotic cytoskeleton.

biochemistry↗

bioRxiv2019

Subunit joining exposes nascent pre-40S rRNA for processing and quality control

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 [A]. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality-control during ribosome assembly.

biochemistry↗

bioRxiv2019

Human 28s rRNA 5’ terminal derived small RNA inhibits ribosomal protein mRNA levels

Recent small RNA (sRNA) high-throughput sequencing studies reveal ribosomal RNAs (rRNAs) as major resources of sRNA. By reanalyzing sRNA sequencing datasets from Gene Expression Omnibus (GEO), we identify 28s rRNA 5 terminal derived sRNA (named 28s5-rtsRNA) as the most abundant rRNA-derived sRNAs. These 28s5-rtsRNAs show a length dynamics with identical 5 end and different 3 end. Through exploring sRNA sequencing datasets of different human tissues, 28s5-rtsRNA is found to be highly expressed in bladder, macrophage and skin. We also show 28s5-rtsRNA is independent of microRNA biogenesis pathway and not associated with Argonaut proteins. Overexpression of 28s5-rtsRNA could alter the 28s/18s rRNA ratio and decrease multiple ribosomal protein mRNA levels. Our results reveal that 28s5-rtsRNA serves as a key regulator in ribosomal protein expression.

biochemistry↗

bioRxiv2019

The structural basis for RNA selectivity by the IMP family of RNA binding proteins

The Igf2 mRNA binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine SELEX and NMR spectroscopy to demonstrate that the major RNA binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

biochemistry↗

bioRxiv2019

Efficient consideration of coordinated water molecules improves computational protein-protein and protein-ligand docking

Highly-coordinated water molecules are frequently an integral part of protein-protein and protein-ligand interfaces. We introduce an updated energy model that efficiently captures the energetic effects of these highly-coordinated water molecules on the surfaces of proteins. A two-stage protocol is developed in which polar groups arranged in geometries suitable for water placement are first identified, then a modified Monte Carlo simulation allows highly coordinated waters to be placed on the surface of a protein while simultaneously sampling amino acid side chain orientations. This \"semi-explicit\" water model is implemented in Rosetta and is suitable for both structure prediction and protein design. We show that our new approach and energy - model yield significant improvements in native structure recovery of protein-protein and protein-ligand docking.

biochemistry↗

bioRxiv2019

An In-Silico Investigation of Menthol Metabolism

Prevalence of mentholated products for consumption has brought great importance to studies on menthols metabolic pathways to ensure safety, design more potent derivatives, and identify therapeutic benefits. Proposed pathways of (-)-menthol metabolism based on metabolites found experimentally in previous works by Yamaguchi, Caldwell & Farmer, Madyastha & Srivatsan and Hiki et al. were not in agreement. This in silico approach is based on the three in vivo studies and aims to resolve the discrepancies. Reactions in the pathways are conjugation with glucuronic acid/sulfate, oxidation to alcohol, aldehyde & carboxylic acid, and formation of a four-membered/five-membered ring. Gas-phase structures, standard Gibbs energies and SMD solvation energies at B3LYP/6-311++G(d,p) level were obtained for 102 compounds in the pathways. This study provides a more complete picture of menthol metabolism by combining information from three experimental studies and filling missing links in previously published pathways.

biochemistry↗

bioRxiv2019

De novo Classification of Mouse B Cell Types using Surfaceome Proteotype Maps

System-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when sample is limiting. We miniaturized and automated the previously described Cell Surface Capture technology increasing sensitivity, reproducibility, and throughput. We used this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and used targeted flow cytometry to uncover developmental cell subpopulations.

biochemistry↗