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Results for “Biochemistry”

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bioRxiv2019

Hypertrophic cardiomyopathy mutations at the folded-back sequestered β-cardiac myosin S1-S2 and S1-S1 interfaces release sequestered heads and increase myosin enzymatic activity

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human {beta}-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human {beta}-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, thereby leading to hypercontractility at the molecular level.

biochemistry↗

bioRxiv2019

Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine

The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various extracellular signals. STAT3 activity was also found to be regulated by acetylation of Lys685. However, the molecular mechanism by which Lys685 acetylation affects the transcriptional activity of STAT3 remains elusive. By genetically encoding the co-translational incorporation of acetyl-lysine into position Lys685 and co-expression with the Elk receptor tyrosine kinase, we were able to biochemically characterize site-specifically acetylated, and simultaneously acetylated and phosphorylated STAT3. We measured the effect of acetylation on the crystal structure, and DNA binding affinity and specificity of Tyr705-phosphorylated and non-phosphorylated STAT3. In addition, we monitored the deacetylation of acetylated Lys685 by reconstituting the mammalian enzymatic deacetylation reaction in live bacteria. Surprisingly, we found that acetylation, per se, had no effect on the crystal structure, and DNA binding affinity or specificity of STAT3, implying that the previously observed acetylation-dependent transcriptional activity of STAT3 involves an additional cellular component. In addition, we discovered that Tyr705-phosphorylation protects Lys685 from deacetylation in bacteria, providing a new possible explanation for the observed correlation between STAT3 activity and Lys685 acetylation.

biochemistry↗

bioRxiv2019

Analysis of solid-state heparin samples by ATR-FTIR spectroscopy

The widely used anticoagulant pharmaceutical, heparin, is a polydisperse, heterogeneous polysaccharide. Heparin is one of the essential medicines defined by the World Health Organisation but, during 2007-2008, was the subject of adulteration. The intrinsic heterogeneity and variability of heparin makes it a challenge to monitor its purity by conventional means. This has led to the adoption of alternative approaches for its analysis and quality control, some of which are based on multivariate analysis of 1H NMR spectra, or exploit correlation techniques. Such NMR spectroscopy-based analyses, however, require costly and technically demanding NMR instrumentation. Here, an alternative approach based on the use of attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR) combined with multivariate analysis is proposed. FTIR-ATR employs more affordable and easy-to-use technology and, when combined with multivariate analysis of the resultant spectra, readily differentiates between glycosaminoglycans of different types, between heparin samples of distinct animal origins and enables the detection of both known heparin contaminants, such as over-sulphated chondroitin sulfate (OSCS), as well as other alien sulphated polysaccharides in heparin samples to a degree of sensitivity comparable to that achievable by NMR. The approach will permit the rapid and cost-effective monitoring of pharmaceutical heparin at any stage of the production process and indeed, in principle, the quality control of any heterogeneous or variable material.

biochemistry↗

bioRxiv2019

Structural and binding studies of human CAMKK2 kinase domain bound to small molecule ligands

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no specific small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homologue, CAMKK1, are thought to have overlapping biological roles. Here we present six novel co-structures of CAMKK2 bound to potent ligands identified from a library of ATP-competitive kinase inhibitors. Isothermal titration calorimetry (ITC) revealed that binding to some of these molecules is enthalpy driven. We expect our results to further advance current efforts to discover small molecule kinase inhibitors specific to each human CAMKK.

biochemistry↗

bioRxiv2019

Effect of flow rate and freezing on cyanocobalamin recovery using a commercial solid phase extraction cartridge.

Analysis of vitamin B12 in sea water is laborious, time consuming, and often requires storage of relatively large-volume water samples. Alleviating these major limitations will increase the throughput of samples and, as a consequence, improve our understanding of the distribution and role of vitamin B12 in the oceans. Previous studies have indicated that target analyte recovery is negatively affected at flow rates exceeding 1 mL min-1 using home-made C18 Solid Phase Extraction (SPE) cartridges. In this study, the effect of flow rate on recovery of vitamin B12 was tested across a range of flow rates between 1 and 37 mL min-1 using a commercial SPE cartridge containing surface-modified styrene divinylbenzene. Recovery of vitamin B12 at flow rates up to the maximum rate tested did not statistically differ from 1 mL min 1. A second study was conducted to determine whether storage of the SPE cartridges at -20{degrees}C had a negative impact on vitamin B12 recovery. Recovery of vitamin B12 from SPE cartridges stored up to 13 days did not differ from unfrozen SPE cartridges. These data suggest that rapid extraction and cold storage of vitamin B12 on commercial SPE cartridges does not negatively affect recovery and offers an economical alternative to field studies.

biochemistry↗

bioRxiv2019

Treatment with 5-aza-dC Induces Apoptotic PARP1-DNA Adducts

The nucleoside analog 5-aza-2-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC.

biochemistry↗

bioRxiv2019

SLC19A1 is an importer of the immunotransmitter cGAMP

23-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral Stimulator of Interferon Genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2'3'-bisphosphosphothioate-cyclic-di-AMP (2'3'-CDAS). These discoveries will provide insight into cGAMPs role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

biochemistry↗

bioRxiv2019

2'3'-cGAMP is an immunotransmitter produced by cancer cells and regulated by ENPP1

23-cyclic GMP-AMP (cGAMP) is characterized as an intracellular second messenger that is synthesized in response to cytosolic dsDNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, using mass spectrometry, we detected that cGAMP is continuously exported as a soluble factor by an engineered cell line but then efficiently cleared by ENPP1, explaining why it has escaped detection until now. By developing a potent, specific, and cell impermeable ENPP1 inhibitor, we detected cGAMP export in cancer cell lines commonly used for mouse tumor models. In tumors, depletion of extracellular cGAMP using neutralizing proteins decreased tumor-associated dendritic cells. Boosting extracellular cGAMP by genetic knockout and pharmacological inhibition of ENPP1 increased tumor-associated dendritic cells, shrunk tumors, and synergized with ionizing radiation and anti-CTLA-4 to cure tumors. In conclusion, cGAMP is an anti-cancer immunotransmitter released by tumors and detected by host innate immunity.

biochemistry↗

bioRxiv2019

Therapeutic Targeting of Casein Kinase 1δ/ε in an Alzheimer's Disease Mouse Model

Sleep disturbances and memory impairment are common symptoms of Alzheimers disease (AD). Given that the circadian clock regulates sleep, hippocampal function, and neurodegeneration, it represents a therapeutic target against AD. Casein kinase 1{delta}/{varepsilon} (CK1{delta}/{varepsilon}) are clock regulators and overexpressed in AD brains, making them viable targets to improve sleep and cognition. We assessed the effects of a small molecule CK1{delta}/{varepsilon} inhibitor (PF-670462) in a cellular model of circadian clocks and in 3xTg-AD mice. Mass spectrometry-based proteomic analyses revealed that PF-670462 treatment in vitro upregulated multiple proteins that are downregulated in AD, while administration in 3xTg-AD mice reversed hippocampal proteomic alterations in diverse AD-associated and clock-regulated pathways, including synaptic plasticity and amyloid precursor protein processing. Furthermore, PF-670462 rescued working memory and normalized behavioural circadian rhythms in 3xTg-AD mice. Our study provides proof of concept for CK1{delta}/{varepsilon} inhibition and direct clock modulation against AD-related proteomic changes, memory impairment, and circadian disturbances.

biochemistry↗

bioRxiv2019

Distinct metabolic states of a cell guide alternate fates of mutational buffering through altered proteostasis

Changes in metabolism can alter the cellular milieu; can this also change intracellular proteostasis? Since proteostasis can modulate mutational buffering, if change in metabolism has the ability to change proteostasis, arguably, it should also alter mutational buffering. Building on this, we find that altered cellular metabolic states in E. coli buffer distinct mutations. Buffered-mutants had folding problems in vivo and were differently chaperoned in different metabolic states. Notably, this assistance was dependent upon the metabolites and not on the increase in canonical chaperone machineries. Additionally, we were able to reconstitute the folding assistance afforded by metabolites in vitro and propose that changes in metabolite concentrations have the potential to alter proteostasis. Collectively, we unravel that the metabolite pools are bona fide members of proteostasis and aid in mutational buffering. Given the plasticity in cellular metabolism, we posit that metabolic alterations may play an important role in the positive or negative regulation of proteostasis.

biochemistry↗

bioRxiv2019

Molecular mechanism of the chitinolytic monocopper peroxygenase reaction

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes, broadly distributed across the Tree of Life. We recently reported that LPMOs can use H2O2 as an oxidant, revealing a novel reaction pathway. Here, we aimed to elucidate the H2O2-mediated reaction mechanism with experimental and computational approaches. In silico studies suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation, and guides the derived hydroxyl radical towards formation of a copper-oxyl intermediate. The initial H2O2 homolytic cleavage and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are suggested to be the main energy barriers. Under single turnover conditions, stopped-flow fluorimetry demonstrates that LPMO-Cu(II) reduction to Cu(I) is a fast process compared to the re-oxidation reactions. We found that re-oxidation of LPMO-Cu(I) is 2000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. In agreement with the notion of ternary complex formation, when chitin is added, re-oxidation by H2O2 is accelerated whereas that by O2 slows. Simulations indicated that Glu60, a highly-conserved residue, gates the access to the confined active site and constrains H2O2 during catalysis, and Glu60 mutations significantly decreased the enzyme performance. By providing molecular and kinetic insights into the peroxygenase activity of chitinolytic LPMOs, this study will aid the development of applications of enzymatic and synthetic copper catalysis and contribute to understanding pathogenesis, notably chitinolytic plant defenses against fungi and insects. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/541292v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@92826org.highwire.dtl.DTLVardef@12b0a19org.highwire.dtl.DTLVardef@a3fa2aorg.highwire.dtl.DTLVardef@17cfa80_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Arenaviridae exoribonuclease presents genomic RNA edition capacity.

The Arenaviridae is a large family of viruses causing both acute and persistent infections and causing significant public health concerns in afflicted regions. A "trademark" of infection is the quick and efficient immuno-suppression mediated in part by a 3-5 RNA exonuclease domain (ExoN) of the Nucleoprotein (NP). Mopeia virus, the eastern African counterpart of Lassa virus, carries such ExoN domain, but does not suppress the host innate immunity. We have recently reported the crystal structure of the Mopeia virus ExoN domain, which presents a conserved fold and active site. In the present study, we show that the ExoN activity rules out a direct link between ExoN activity and alteration of the host innate immunity. We found that the Arenavirus ExoN, however, is able to excise mis-incorporated bases present at the 3-end of double stranded RNA. ExoN(-) arenaviruses cultured in cells dampened in innate immunity still replicated in spite of a significant reduction in the viral charge over several passages. The remaining ExoN(-) virus population showed an increased base substitution rate on a narrow nucleotide spectrum, linking the ExoN activity to genome editing. Since, the Arenavirus ExoN belongs to the same nuclease family as that of the nsp14 coronavirus ExoN ; which has been recently shown to promote viral RNA synthesis proofreading; we propose that Arenavirus ExoN is involved in a "limited RNA editing" mechanism mainly controlled by structural constraints and a low mutational/fitness ratio. Author summaryOnly Arenaviridae and Coronaviridae encode a 3-5 RNA exonuclease domain (ExoN) in their genome. This activity is either used to counteract the innate immunity response during viral infection or to ensure genome stability during replication. Mopeia virus (MOPV), the eastern African counterpart of Lassa virus, carries such ExoN domain, but does not suppress the host innate immunity. We studied MOPV ExoN activity both in vitro and in cellula to assess the role of ExoN MOPV and found that the Arenaviral ExoN is fully active on dsRNA, and is able like the one of Coronaviridae to excise a mismatched base. We measured genetic stability and found evidence of a limited spectrum of RNA synthesis proofreading mechanism, together with a strongly impacted viral replication. We propose that the Arenaviral ExoN is involved in a functional check of the conserved RNA structures of the viral genome.

biochemistry↗

bioRxiv2019

Characterization of the workplace chemical exposome using untargeted LC-MS/MS: a case study

Western people now spend close to 90% of their time indoors, one-quarter of which occurs at their place of employment. As such, interactions between employees and the workplace built environment have significant potential impact on employee health and safety. However, the range of workers daily chemical exposures is still poorly understood. Likewise, the influence of workers themselves and of worker behavior on the chemical composition of the workplace is still unknown. In this case study, we used untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the chemical signatures of three different types of workplaces: scientific research buildings, office buildings, and one mixed-purpose building. Our results identified differential signatures of public building surfaces based on building purpose, sampling location and surface materials. Overall, these results are helping define the influence of human behavior on the workplace chemical environment and identify the chemical hazards to which people are exposed throughout their workday. HighlightsO_LIImplementation of untargeted liquid chromatography-tandem mass spectrometry to study workplace chemical exposures. C_LIO_LIShared chemical signatures were identified based on building purpose. C_LIO_LIDifferential chemical signatures were identified based on surface material and sampling location. C_LIO_LIAnnotated molecules include pharmaceuticals, illicit drugs, food chemicals, constituents of paints and stains, and cleaning products. C_LI

biochemistry↗

bioRxiv2019

The structure of a highly conserved picocyanobacterial protein reveals a Tudor domain with a novel tRNA binding function

Cyanobacteria of the Prochlorococcus and marine Synechococcus genera are the most abundant photosynthetic microbes in the ocean. Intriguingly, the genomes of these bacteria are very divergent even within each genus, both in gene content and at amino acid level of the encoded proteins. One striking exception to this is a 62 amino acid protein, termed Prochlorococcus/Synechococcus Hyper Conserved Protein (PSHCP). PSHCP is not only found in all sequenced Prochlorococcus and marine Synechococcus genomes but it is also nearly 100% identical in its amino acid sequence across all sampled genomes. Such universal distribution and sequence conservation suggests an essential cellular role of the encoded protein in these bacteria. However, the function of PSHCP is unknown. We used Nuclear Magnetic Resonance (NMR) spectroscopy to determine its structure. We found that 52 of the 62 amino acids in PSHCP form a Tudor domain, while the remainder of the protein is disordered. NMR titration experiments revealed that PSHCP has only a weak affinity for DNA, but an 18.5 fold higher affinity for tRNA, hinting at an involvement of PSHCP in translation. Computational docking and mutagenesis studies identified a positively charged patch surrounding residue K30 that serves as the primary docking site for tRNA on PSHCP. These results provide the first insight into the structure and function of PSHCP and suggest a new function for Tudor domains in recognizing tRNA.

biochemistry↗

bioRxiv2019

Structures of the Otopetrin Proton Channels Otop1 and Otop3

Otopetrins (Otop1-Otop3) comprise one of only two known eukaryotic proton-selective channel families. Otop1 is required for formation of otoconia and is a candidate mammalian sour taste receptor. Here, we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture of Otopetrins with each subunit consisting of twelve transmembrane helices divided into structurally related N and C domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a cavernous central tunnel. Two hydrophilic vestibules, as well as the intrasubunit interface between N and C domains, form conduits for water entry into the membrane plane in molecular dynamics simulations, suggesting they each could provide pathways for proton conduction. We also demonstrate the functional relevance of a salt bridge in the C domain vestibule by mutagenesis. Our results provide a structural basis for understanding the function of the Otopetrin proton channel family.

biochemistry↗

bioRxiv2019

Exploring DCAF15 for reprogrammable targeted protein degradation

The targeted degradation of proteins by reprogramming E3 ligases with bifunctional small molecules is an exciting area of chemical biology because it promises a set of chemical tools for achieving the same task as siRNA or CRISPR/Cas9. Although there are hundreds of E3 ligases in the human proteome only a few have been shown to be reprogrammable to target new proteins. Recently various arylsulfonamides were shown to induce degradation of the splicing factor RBM39 via the RING type E3 ligase CRL4DCAF15. Here we identify the arylsulfonamide most amenable to chemical modifications and demonstrate its behaviour in bifunctional reprogramming.

biochemistry↗

bioRxiv2019

Processing cookies formulated with goat cream enriched with conjugated linoleic acid

Goat fat is one of the most important sources of conjugated linoleic acid (CLA), a fatty acid which has health benefits. However, CLA consumption is limited to meats and milk products as CLA is generated in ruminants. This study aimed to replace vegetable fat by goat cream enriched with CLA. Four cookie recipes were developed with only the fat source being different: CVF - vegetable fat; CB - butter; CGC - goat cream without CLA; CGCLA - goat cream with CLA. Cookies were evaluated according to physical (color and texture) and physical-chemical parameters (lipids, proteins, total sugars, fiber, ash, moisture and Aw), Consumer Testing (n = 123) and lipid profile. The CGCLA presented higher values in the color parameters, and the higher and the lower scores in relation to hardness were 5.54 (CB) and 2.21 (CVF), respectively. Lipids and total sugars varied inversely, and the highest percentages of lipids were in the CVF and CG samples, which obtained lower total sugar content. There was no difference in the acceptance and preference of the four formulations, and the formulations with the goat creams (CG and CGCLA) were as accepted as CFV. The lipid profile of the cookies presented CFV with the highest percentage of trans fatty acids (TFA) with 16.76 %. CGCLA presented 70 % more CLA in relation to CB and CGC, thus certifying that CLA was present in relevant quantities in the CGCLA, even after cooking. The CGCLA is a biscuit with higher levels of CLA, and in this study it was possible to verify that the goat milk cream enriched with CLA can be used in producing cookies which adds functional and nutritional properties to them and offers other alternatives to produce food from goat's milk cream.

biochemistry↗

bioRxiv2019

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling

F1Fo-ATP synthases play a central role in cellular metabolism, making the energy of the proton-motive force across a membrane available for a large number of energy-consuming processes. We determined the single-particle cryo-EM structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at 2.7- 2.8 [A] resolution. Separation of 13 well-defined rotary substates by 3D classification provides a detailed picture of the molecular motions that accompany c-ring rotation and result in ATP synthesis. Crucially, the F1 head rotates along with the central stalk and c-ring rotor for the first ~30{degrees} of each 120{degrees} primary rotary step. The joint movement facilitates flexible coupling of the stoichiometrically mismatched F1 and Fo subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit, a well-established target of physiologically important inhibitors. Our maps provide atomic detail of the c-ring/a-subunit interface in the membrane, where protonation and deprotonation of c-ring cGlu111 drives rotary catalysis. An essential histidine residue in the lumenal proton access channel binds a strong non-peptide density assigned to a metal ion that may facilitate c-ring protonation, as its coordination geometry changes with c-ring rotation. We resolve ordered water molecules in the proton access and release channels and at the gating aArg239 that is critical in all rotary ATPases. We identify the previously unknown ASA10 subunit and present complete de novo atomic models of subunits ASA1-10, which make up the two interlinked peripheral stalks that stabilize the Polytomella ATP synthase dimer.

biochemistry↗