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Results for “Biochemistry”

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At least 1,117 records · Page 62Linked to original sources

bioRxiv2018

Optimized fragmentation improves the identification of peptides cross-linked using MS-cleavable reagents

Cross-linking mass spectrometry (XLMS) is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by non-specialist laboratories. Specifically, the use of novel mass spectrometry-cleavable (MS-cleavable) reagents dramatically reduces complexity of the data by providing i) characteristic reporter ions and ii) the mass of the individual peptides, rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone is yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl sulfoxide (DSSO)- based cross-links on HCD-equipped mass spectrometers. From the comparison of 9 different fragmentation energies we chose several stepped-HCD fragmentation methods that were evaluated on a variety of cross-linked proteins. The optimal stepped-HCD-method was then directly compared with previously described methods using an Orbitrap Fusion Lumos TribridTM instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multistage MS (MSn) enabled instrumentation and alternative dissociation techniques.

biochemistry↗

bioRxiv2018

Gentamicin B1 is not a minor gentamicin component with major nonsense mutation suppression activity

Nonsense mutations are single base substitutions that introduce a premature termination codon (PTC) preventing the formation of full-length protein. They are the causative mutations in about 10% of patients in a large number of rare genetic diseases. High concentrations of the antibiotic gentamicin can induce the incorporation of an amino acid at a PTC and formation of full-length protein, a process called PTC readthrough. Gentamicin is composed of several related aminoglycosides. We recently reported (doi/10.1073/pnas.1620982114) that the major gentamicin components that are responsible for its antibacterial activity showed weak to no PTC readthrough activity but that the minor component gentamicin B1 was a potent readthrough inducer. We have now determined that gentamicin B1 acquired from the sole supplier at the time the study was carried out was not gentamicin B1 but instead the closely related aminoglycoside G418. Gentamicin B1 recently became available from a second commercial source. Here, we provide nuclear magnetic resonance (NMR) assignment data for the two commercial compounds and verify only the second is indeed gentamicin B1. We show that gentamicin B1 lacks PTC readthrough activity in HDQ-P1 and DMS-114 cells homozygous for the TP53 R213X nonsense mutation, as well as in a cell-free translation assay.

biochemistry↗

bioRxiv2018

Structural insights into SETD3-mediated histidine methylation on β-actin

SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. In a previous paper (Kwiatkowski et al. 2018), we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on {beta}-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified {beta}-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of {beta}-actin by SETD3 is highly sequence specific, and both SETD3 and {beta}-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the structural research uncovers the molecular mechanism of sequence-selective histidine methylation by SETD3, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.

biochemistry↗

bioRxiv2018

Beyond the seed: structural basis for supplementary mircoRNA targeting

microRNAs (miRNA) guide Argonaute proteins to mRNAs targeted for repression. Target recognition occurs primarily through the miRNA seed region, composed of guide (g) nucleotides g2-g8. However, nucleotides beyond the seed are also important for some known miRNA-target interactions. Here, we report the structure of human Argonaute2 (Ago2) engaged with a target RNA recognized through both miRNA seed and supplementary (g13-g16) regions. Ago2 creates a \"supplementary chamber\" that accommodates up to 5 miRNA-target base pairs. Seed and supplementary chambers are adjacent to each other, and can be bridged by an unstructured target loop of 1-15 nucleotides. Opening of the supplementary chamber may be constrained by tension in the miRNA 3' tail as increases in miRNA length stabilize supplementary interactions. Contrary to previous reports, we demonstrate optimal supplementary interactions can increase target affinity >20-fold. These results provide a mechanism for extended miRNA-targeting, suggest a function for 3' isomiRs in tuning miRNA targeting specificity, and indicate that supplementary interactions may contribute more to target recognition than is widely appreciated.

biochemistry↗

bioRxiv2018

Lipid Lowering Oxopropanylindole Hydrazone Derivatives with Antioxidant and Antihyperglycemic Activity

A series of substituted oxopropanylindole hydrazone derivatives was synthesized and evaluated for anti-oxidant and anti-dyslipidemic activity. Among these 12 compounds the three compounds 6c, 7b and 7d showed good anti-oxidant activity and the compound 6c attenuataed LDL oxidation by 32%. The compounds 6c and 7d also showed good anti-dyslipidemic activity by reducing serum levels of total cholesterol (TC), phospholipids (PL) and triglycerides (TG). These two compounds were further evaluated for anti-adipogenic and anti-hyperglycemic activity, where 6c was found most active compound with 44% reduction in lipid accumulation and 20.5% and 24.3% reduction in blood glucose at 5h and 24h respectively, as compared to standard drug metformin.

biochemistry↗

bioRxiv2018

Reconciling in vitro and in vivo activities of engineered, LacI-based repressor proteins: Contributions of DNA looping and operator sequence variation

One way to create new components for synthetic transcription circuits is to re-purpose naturally occurring transcription factor proteins and their cognate DNA operators. For the proteins, re-engineering can be accomplished via domain recombination (to create chimeric regulators) and/or amino acid substitutions. The resulting activities of new protein regulators are often assessed in vitro using a representative operator. However, when functioning in vivo, transcription factors can interact with multiple operators. We compared in vivo and in vitro results for two LacI-based transcription repressor proteins, their mutational variants, and four operator sequences. The two sets of repressor variants differed in their overall in vivo repression, even though their in vitro binding affinities for the primary operator spanned the same range. Here, we show that the offset can be explained by different abilities to simultaneously bind and \"loop\" two DNA operators. Further in vitro studies of the looping-competent repressors were carried out to measure binding to a secondary operator sequence. Surprisingly, binding to this operator was largely insensitive to amino acid changes in the repressor protein. In vitro experiments with additional operators and analyses of published data indicates that amino acid changes in these repressor proteins leads to complicated changes in ligand specificity. These results raise new considerations for engineering components of synthetic transcription circuits and - more broadly - illustrate difficulties encountered when trying to extrapolate information about specificity determinant positions among protein homologs.

biochemistry↗

bioRxiv2018

Live-cell single RNA imaging reveals bursts of translational frameshifting

Ribosomal frameshifting during the translation of RNA is implicated in both human disease and viral infection. While previous work has uncovered many mechanistic details about single RNA frameshifting kinetics in vitro, very little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames. Applying this technology to RNA encoding the HIV-1 frameshift sequence reveals a small subset (~8%) of the translating pool robustly frameshift in living cells. Frameshifting RNA are preferentially in multi-RNA \"translation factories,\" are translated at about the same rate as non-frameshifting RNA (~2 aa/sec), and can continuously frameshift for more than four rounds of translation. Fits to a bursty model of frameshifting constrain frameshifting kinetic rates and demonstrate how ribosomal traffic jams contribute to the persistence of the frameshifting state. These data provide novel insight into retroviral frameshifting and could lead to new strategies to perturb the process in living cells.

biochemistry↗

bioRxiv2018

Molecular complexity of the major urinary protein system of the Norway rat, Rattus norvegicus

Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Detailed sequencing of the proteins reveals a less complex pattern of primary sequence polymorphism than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications including phosphorylation and exoproteolytic trimming of specific isoforms. The possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse is also discussed.

biochemistry↗

bioRxiv2018

Resilience of BST-2/Tetherin structure to single amino acid substitutions

Human Tetherin, also known as BST-2 or CD317, is a dimeric, extracellular membrane-bound protein that consists of N and C terminal membrane anchors connected by an extracellular domain. BST-2 is involved in binding enveloped viruses, such as HIV, and inhibiting viral release in addition to a role in NF-kB signaling. Viral tethering by Tetherin can be disrupted by the interaction with Vpu in HIV-1 in addition to other viral proteins. The structural mechanism of Tetherin function is not clear and the effects of human Tetherin mutations identified by sequencing consortiums are not known. To address this gap in the knowledge, we used data from the Ensembl database to construct and model known human missense mutations within the ectodomain to investigate how the structure of the ectodomain influences function. From the data, we identified an island of sequence stability within the ectodomain, which corresponds to functionally or structurally important region identified in previous biochemical and biophysical studies. Additionally, most mutations have little effect on structure, suggesting that they would not affect function. These findings are in agreement with biochemical and cellular studies which suggest that mutations that do not disrupt the alpha helices of Tetherin have little apparent effect on function. Thus, Tetherin sequence is likely less important than structure and this apparent flexibility may allow for greater anti-viral activities with a larger number of viruses.

biochemistry↗

bioRxiv2018

6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion

The proteinaceous extracellular matrix (ECM) is vital for cancer cell survival, proliferation, migration, and differentiation. However, little is known regarding metabolic pathways required in the ECM secretion process. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for endoplasmic reticulum (ER) structural integrity and protein secretion. Chemical inhibition or genetic suppression of its activity led to cell stress accompanied by significantly expanded ER volume and can be rescued by compensating glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD exemplifies a nexus of cytosolic carbohydrate metabolism and protein secretion.

biochemistry↗

bioRxiv2018

Structural basis for ion selectivity in TMEM175 K+ channels

The TMEM175 family constitutes recently discovered K+ channels that lack signatures for a P-loop selectivity filter, a hallmark of all known K+ channels. This raises the question how selectivity in TMEM175 channels is achieved. Here we report the X-ray structure of a bacterial TMEM175 family member in complex with a novel chaperone built of a nanobody fusion-protein. The structure of the channel in a non-conductive conformation was solved at 2.4 [A] and revealed bound K+ ions along the channel pore. A hydrated K+ ion at the extracellular pore entrance that could be substituted with Cs+ and Rb+ is coordinated by backbone-oxygens forming a cation-selective filter at the tip of the pore-lining helices. Another K+ ion within the pore indicates the passage of dehydrated ions. Unexpectedly, a highly conserved threonine residue deeper in the pore conveys the K+ selectivity. The position of this threonine in the non-conductive state suggests major conformational rearrangements of the pore-lining helices for channel opening, possibly involving iris-like motions.

biochemistry↗

bioRxiv2018

Unprocessed serum glycosylphosphatidylinositol-anchored proteins are correlated to metabolic states

To study the possibility that components of eukaryotic plasma membranes are released in spontaneous or controlled fashion, a chip-based sensor was developed for complete glycosylphosphatidylinositol-anchored proteins (GPI-AP), which may form together with (phospho)lipids so far unknown (non-vesicular) extracellular complexes (GLEC). The sensor relies on changes in phase shift and amplitude of surface acoustic waves propagating over the chip surface upon specific capturing of the GPI-AP and detection of associated phospholipids and renders isolation of the labile GLEC unnecessary. GLEC were found to be released from isolated rat adipocyte plasma membranes immobilized on the chip, dependent on the flow rate and composition of the buffer stream. Moreover, incubation medium of isolated adipocytes and serum of rats are sources for GLEC which enables their differentiation according to cell size and genotype or body weight, respectively, as well as human serum.

biochemistry↗

bioRxiv2018

S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate desmosome assembly and cell adhesion.

S-acylation is the only fully reversible lipid modification of proteins however little is known about how protein S-acyltransferases (PATs) that mediate it are regulated. DHHC5 is a plasma membrane-localised PAT with roles in synaptic plasticity, massive endocytosis and cancer cell growth/invasion. Here we demonstrate that stabilisation of DHHC5 at the plasma membrane requires binding to and palmitoylation of an accessory protein Golga7b. This interaction requires the palmitoylation of the C-terminus of DHHC5 which regulates the internalisation of DHHC5 from the plasma membrane. Proteomic analysis of DHHC5/Golga7b-associated protein complexes reveals an enrichment in adhesion proteins, particularly components of desmosomes. We show that Desmoglein-2 and Plakophilin-3 are substrates of DHHC5 and that DHHC5/Golga7b are required for localisation of Desmoglein-2 to the plasma membrane and desmosomal patterning. Loss of DHHC5/Golga7b causes functional impairments in cell adhesion suggesting these proteins have a wider role in cell adhesion beyond desmosome assembly. This work uncovers a novel mechanism of DHHC5 regulation by Golga7b and demonstrates a role for the DHHC5/Golga7b complex in the regulation of cell adhesion.

biochemistry↗

bioRxiv2018

Effect of partial substitution of rice with sorghum and inclusion of hydrolyzable tannins on digestibility and postprandial glycemia in adult dogs

Sorghum is used as a substitute of rice in dog food, owing to its nutritional similarity and low cost. However, its use has been associated with negative effects, like a reduction in palatability, digestibility, and enzyme activity, which can decrease nutrient absorption. The presence of condensed tannins (CT) in sorghum may cause these effects. Another tannin group, the hydrolysable tannins (HT), is known for its antioxidant properties. Research has shown the nutritional effects of sorghum on dogs, but the effect of HT on dogs remains unknown. We evaluated the effects of substituting rice with sorghum containing CT and inclusion of commercial extract of HT on digestibility, fecal and urinary characteristics, and postprandial blood glucose levels in adult dogs. Nine adult Beagle were randomly subjected to 4 treatments: 50% rice; (RS) 25% rice + 25% sorghum; (RHT) 50% rice + 0.10% HT; (RSHT) 25% rice + 25% sorghum + 0,10% HT. Tannins did not affect food intake. The digestibility of dry matter, organic matter, crude protein, acid hydrolyzed fat, gross energy, and metabolizable energy (ME) decreased with sorghum inclusion (P < 0.05). Sorghum also decreased protein digestibility (P < 0.05). Greater fecal dry matter was observed with the RHT diet. HT associated with sorghum reduced ME (P < 0.05). Sorghum inclusion enhanced fecal output, without altering fecal score (P > 0.05). No alterations in urinary characteristics were observed. Sorghum and HT did not affect the postprandial blood glucose response measured by the area under the curve (P > 0.05). The substitution of rice by sorghum negatively affected protein absorption and ME of the diets. Sorghum can be considered as a good source of carbohydrates in therapeutic diets for weight control. HT may potentiate the effect of CT, but more research is needed to evaluate its potential use in dog nutrition.

biochemistry↗

bioRxiv2018

Vernonia Amygdalina Del (Bitter Leaf) extract ameliorates isoniazid (INH) induced liver injury in Swiss Albino Mice

Liver plays a central role in the metabolism of drugs. Drug clearance and transformation exposes liver to toxic injury. Antitubercular drugs have been found to be hepatotoxic and potentially lead to drug-induced liver injury. Isoniazid is one of the most hepatotoxic first line antitubercular drugs. Conventional drugs used in the treatment of liver disease are often inadequate and a search for supplementation or alternative drugs for the treatment of hepatic damage is indispensible. Therefore our study aims to investigate the hepatoprotective potential of Vernonia Amygdalina Del (bitter leaf) extract against Isoniazid-induced liver injury in Swiss Albino Mice. Treatment of Mice orally with Vernonia Amygdalina Del extract at dose of 250mg/kg and 375 mg/kg significantly lowered (P<0.05) the serum level of liver enzymes in Isoniazid pretreated mice. The hepatoptotective activity of the extract found to be comparable with the standard drug, Silymarin (100 mg/kg, P.o.). Moreover, treatment with the extract significantly alleviated Isoniazid induced hepatic injury as supported by the photomicrographs of liver section of mice. The data shows aqueous Vernonia Amygdalina Del extract has a very promising hepatoprotective potential against isoniazid-induced liver injury.

biochemistry↗

bioRxiv2018

Molecular fingerprints for a novel glucosamine kinase family in Actinobacteria

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine-binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production.

biochemistry↗

bioRxiv2018

Structural basis of Cullin-2 RING E3 ligase regulation by the COP9 signalosome

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 (N8) from activated Cullins. The structure of stable CSN-CRL can be used to understand this mechanism of regulation. Here we present the first structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (MS). These structures reveal a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen deuterium exchange-MS we show that CRL2 binding and conformational activation of CSN5/CSN6 occur in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identified novel sensory regions of the CSN that mediate its stepwise activation mechanism and provide a framework for better understanding the regulatory mechanism of other Cullin family members.\n\nOne sentence summaryStructure and dynamics of the CSN-CRL2 complexes assessed by cryo-electron microscopy and structural mass spectrometry.

biochemistry↗

bioRxiv2018

Genetic, structural, and functional analysis of mutations causing methylmalonyl-CoA epimerase deficiency

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of D-methylmalonyl-CoA and L-methylmalonyl-CoA in propionate catabolism. Autosomal recessive mutations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with mutations in MCEE. Nine patients were homozygous for the known nonsense mutation p.Arg47* (c.139C>T), and one for the novel missense mutation p.Ile53Arg (c.158T>G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described patient mutations p.Lys60Gln and p.Arg143Cys, onto our 1.8 [A] structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this mutation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. We solved the structure of MCEE-Arg143Cys to 1.9 [A] and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.

biochemistry↗