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Results for “Biochemistry”

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At least 1,621 records · Page 90Linked to original sources

bioRxiv2018

Artefactual formation of pyruvate from in-source conversion of lactate

RationaleLactate and pyruvate are high abundance products of glucose metabolism. Analysis of both molecules as part metabolomics studies in cellular metabolism and physiology have been aided by advances in liquid chromatography-mass spectrometry (LC-MS).\n\nMethodsWe used ion pairing-chromatography and negative ion mode ESI on an QExactive HF to perform stable isotope assisted metabolomics profiling of lactate and pyruvate metabolism.\n\nResultsUsing an LC-MS method for polar metabolite analysis we discovered an artefactual formation of pyruvate from in-source fragmentation of lactate. Surprisingly, this in-source fragmentation has not been previously described, thus we report this identification to warn other investigators. This artefact was detected by baseline chromatographic resolution of lactate and pyruvate by LC with confirmation of this artefact by stable isotope labeling of lactate and pyruvate.\n\nConclusionsThese findings have immediate implications for metabolomics studies by LC-MS and direct infusion MS, especially in negative ion mode, whereby users should resolve lactate from pyruvate or robustly quantify the potential formation of pyruvate from higher abundance lactate in their assays.

biochemistry↗

bioRxiv2018

Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

The nick translation property of DNA polymerase I (Pol I) ensures the maturation of Okazaki fragments by removing primer RNAs and facilitating ligation. However, prolonged nick translation traversing downstream DNA is an energy wasting futile process, as Pol I simultaneously polymerizes and depolymerizes at the nick sites utilizing energy-rich dNTPs. Using an in vitro assay system, we demonstrate that the {beta}-clamp of the Escherichia coli replisome strongly inhibits nick translation on the DNA substrate. To do so, {beta}-clamp inhibits the strand displacement activity of Pol I by interfering with the interaction between the finger subdomain of Pol I and the downstream primer-template junction. Conversely, {beta}-clamp stimulates the 5 exonuclease property of Pol I to cleave single nucleotides or shorter oligonucleotide flaps. This single nucleotide flap removal at high frequency increases the probability of ligation between the upstream and downstream DNA strands at an early phase, terminating nick translation. Besides {beta}-clamp-mediated ligation helps DNA ligase to seal the nick promptly during the maturation of Okazaki fragments.

biochemistry↗

bioRxiv2018

Generative adversarial networks as a tool to recover structural information from cryo-electron microscopy data

Cryo-electron microscopy (cryo-EM) is a powerful structural biology technique capable of determining atomic-resolution structures of biological macromolecules. Despite this ability, the low signal-to-noise ratio of cryo-EM data continues to remain a hurdle for assessing raw cryo-EM micrographs and subsequent image analysis. To help address this problem, we have performed proof-of-principle studies with generative adversarial networks, a form of artificial intelligence, to denoise individual particles. This approach effectively recovers global structural information for both synthetic and real cryo-EM data, facilitating per-particle assessment from noisy raw images. Our results suggest that generative adversarial networks may be able to provide an approach to denoise raw cryo-EM images to facilitate particle selection and raw particle interpretation for single particle and tomography cryo-EM data.

biochemistry↗

bioRxiv2018

Intron-mediated enhancement boosts Rtn4 circRNA expression: A robust method for exploring circRNA function

CircRNAs are expressed in many important biological processes. Studying their function requires an effective expression method. When we used intron-mediated enhancement (IME) to improve circRNA expression of mouse Rtn4 (Nogo, a key protein in Nogo-Rho pathways) circRNA as a test case, we achieved a 4-6-fold improvement compared to an existing method. We further developed this approach into a general circRNA expression vector pCircRNA-DMo. An unexpected feature of our approach is its ability to promote translation of circRNA into detectable amounts of proteins. Intriguingly, both monomer and multimer repeating peptides can be observed as a result of rolling circle translation of RTN4 circRNA. We also confirmed the presence of both peptide forms in human and mouse brains, highlighting the significance of circRNA translation in vivo. In summary, we demonstrate the significant advantage of IME in enhancing circRNA biogenesis and hence our vector offers a robust platform for exploring potential circRNA peptide-encoding functions.

biochemistry↗

bioRxiv2018

Structural and histone binding studies of the chromo barrel domain of TIP60

TIP60 consists of an N-terminal chromo barrel domain (TIP60-CB) and a C-terminal acetyltransferase domain and acetylates histone and non-histone proteins within diverse cellular processes. Whereas the TIP60-CB is thought to recognize histone tails, molecular details of this interaction remain unclear. Here we attempted a quantitative analysis of the interaction between the TIP60-CB and histone peptides, but did not observe any binding through either fluorescence polarization or isothermal titration calorimetry. We solved a crystal structure of the TIP60-CB alone. Analysis of the crystal structure demonstrates a putative peptide binding site that may be occluded by the basic side chain of a residue in a unique {beta} hairpin between the two N-terminal strands of the {beta} barrel.

biochemistry↗

bioRxiv2018

Crystal structure of chromo barrel domain of RBBP1

RBBP1 is a retinoblastoma protein (pRb) binding protein acting as a repressor of gene transcription. RBBP1 is a multidomain protein including a chromo barrel domain, and its chromo barrel domain has been reported to recognize histone H4K20me3 weakly, and this binding is enhanced by the simultaneous binding of DNA. However, the molecular basis of this DNA-mediated histone binding by the chromo barrel domain of RBBP1 is unclear. Here we attempted to co-crystallize the chromo barrel domain of RBBP1 with either a histone H4K20me3 peptide alone or with both a histone H4K20me3 peptide and DNA, but only solved the peptide/DNA unbound crystal structure. Our structural analysis indicates that RBBP1 could interact with histone H4K20me3 similar to other histone binding chromo barrel domains, and the surface charge representation analysis of the chromo barrel domain of RBBP1 suggests that the chromo barrel domain of RBBP1 does not have a typical DNA binding surface, indicating that it might not bind to DNA. Consistently, our ITC assays also showed that DNA does not significantly enhance the histone binding ability of the chromo barrel domain of RBBP1.

biochemistry↗

bioRxiv2018

Analysis of global and site-specific radiation damage in cryo-EM

Micro-crystal electron diffraction (MicroED) is an emerging method in cryo-EM for structure determination using nanocrystals. It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

biochemistry↗

bioRxiv2018

Structure of Zip2:Spo16, a conserved XPF:ERCC1-like complex critical for meiotic crossover formation

In eukaryotic meiosis, generation of haploid gametes depends on the formation of inter-homolog crossovers, which enable the pairing, physical linkage, and eventual segregation of homologs in the meiosis I division. A class of conserved meiosis-specific proteins, collectively termed ZMMs, are required for formation and spatial control of crossovers throughout eukaryotes. Here, we show that three S. cerevisiae ZMM proteins - Zip2, Zip4, and Spo16 - interact with one another and form a DNA-binding complex critical for crossover formation and control. We determined the crystal structure of a Zip2:Spo16 subcomplex, revealing a heterodimer structurally related to the XPF:ERCC1 endonuclease complex. Zip2:Spo16 lacks an endonuclease active site, but binds specific DNA structures found in early meiotic recombination intermediates. Mutations in multiple DNA-binding surfaces on the Zip2:Spo16 complex severely compromise DNA binding, supporting a model in which the complexs central and HhH domains cooperate to bind DNA. Overall, our data support a model in which the Zip2:Zip4:Spo16 complex binds and stabilizes early meiotic recombination intermediates, then coordinates additional factors to promote crossover formation and license downstream events including synaptonemal complex assembly.

biochemistry↗

bioRxiv2018

Doxorubicin induces caspase-mediated proteolysis of KV7.1

The voltage-gated potassium channel Kv7.1 (KCNQ1) co-assembles with KCNE1 to generate the cardiac potassium current IKs. Gain- and loss-of-function mutations in KCNQ1 are associated with atrial fibrillation and long-QT (LQT) syndrome, respectively, highlighting the importance of modulating IKS activity for proper cardiac function. On a post-translational level, IKS can be regulated by phosphorylation, ubiquitination and sumoylation. Here, we report proteolysis of Kv7.1 as a novel, irreversible posttranslational modification. The identification of two C-terminal fragments (CTF1 and CTF2) of Kv7.1 led us to identify an aspartate critical for the generation of CTF2 and caspases as responsible for mediating Kv7.1 proteolysis. Activating caspases by apoptotic stimuli significantly reduced Kv7.1/KCNE1 currents, which was abrogated in cells expressing caspase-resistant Kv7.1 D459A/KCNE1 channels. An increase in cleavage of Kv7.1 could be detected in the case of LQT mutation G460S, which is located adjacent to the cleavage site. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provoked caspase-mediated cleavage of endogenous Kv7.1 in human cardiomyocytes. In summary, our findings establish caspases as novel regulatory components for modulating Kv7.1 activity which may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity.\n\nNon-standard Abbreviations and Acronyms

biochemistry↗

bioRxiv2018

Acetate is generated de novo from glucose metabolism in mammals and is coupled to central carbon metabolism

In cases of nutritional excess, there is incomplete catabolism of a nutritional source and secretion of a waste product (overflow metabolism), such as the conversion of glucose to lactate (the Warburg Effect) in tumors. Here we report that excess glucose metabolism generates acetate, a key nutrient whose source has been unclear. Conversion of pyruvate, the product of glycolysis, to acetate occurs through two mechanisms: 1) coupling to reactive oxygen species (ROS), and 2) a neomorphic enzyme activity from keto acid dehydrogenases that enable it to function as a pyruvate decarboxylase. Furthermore, we demonstrate that glucose-derived acetate is sufficient to maintain acetyl-coenzyme A (Ac-CoA) pools and cell proliferation in certain limited metabolic environments such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. Thus, de novo acetate production is coupled to the activity of central carbon metabolism providing possible regulatory mechanisms and links to pathophysiology.

biochemistry↗

bioRxiv2018

Crystallizing the Parkinson’s Disease Protein LRRK2 Under Microgravity Conditions

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a considerable cause for Parkinsons disease (PD). However, the high- resolution 3D structure of the protein is still lacking. This structure will not only help to understand PD etiology but will also enable rational drug design. We have established a reliable method to produce LRRK2 crystals for the first time. However, the limited resolution of the diffraction data prevented structure determination using crystallographic methods. Herein we describe our efforts to improve the crystal quality by crystallizing under microgravity conditions aboard the International Space Station (ISS). Our method features diffusive sample mixing in capillaries and controlled crystal formation by transporting the samples in a frozen state. The crystallisation was successfully repeated under microgravity conditions. However, comparison of earth-grown and microgravity-grown LRRK2 crystals did not reveal any differences in diffraction quality. Here we present the established protocol and our experience adapting crystallization condition to the requirements necessary for successful crystallization of large and sensitive biomolecules under microgravity.

biochemistry↗

bioRxiv2018

Defective RNA Polymerase III is negatively regulated by the SUMO-Ubiquitin-Cdc48 Pathway

Transcription by RNA polymerase III (Pol III) is an essential cellular process, and mutations in Pol III can cause neurodegenerative disease in humans. However, in contrast to Pol II transcription, which has been extensively studied, the knowledge of how Pol III is regulated is very limited. We report here that in budding yeast, Saccharomyces cerevisiae, Pol III is negatively regulated by the Small Ubiquitin-like MOdifier (SUMO), an essential post-translational modification pathway. Besides sumoylation, Pol III is also targeted by ubiquitylation and the Cdc48/p97 segregase, the three of which likely act in a sequential manner and eventually lead to proteasomal degradation of Pol III subunits, thereby repressing Pol III transcription. This study not only uncovered a regulatory mechanism for Pol III, but also suggests that the SUMO and ubiquitin modification pathways and the Cdc48/p97 segregase can be potential therapeutic targets for Pol III-related human diseases.

biochemistry↗

bioRxiv2018

The N-terminus of Sec61p plays key roles in ER protein import and ERAD

Sec61p is the channel-forming subunit of the heterotrimeric Sec61 complex that mediates co-translational protein import into the endoplasmic reticulum (ER). In yeast, proteins can also be post-translationally translocated by the hetero-heptameric Sec complex, composed of the Sec61 and the Sec63 complexes. The Sec61 channel is also a candidate for the dislocation channel for misfolded proteins from the ER to the cytosol during ER-associated degradation (ERAD). The structure of the Sec61 complex is highly conserved, but the roles of its N-terminal acetylation and its amphipathic N-terminal helix are unknown so far. To gain insight into the function of the Sec61p N-terminus, we mutated its N-acetylation site, deleted its amphipathic helix, or both the helix and the N-acetylation site. Mutation of the N-acetylation site on its own had no effect on protein import into the ER in intact cells, but resulted in an ERAD defect. Yeast expressing sec61 without the N-terminal amphipathic helix displayed severe growth defects and had profound defects in post-translational protein import into the ER. Nevertheless the formation of the hetero-heptameric Sec complex was not affected. Instead, the lack of the N-terminal amphipathic helix compromised the integrity of the heterotrimeric Sec61 complex. We conclude that the N-terminal helix of Sec61p is required for post-translational protein import into the ER and Sec61 complex stability, whereas N-terminal acetylation of Sec61p plays a role in ERAD.

biochemistry↗

bioRxiv2018

Scopoletin Augments DJ1/Nrf2 Signalling and Prevents Protein Aggregation in Parkinson’s disease

Given the role of oxidative stress in PD pathogenesis and off-target side effects of currently available drugs, several natural phytochemicals seems to be promising in the management of PD. Here, we tested the hypothesis that scopoletin, an active principle obtained from Morinda citrifolia (MC), efficiently quenches oxidative stress through DJ-1/Nrf2 signalling and ameliorates rotenone-induced PD. Despite reducing the oxidative stress, administration of MC extract (MCE) has lessened the protein aggregation as evident from decreased levels of nitrotyrosine and -Synuclein. In vitro studies revealed that scopoletin lessened rotenone-induced apoptosis in SH-SY5Y cells through preventing oxidative injury. Particularly, scopoletin markedly up-regulated DJ-1, which then promoted the nuclear translocation of Nrf2 and transactivation of antioxidant genes. Furthermore, we found that scopoletin prevent the nuclear exportation of Nrf2 by reducing the levels of Keap1 and thereby enhancing the neuronal defence system. Overall, our findings suggest that scopoletin acts through DJ-1 mediated Nrf2 signalling to protect the brain from rotenone-induced oxidative stress and PD. Thus, we postulate that scopoletin could be a potential drug to treat PD.

biochemistry↗

bioRxiv2018

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein prior to its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data are only available for MurJ from the extremophile Thermosipho africanus. Here, we report the crystal structure of substrate-free MurJ from the Gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Finally, through the use of sequence co-evolution analysis we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

biochemistry↗

bioRxiv2018

A Chemical Biology Toolbox for the Study of Protein Methyltransferases and Epigenetic Signaling

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4+ T cell differentiation assays revealing the remarkable potential of individual probes to alter multiple T cell subpopulations with important implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity.

biochemistry↗

bioRxiv2018

Zinc(II) binding on human wild-type ISCU and Met140 variants modulates Fe-S complex activity

The human de novo iron-sulfur (Fe-S) assembly complex consists of the cysteine desulfurase NFS1, accessory protein ISD11, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN has been shown to bind the NFS1-ISD11-ISCU complex (SDU), to activate the desulfurase activity and thus Fe-S cluster biosynthesis. Conversely, in the absence of FXN, the NFS1-ISD11 (SD) complex was reported to be inhibited by the binding of recombinant ISCU. Here, we show that recombinant ISCU binds zinc(II) ion, and that the presence of zinc in as-isolated ISCU has impacts on the SDU desulfurase activity as measured by sulfide production. Indeed, the removal of this zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SD alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SD. Recent yeast studies have reported a substitution on the yeast ISCU orthologue Isu, at position Met141 (Met140 in human numbering of precursor protein) to Ile, Leu, Val, or Cys that could bypass the requirement of FXN for Fe-S cluster assembly and cell viability. Using recombinant human proteins, we report no significant differences in the biochemical and biophysical properties observed between wild-type and variants M140I, M140 L, and M140 V of ISCU. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SD complex. This study therefore identifies an important regulatory role for ISCU-bound zinc in modulation of the human Fe-S assembly system in vitro but no FXN bypass effect on mutations at position Met140 in human ISCU.\n\nABBREVIATIONS

biochemistry↗

bioRxiv2018

Structural insights into the recognition of mono- and di-acetyllysine by the ATAD2B bromodomain

Bromodomains are chromatin reader modules that recognize acetylated lysine. Different bromodomains exhibit a preference for specific patterns of lysine acetylation marks on core and variant histone proteins, however, the functional relationships that exist between histone acetyllysine ligands and bromodomain recognition remain poorly understood. In this study, we examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain selects for mono- and di-acetylated histones, and structural analysis identified key residues in the acetyllysine binding pocket that dictate ligand binding specificity. The X-ray crystal structure of the ATAD2B bromodomain in complex with an ATAD2 bromodomain inhibitor was solved at 2.4 [A] resolution. This structure demonstrated that critical contacts required for bromodomain inhibitor coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized a variant of the ATAD2B bromodomain that through alternative splicing loses critical amino acids required for histone ligand and inhibitor coordination. Altogether our results outline the structural and functional features of the ATAD2B bromodomain and identify a novel mechanism important for regulating the interaction of the ATAD2B protein with chromatin. HIGHLIGHTSO_LIThe ATAD2B bromodomain recognizes mono- and di-acetylated histone ligands. C_LIO_LIChemical shift perturbations outline the ATAD2B bromodomain acetyllysine binding pocket. C_LIO_LIAn ATAD2B bromodomain-inhibitor complex reveals important binding contacts. C_LIO_LIAn alternate splice variant in the ATAD2B bromodomain abolishes histone and inhibitor binding. C_LI

biochemistry↗