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bioRxiv2018

A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

Nonsense suppressors (NonSups) induce \"readthrough\", i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly-purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.\n\nTable of Contents artwork\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC=\"FIGDIR/small/330506_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@a548dborg.highwire.dtl.DTLVardef@15237bcorg.highwire.dtl.DTLVardef@c93d3forg.highwire.dtl.DTLVardef@1d40e2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2018

Grape polyphenols reduce gut-localized reactive oxygen species associated with the development of metabolic syndrome in mice.

High-fat diet (HFD)-induced leaky gut syndrome combined with low-grade inflammation increase reactive oxygen species (ROS) in the intestine and may contribute to dysbiosis and metabolic syndrome (MetS). Poorly bioavailable and only partially metabolizable dietary polyphenols, such as proanthocyanidins (PACs), may exert their beneficial effects on metabolic health by scavenging intestinal ROS. To test this hypothesis, we developed and validated a novel, noninvasive, in situ method for visualizing intestinal ROS using orally administered ROS-sensitive indocyanine green (ICG) dye. C57BL/6J mice fed HFD for 10 weeks accumulated high levels of intestinal ROS compared to mice fed low-fat diet (LFD). Oral administration of poorly bioavailable grape polyphenol extract (GPE) and {beta}-carotene decreased HFD-induced ROS in the gut to levels comparable to LFD-fed mice, while administration of more bioavailable dietary antioxidants (- lipoic acid, vitamin C, vitamin E) did not. Forty percent of administered GPE antioxidant activity was measured in feces collected over 24 h, confirming poor bioavailability and persistence in the gut. The bloom of beneficial anaerobic gut bacteria, such as Akkermansia muciniphila, associated with improved metabolic status in rodents and humans may be directly linked to protective antioxidant activity of some dietary components. These findings suggest a possible mechanistic explanation for the beneficial effects of poorly bioavailable polyphenols on metabolic health.

biochemistry↗

bioRxiv2018

MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions1-3. The maximum catalytic activity of hydroxylase (MMOH) is achieved via interplay of its regulatory protein (MMOB) and reductase4-6. An additional auxiliary protein, MMOD, is believed to function as an inhibitor of the catalytic activity of MMOH; however, the mechanism of its action remains unknown7,8. Herein, we report the crystal structure of MMOH-MMOD complex from Methylosinus sporium strain 5 (2.6 [A]), which illustrates that two molecules of MMOD associate symmetrically with the canyon region of MMOH in a manner similar to MMOB, indicating that MMOD competes with MMOB for MMOH recognition. Further, MMOD binding disrupts the geometry of the di-iron centre and opens the substrate access channel. Notably, the electron density of 1,6-hexanediol at the substrate access channel mimics products of sMMO in hydrocarbon oxidation. The crystal structure of MMOH-MMOD unravels the inhibitory mechanism by which MMOD suppresses the MMOH catalytic activity, and reveals how hydrocarbon substrates/products access to the di-iron centre.

biochemistry↗

bioRxiv2018

Limits for resolving tandem mass tag reporter ions with identical integer mass using phase constrained spectrum deconvolution

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT) which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements. This contrasts with faster peptide sequencing capabilities enabled by lowered mass resolution on Orbitrap instruments. It is therefore important to determine the mass resolution limits for highly multiplexed quantification when maximizing proteome depth. Here we defined the lower boundaries for resolving TMT reporter ions with 0.0063 Da mass differences using an ultra-high-field Orbitrap mass spectrometer. We found the optimal method depends on the relative ratio between closely spaced reporter ions and that 64 ms transient acquisition time provided sufficient resolving power for separating TMT reporter ions with absolute ratio changes up to 16-fold. Furthermore, a 32 ms transient processed with phase-constrained spectrum deconvolution provides >50% more identifications with >99% quantified, but with a slight loss in quantification precision and accuracy. These findings should guide decisions on what Orbitrap resolution settings to use in future proteomics experiments relying on TMT reporter ion quantification with identical integer masses.

biochemistry↗

bioRxiv2018

Re-targeting of macroH2A following mitosis to cytogenetic-scale heterochromatic domains

The heritability of chromatin states through cell division is a potential contributor to the epigenetic maintenance of cellular memory of prior states. The macroH2A histone variant has properties of a regulator of epigenetic cell memory, including roles controlling gene silencing and cell differentiation. Its mechanisms of regional genomic targeting and maintenance through cell division are unknown. Here we combined in vivo imaging with biochemical and genomic approaches to show that human macroH2A is incorporated into chromatin in the G1 phase of the cell cycle following DNA replication. The newly-incorporated macroH2A re-targets the same, large heterochromatic domains where macroH2A was already enriched in the previous cell cycle. It remains heterotypic, targeting individual nucleosomes that do not already contain a macroH2A molecule. The pattern observed resembles that of new deposition of centromeric histone variants during the cell cycle, indicating mechanistic similarities for macrodomain-scale regulation of epigenetic properties of the cell.

biochemistry↗

bioRxiv2018

Tracing a proteins folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange

The conformations populated during protein folding have been studied for decades; yet, their evolutionary importance remains largely unexplored. Ancestral sequence reconstruction allows access to proteins across evolutionary time, and new methods such as pulsed-labeling hydrogen exchange coupled with mass spectrometry allow determination of folding intermediate structures at near amino-acid resolution. Here, we combine these techniques to monitor the folding of the ribonuclease H family along the evolutionary lineages of T. thermophilus and E. coli RNase H. All homologs and ancestral proteins studied populate a similar folding intermediate despite being separated by billions of years of evolution. Even though this conformation is conserved, the pathway leading to it has diverged over evolutionary time, and rational mutations can alter this trajectory. Our results demonstrate that evolutionary processes can affect the energy landscape to preserve or alter specific features of a proteins folding pathway.

biochemistry↗

bioRxiv2018

Domain Insertion Permissibility is a Measure of Engineerable Allostery in Ion Channels

Allostery is a fundamental principle of protein regulation that remains poorly understood and hard to engineer, in particular in ion channels. Here we use human Inward Rectifier K+ Channel Kir2.1 to establish domain insertion permissibility as a new experimental paradigm to identify engineerable allosteric sites. We find that permissibility is best explained by dynamic protein properties, such as conformational flexibility. Many allosterically regulated sites in Kir2.1 or sites equivalent to those regulated in homologs, such as G-protein-gated inward rectifier K+ channels (GIRK), have differential permissibility; that is, for these sites permissibility depends on the structural properties of the inserted domain. Our data and the well-established link between protein dynamics and allostery led us to propose that differential permissibility is a metric of both existing and latent allostery in Kir2.1. In support of this notion, inserting light-switchable domains into either existing or latent allosteric sites, but not elsewhere, renders Kir2.1 activity sensitive to light.

biochemistry↗

bioRxiv2018

Identification of a novel tetrameric structure for human apolipoprotein-D

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded {beta}-barrel with a single adjacent -helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimers disease, where apolipoprotein-D overexpression reduces the amyloid-{beta} burden in Alzheimers disease mouse models.\n\nOligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids.\n\nHere, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a {bsim}95 to {bsim}100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

biochemistry↗

bioRxiv2018

Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation

The human dopamine transporter(hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the molecular architecture of hDAT and its various conformational states are poorly understood. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, first we identified ligands that stabilize hDAT in detergent micelles. Then, we screened [~]200 single residue mutants of hDAT using high-throughput scintillation proximity assay, and identified a thermostable variant(I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.

biochemistry↗

bioRxiv2018

A non-canonical metal center drives activity of the Sediminispirochaeta smaragdinae metallo-β- lactamase SPS-1

In an effort to evaluate whether a recently reported putative metallo-{beta}-lactamase (M{beta}L) contains a novel M{beta}L active site, SPS-1 from Sediminispirochaeta smaragdinae was over-expressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equivalents of Zn (II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not {beta}-lactam substrates with bulky substituents in the 6-7 position. Spectroscopic studies on Co (II)-substituted SPS-1 suggest a novel metal center in SPS-1, with reduced spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar that that in NDM-1 and other subclass B1 M{beta}Ls; however, the Zn1 metal ion is coordinated by 2 histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 [A] from the position reported in other M{beta}Ls. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents in the 6/7 position of {beta}-lactam antibiotics. This study reveals a novel metal binding site in M{beta}Ls, and suggests that the targeting of metal binding sites in M{beta}Ls with inhibitors is now more challenging with the identification of this new M{beta}L.

biochemistry↗

bioRxiv2018

The Expression of Inflammatory Genes in 3T3-L1 Adipocytes Exhibits a Memory to Stimulation by Macrophage Secretions

Obesity is characterized by increased output of inflammatory compounds from adipose tissue. Whilst the relative contribution of adipocytes and resident macrophages to this phenomenon is debated, there is no doubt that the secretions of each cell type can stimulate the expression of inflammatory genes in the other. We hypothesized that mechanisms must exist to prevent an escalating positive feedback loop between the two cell types, so that after an initial exposure to macrophage secretions, adipocytes would become desensitized to subsequent inflammatory stimulation.\n\nWe used microarrays to investigate the response of 3T3-L1 adipocytes to macrophage secretions (macrophage conditioned medium, MCM). MCM caused a rapid (<4 hours) and high amplitude (over 100-fold) rise in the expression of several inflammatory genes. For some genes, generally cytokines, expression returned to basal levels within 24 h following removal of the MCM, but other transcripts, notably those for acute phase proteins and extracellular matrix remodeling proteins, remained highly expressed even during the washout period.\n\nUnexpectedly, some cytokine genes (e.g., iNOS, IL-6) showed an enhanced expression to a second exposure of MCM, illustrating that the transcriptome response of 3T3-L1 adipocytes retains a memory to the first stimulus. We characterized the parameters that give rise to the memory phenomenon, finding that additional stimuli do not augment or abrogate the effect. The memory is preserved for several days after the initial exposure and it is not due to a change in sensitivity to the MCM but, rather, a change in the capacity of the signal-target system. The possible mechanisms of the memory are discussed, along with the physiological ramifications should the phenomenon be replicated in vivo.

biochemistry↗

bioRxiv2018

A skin pigment compound that also functions as sunglasses for birds

An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as "compound X", was fractionated and examined using a variety of spectroscopic techniques including LC-MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective "compound X". It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.

biochemistry↗

bioRxiv2018

Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice

The recent discovery that myosin has two distinct states in relaxed muscle - disordered relaxed (DRX) and super-relaxed (SRX) - provides another factor to consider in our fundamental understanding of the aging mechanism in skeletal muscle, since myosin is thought to be a potential contributor to dynapenia. The primary goal of this study was to determine the effects of age on DRX and SRX states and to examine their sex specificity. We have used quantitative fluorescence microscopy of the fluorescent nucleotide analog 2'/3'-O-(N-methylanthraniloyl) ATP (mantATP) to measure single-nucleotide turnover kinetics of myosin in skinned skeletal muscle fibers under relaxing conditions. We examined changes in DRX and SRX in response to the natural aging process by measuring the turnover of mantATP in skinned fibers isolated from psoas muscle of adult young (3-4 months old) and aged (26-28 months old) C57BL/6 female and male mice. Fluorescence decays were fitted to a multi-exponential decay function to determine both the time constants and mole fractions of fast and slow turnover populations, and significance was analyzed by a t-test. We found that in females, both the DRX and SRX lifetimes of myosin ATP turnover at steady state were shorter in aged muscle fibers compared to young muscle fibers (p[≤]0.033). However, there was no significant difference in relaxation lifetime of either DRX (p=0.202) or SRX (p=0.804) between young and aged male mice. No significant effects were measured on the mole fractions (populations) of these states, as a function of sex or age (females, p=0.100; males, p=0.929). The effect of age on the order of myosin heads at rest and their ATPase function is sex specific, affecting only females. These findings provide new insight into the molecular factors and mechanisms that contribute to aging muscle dysfunction in a sex-specific manner.

biochemistry↗

bioRxiv2018

Proteomic Analysis of Urine from California Sea Lions (Zalophus californianus): a Resource for Urinary Biomarker Discovery

Urinary markers for the assessment of kidney diseases in wild animals are limited, in part, due to the lack of urinary proteome data, especially for marine mammals. One of the most prevalent kidney diseases in marine mammals is caused by Leptospira interrogans, which is the second most common etiology linked to stranding of California sea lions (Zalophus californianus). Urine proteins from eleven sea lions with leptospirosis kidney disease and eight sea lions without leptospirosis or kidney disease were analyzed using shotgun proteomics. In total, 2694 protein groups were identified and 316 were differentially abundant between groups. Major urine proteins in sea lions were similar to major urine proteins in dogs and humans except for the preponderance of resistin, lysozyme C, and PDZ domain containing 1, which appear to be over-represented. Previously reported urine protein markers of kidney injury in humans and animals were also identified. Notably, neutrophil gelatinase-associated lipocalin, osteopontin, and epidermal fatty acid binding protein were elevated over 20-fold in the leptospirosis-infected sea lions. Consistent with leptospirosis infection in rodents, urinary proteins associated with the renin-angiotensin system were depressed, including neprilysin. This study represents a foundation from which to explore the clinical use of urinary protein markers in California sea lions.\n\nAbbreviations

biochemistry↗

bioRxiv2018

O-GlcNAc Homeostasis Controls Cell Fate Decisions During Hematopoiesis

The addition of O-GlcNAc (a single {beta}-D-N-acetylglucosamine sugar at serine and threonine residues) by O-GlcNAc transferase (OGT) and removal by O-GlcNAcase (OGA) maintains homeostatic levels of O-GlcNAc. We investigated the role of O-GlcNAc homeostasis in hematopoiesis utilizing G1E-ER4 cells carrying a GATA-1 transcription factor fused to the estrogen receptor (GATA-1ER) that undergo erythropoiesis following the addition of {beta}-estradiol (E2) and myeloid leukemia cells that differentiate into neutrophils in the presence of all-trans retinoic acid. During G1E-ER4 differentiation, a decrease in overall O-GlcNAc levels and an increase in GATA-1 interactions with OGT and OGA were observed. Transcriptome analysis on G1E-ER4 cells differentiated in the presence of Thiamet-G (TMG), an OGA inhibitor, identified expression changes in 433 GATA-1 target genes. Chromatin immunoprecipitation demonstrated that the occupancy of GATA-1, OGT, and OGA at Laptm5 gene GATA site was decreased with TMG. Myeloid leukemia cells showed a decline in O-GlcNAc levels during differentiation and TMG reduced the expression of genes involved in differentiation. Sustained treatment with TMG in G1E-ER4 cells prior to differentiation caused a reduction of hemoglobin positive cells during differentiation. Our results show that alterations in O-GlcNAc homeostasis disrupt transcriptional programs causing differentiation errors suggesting a vital role of O-GlcNAcylation in control of cell fate.

biochemistry↗

bioRxiv2018

Practical model selection for prospective virtual screening

Virtual (computational) high-throughput screening provides a strategy for prioritizing compounds for experimental screens, but the choice of virtual screening algorithm depends on the dataset and evaluation strategy. We consider a wide range of ligand-based machine learning and docking-based approaches for virtual screening on two protein-protein interactions, PriA-SSB and RMI-FANCM, and present a strategy for choosing which algorithm is best for prospective compound prioritization. Our workflow identifies a random forest as the best algorithm for these targets over more sophisticated neural network-based models. The top 250 predictions from our selected random forest recover 37 of the 54 active compounds from a library of 22,434 new molecules assayed on PriA-SSB. We show that virtual screening methods that perform well in public datasets and synthetic benchmarks, like multi-task neural networks, may not always translate to prospective screening performance on a specific assay of interest.

biochemistry↗

bioRxiv2018

Crystal structure of the MBD domain of MBD3 in complex with methylated CG DNA

MBD3 is a core subunit of the Mi-2/NuRD complex, and has been previously reported to lack methyl-CpG binding ability. However, recent reports show that MBD3 recognizes both mCG and hmCG DNA with a preference for hmCG, and is required for the normal expression of hmCG marked genes in ES cells. Nevertheless, it is not clear how MBD3 recognizes the methylated DNA. In this study, we carried out structural analysis coupled with isothermal titration calorimetry (ITC) binding assay and mutagenesis studies to address the structural basis for the mCG DNA binding ability of the MBD3 MBD domain. We found that the MBD3 MBD domain prefers binding mCG over hmCG through the conserved arginine fingers, and this MBD domain as well as other mCG binding MBD domains can recognize the mCG duplex without orientation selectivity. Furthermore, we found that the tyrosine-to-phenylalanine substitution at Phe34 of MBD3 is responsible for its weaker mCG DNA binding ability compared to other mCG binding MBD domains. In summary, our study demonstrates that the MBD3 MBD domain is a mCG binder, and also illustrates its binding mechanism to the methylated CG DNA.

biochemistry↗

bioRxiv2018

Pathogenic mutations in NUBPL affect complex I activity and cold tolerance in the yeast model Yarrowia lipolytica

Complex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in NUBPL, an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica. L102P destabilized the Ind1 protein, leading to a null-mutant phenotype. D103Y, L191F and G285C affected complex I assembly to varying degrees, whereas the G138D variant did not impact on complex I levels or dNADH:ubiquinone activity. Blue-native PAGE and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q-module intermediate of complex I. In the D103Y variant the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 G285C variant was able to support complex I assembly at 28{degrees}C, but not at 10{degrees}C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.

biochemistry↗