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Results for “Biochemistry”

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bioRxiv2019

Imaging of Morphological and Biochemical Hallmarks of Apoptosis with Optimized Optogenetic Actuators

The creation of pathway-specific optogenetic switches for the activation of cell death pathways can provide insight into the mechanisms of apoptosis and also form a basis for non-invasive, next generation therapeutic strategies. Previous work has employed the Cryptochrome 2 (Cry2)/CIB, a blue light activated protein - protein dimerization module from A. thaliana in conjunction with BAX, an OMM targeting pro-apoptotic protein, for light-mediated initiation of mitochondrial outer membrane permeabilization (MOMP) and downstream apoptosis. In this work, we have further developed our original light activated Cry2-BAX system (henceforth referred to as "OptoBAX") by improving the photophysical properties and light-independent interaction of our optogenetic switch. The resulting optogenetic constructs have significantly reduced the frequency of light exposure required for the activation of membrane permeabilization, in addition to reducing dark state cytotoxicity. Furthermore, we have utilized OptoBAX in a series of experiments designed to measure the timing of the dramatic morphological and biochemical changes that occur in apoptotic cells following light-induced permeabilization of the outer mitochondrial membrane. Utilizing this data, we construct a timeline of biochemical and morphological events in early apoptosis, in addition to tracking the MOMP-induced redistribution of actin, a protein critical to apoptotic progression.

biochemistry↗

bioRxiv2019

Visualizing Conformational Transitions of the Lipid II Flippase MurJ

The biosynthesis of many polysaccharides, including bacterial peptidoglycan and eukaryotic N-linked glycans, requires transport of lipid-linked oligosaccharide (LLO) precursors across the membrane by specialized flippases. MurJ is the flippase for the lipid-linked peptidoglycan precursor Lipid II, a key player in bacterial cell wall synthesis, and a target of recently discovered antibacterials. However, the flipping mechanism of LLOs including Lipid II remains poorly understood due to a dearth of structural information. Here we report crystal structures of MurJ captured in inward-closed, inward-open, inward-occluded and outward-facing conformations. Together with cysteine accessibility, mass spectrometry, and complementation studies, we elucidate the conformational transitions in MurJ that mediate lipid flipping, identified the key ion for function, and provide a framework for the development of inhibitors.

biochemistry↗

bioRxiv2019

A quantitative tri-fluorescent yeast two-hybrid system: from flow cytometry to in-cellula affinities

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on PPIs with different affinities. After only two hours of reaction, expression of the reporter can easily be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflects the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated high-throughput analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.

biochemistry↗

bioRxiv2019

Dimerization of human Rio2 kinase/ATPase locks its ATP-binding site in an apo state

Rio proteins form a conserved family of atypical protein kinases. Rio2 is a serine/threonine protein kinase/ATPase involved in pre-40S ribosomal maturation. Current crystal structures of archaeal and fungi Rio2 proteins report a monomeric form of the protein. Here, we describe three atomic structures of the human Rio2 kinase showing that it forms a homodimer. Upon self-association, the ATP-binding pocket is hidden from the solvent and the protein is locked in an apo state corresponding to an inactive form of the kinase. The homodimerization is mediated by key residues previously shown to be responsible for ATP binding and catalysis. This unusual protein kinase dimer reveals an intricate mechanism of mutually exclusive substrate binding and oligomeric state formation. We propose that this oligomeric state could serve a dual function in maintaining the protein in an inactive state and being a novel type of nuclear import signal. Significance StatementRio kinases form a family of atypical protein kinases that are believed to be ATPases rather than kinases. The three members of the Rio family are involved in ribosome biogenesis. We show here that contrarily to what was reported so far, Rio2 is able homodimerize in a conformation that locks it in an apo state, preventing its (re)association to pre-mature ribosomes. This unconventional self-association is not seen in any other protein kinase. This mechanism is likely to be transient and could used to efficiently re-import the protein to the nucleus.

biochemistry↗

bioRxiv2019

The carboxyl-terminal sequence of Bim enables Bax activation and killing of unprimed cells

The Bcl-2 family BH3 protein Bim promotes apoptosis at mitochondria by activating the pore forming proteins Bax and Bak and by inhibiting the anti-apoptotic proteins Bcl-XL, Bcl-2 and Mcl-1. Bim binds to these proteins via its BH3 domain and to the mitochondrial membrane by a carboxyl-terminal sequence (CTS). In cells killed by Bim, the expression of a Bim mutant in which the CTS was deleted (BimL-dCTS) triggered variable amounts of apoptosis that correlated with inhibition of anti-apoptotic proteins being sufficient to permeabilize mitochondria isolated from the same cells. Detailed analysis of the molecular mechanism demonstrated that BimL-dCTS inhibited Bcl-XL but did not activate Bax. Our examination of additional point mutants unexpectedly revealed that the CTS of Bim is required for physiological concentrations of Bim to activate Bax and that different residues in the CTS enable Bax activation and binding to membranes.

biochemistry↗

bioRxiv2019

Metabolic Modulation Predicts Heart Failure Tests Performance

Cardiopulmonary testing (CPET) and biomarkers such as NT-proBNP, Galectin-3, and C-reactive protein (CRP) have shown great promise for characterizing the heart failure (HF) phenotypes. However, the underlying metabolic changes that cause, predict and accompany changes in these parameters are not well known. In depth knowledge of these metabolic changes has the ability to provide new insights towards the clinical management of HF as well as providing better biomarkers for the assessment of disease progression and measurement of success from clinical interventions. To address this knowledge gap, we undertook a deep metabolomic and lipidomic phenotyping of a cohort of HF patients and utilized Multiple Regression Analysis (MRA) to identify associations between the patients plasma metabolome and the lipidome to parameters of CPET and the above mentioned HF biomarkers (HFBio). A total of 49 subjects were submitted to CPET and HFBio evaluation with deep metabolomic and lipidomic profiling of the plasma. Stepwise MRA and Standard Least Squares methods were used to determine models of best fit. Metabolic Pathway Analysis was utilized to detect what metabolites were over-represented and significantly enriched, and Metabolite Set Enrichment Analysis was used to explore pre-existing biological knowledge from studies of human metabolism. The study cohort was predominantly males of African American decent, presenting a high frequency of diabetes, hyperlipidemia, and hypertension with a low mean left ventricular ejection fraction and a high left ventricular end-diastolic and end-systolic volume. VE/VCO2 and Peak VO2 (CV=0.07 and 0.08, respectively) showed the best metabolic predictive model for test performance, while CRP and NT-ProBNP (CV=0.31 and 0.29, respectively) were the least fitted models. Aminoacyl-tRNA and amino acid biosynthesis, amino acid metabolism, nitrogen metabolism, pantothenate and CoA biosynthesis, sphingolipid and glycerolipid metabolism, fatty acid biosynthesis, glutathione metabolism, and pentose phosphate pathway (PPP) were revealed to be the most relevant pathways. Principal related diseases were brain dysfunction and enzyme deficiencies associated with lactic acidosis. Considering the modulations in HF poor test performance, our results indicate an overall profile of oxidative stress, lactic acidosis, and metabolic syndrome coupled with mitochondria dysfunction. Overall, the insights resulting from this studys MRA coincides with what has previously been discussed in parts in existing literature thereby confirming the validity of our findings while at the same time thoroughly characterizing the metabolic underpinning of CPET and HFBio results.

biochemistry↗

bioRxiv2019

Murburn concept explains the acutely lethal effect of cyanide

Cyanide (CN) toxicity is traditionally understood to result from its binding of hemeFe centers, thereby disrupting mitochondrial cytochrome oxidase function and oxygen utilization by other globin proteins. Recently, a diffusible reactive oxygen species (DROS) mediated reaction mechanism called murburn concept was proposed to explain mitochondrial ATP-synthesis and heat generation. Per this purview, it was theorized that CN ion-radical equilibrium dissipates the catalytically vital DROS into futile cycles, producing water. In the current study, a comparative quantitative assessment of the above two explanations is made for: (i) lethal dosage or concentrations of CN, (ii) thermodynamics and kinetics of the binding/reaction, and (iii) correlation of CN with the binding data and reaction chemistry of H2S/CO. The quantitative findings suggest that the hemeFe binding-based toxicity explanation is untenable. CN also inhibited the experimental in vitro DROS-mediated coupling of inorganic phosphate with ADP. Further, pH-dependent inhibition profiles of heme enzyme catalyzed oxidation of a phenolic (wherein an -OH group reacts with DROS to form water, quite akin to the murburn model of ATP synthesis) indicated that- (i) multiple competitive reactions in milieu controlled outcomes and (ii) low concentrations of CN cannot disrupt activity via a coordination (binding) of cyanide at the distal hemeFe. Therefore, the M-level IC50 and the acutely lethal effect of CN on cellular respiration could be explained by the deleterious interaction of CN ion-radical equilibrium with DROS in matrix, disrupting mitochondrial ATP synthesis. This work supports the murburn explanation for cellular respiration.

biochemistry↗

bioRxiv2019

Oxidation state dependent conformational changes of HMGB1 regulates the formation of the CXCL12/HMGB1 heterocomplex

High-mobility Group Box 1 (HMGB1) is an abundant protein present in all mammalian cells and involved in several processes. During inflammation or tissue damage, HMGB1 is released in the extracellular space and, depending on its redox state, can form a heterocomplex with CXCL12. The heterocomplex acts exclusively on the chemokine receptor CXCR4 enhancing leukocyte recruitment. Here, we used multi-microsecond molecular dynamics (MD) simulations to elucidate the effect of the disulfide bond on the structure and dynamics of HMGB1. The results of the MD simulations show that the presence or lack of the disulfide bond between Cys23 and Cys45 modulates the conformational space explored by HMGB1, making the reduced protein more suitable to form a complex with CXCL12.

biochemistry↗

bioRxiv2019

Molecular architecture of a membrane-spanning hormone acyltransferase required for metabolic regulation

Integral membrane proteins represent a large and essential portion of the proteome that often prove challenging for structural studies. We demonstrate a synergistic approach to structurally model topologically complex integral membrane proteins by combining co-evolutionary constraints and computational modeling with biochemical validation. We report the first structural model of a eukaryotic membrane-bound O-acyltransferase (MBOAT), ghrelin O-acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure suggests an unanticipated strategy for trans-membrane protein acylation, with catalysis occurring in an internal channel as GOAT acts as an "enzyme inside a pore". Our structure opens the door to structure-guided inhibitor design targeting GOAT and other MBOAT family members while validating the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins.

biochemistry↗

bioRxiv2019

PhoX - an IMAC-enrichable Crosslinking Reagent

Chemical crosslinking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, sub-stoichiometric reaction efficiencies render routine detection of crosslinked peptides problematic. Here we present a new tri-functional crosslinking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the crosslinked peptides amenable to the well-established IMAC enrichment. The handle allows for 300x enrichment efficiency and 97% specificity, dramatically reducing measurement time and improving data quality. We exemplify the approach on various model proteins and protein complexes, e.g. resulting in a structural model of the LRP1/RAP complex. PhoX is also applicable to whole cell lysates. When focusing the database search on ribosomal proteins, our first attempt resulted in 355 crosslinks, out-performing current efforts in less measurement time.

biochemistry↗

bioRxiv2019

Is testosterone responsible for athletic success in female athletes?

The aim of this study is to determine the interrelationship between the resting serum testosterone (T) levels (an inherited trait) of female athletes from different types of sporting events and their athletic success. The study involves 599 Russian international-level female athletes (95 highly elite, 190 elite, and 314 sub-elite) and 298 age-matched female controls. All subjects were age 16-35 years old and to the best of our knowledge have always tested negative for performance enhancing substances. The athlete cohort was stratified into four groups according to event duration, distance, and type of activity: 1) endurance athletes, 2) athletes with mixed activity, 3) speed/strength athletes, and 4) sprinters. Athletic success was measured by determining the level of achievement of each athlete. The mean (SD) T levels of athletes and controls were 1.65 (0.87) and 1.76 (0.6) nmol/L (P=0.057) with ranges of 0.08-5.80 and 0.38-2.83 nmol/L in athletes and controls, respectively. No significant differences in T levels were found between different groups of athletes. T levels were positively correlated (r=0.62, P<0.0001) with athletic success in sprinters (runners, cyclists, kayakers, speed skaters, swimmers). Moreover, none of the sub-elite sprinters had T > 1.9 nmol/L, while 50% of elite and highly elite sprinters had T > 1.9 nmol/L (95% CI: 2.562-862.34; OR=47.0; P<0.0001). We do not observe the benefits of having high T levels for success in other groups of athletes. Conversely, highly elite middle-distance (P=0.235) and mixed activity athletes (P=0.096) tended to have lower T levels than less successful athletes. Our data suggest that the measurement of the serum T levels significantly correlates with athletic success in sprinters but not other types of athletes and in the future may be useful in the prediction of sprinting ability.

biochemistry↗

bioRxiv2019

Can one trust kinetic and thermodynamic observables from biased metadynamics simulations: detailed quantitative benchmarks on millimolar drug fragment dissociation

Obtaining atomistic resolution of ligand dissociation from a protein is a much sought after experimental and computational challenge. Structural details of the dissociation process are in general hard to capture in experiments, while the relevant timescales are far beyond molecular dynamics (MD) simulations even with the most powerful super-computers. As such many different specialized enhanced sampling methods have been proposed that make it possible to efficiently calculate the dissociation mechanisms in protein-ligand systems. However, accurate benchmarks against long unbiased MD simulations are either not reported yet or simply not feasible due to the extremely long timescales. In this manuscript, we consider one such recent method "infrequent metadynamics", and benchmark in detail the various thermodynamics and kinetic information obtained from this method against extensive unbiased MD simulations for the dissociation dynamics of two different millimolar fragments from the protein FKBP in explicit water with residence times in nanoseconds to microseconds regime. We find that the metadynamics approach gives the same binding free energy profile, dissociation pathway and ligand residence time as the unbiased MD, albeit using only 6 to 50 times lower computational resources. Furthermore, we demonstrate how the metadynamics approach can self-consistently be used to ascertain whether the reweighted kinetic constants are reliable or not. We thus conclude that the answer to the question posed in the title of this manuscript is: statistically speaking, yes.

biochemistry↗

bioRxiv2019

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Aminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with -ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 {degrees}C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction. IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

biochemistry↗

bioRxiv2019

In vitro characterization of the colibactin-activating peptidase ClbP enables development of a fluorogenic activity probe

The gut bacterial genotoxin colibactin has been linked to the development of colorectal cancer. In the final stages of colibactins biosynthesis, an inactive precursor (pre-colibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.

biochemistry↗

bioRxiv2019

Structure-guided function discovery of an NRPS-like glycine betaine reductase for choline biosynthesis in fungi

Nonribosomal peptide synthetases (NRPS) and NRPS-like enzymes have diverse functions in primary and secondary metabolism. By using a structure-guided approach, we uncovered the function of an NRPS-like enzyme with unusual domain architecture, catalyzing two sequential two-electron reductions of glycine betaine to choline. Structural analysis based on homology model suggests cation-{pi} interactions as the major substrate specificity determinant, which was verified using substrate analogs and inhibitors. Bioinformatic analysis indicates this NRPS-like glycine betaine reductase is highly conserved and widespread in fungi kingdom. Genetic knockout experiments confirmed its role in choline biosynthesis and maintaining glycine betaine homeostasis in fungi. Our findings demonstrate that the oxidative choline-glycine betaine degradation pathway can operate in a fully reversible fashion and provide new insights in understanding fungal choline metabolism. The use of an NRPS-like enzyme for reductive choline formation is energetically efficient compared to known pathways. Our discovery also underscores the capabilities of structure-guided approach in assigning function of uncharacterized multidomain proteins, which can potentially aid functional discovery of new enzymes by genome mining.

biochemistry↗

bioRxiv2019

Assessing a commercial capillary electrophoresis interface (ZipChip) for shotgun proteomic applications

Capillary electrophoresis coupled electrospray ionization mass spectrometry (CE-MS) has long existed as a theoretical alternative to liquid chromatography coupled mass spectrometry (LC-MS). Until recently, however, the coupling of these technologies has occupied only a small niche within specific applications. A recent innovation in CE-MS is the ZipChip interface system from 908 devices that was pioneered by the Ramsay lab at NC State. This newly available source offers advantages over previous CE-MS interfaces including both relative ease of use and direct compatibility to thousands of mass spectrometers currently in use throughout the world with no hardware alterations. The ZipChip CE-MS has been demonstrated in recent studies to provide high resolution and rapid separations for the analysis of intact proteins, glycoproteins and glycosylated peptides, with more applications likely on the way. In this study we assess the capabilities of the ZipChip system in the context of high throughput global shotgun proteomics experiments. We find that on a high field Orbitrap system we can repeatedly identify as many as 800 unique protein groups in an experiment using a run time of 12 minutes. We find the ZipChip CE-MS system to be widely applicable for both data dependent and data independent acquisition experiments as well as targeted experiments. We conclude that the ZipChip is an attractive alternative solution to traditional nanoflow ESI-MS/MS for the analysis of the genomes of single celled organisms and for offline fractionation of eukaryotic proteomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/559591v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@6a46eforg.highwire.dtl.DTLVardef@156fc4dorg.highwire.dtl.DTLVardef@1ce562eorg.highwire.dtl.DTLVardef@17c1932_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

CHOLESTEROL MYTH AND THE IMPERATIVE ROLE OF FUNCTIONAL EVALUATION OF LIPOPROTEINS IN ATHEROSCLEROTIC RISK PREDICTION STRATEGIES

ObjectiveTo arrive at apposite risk prediction strategies in identifying markers that could delineate patients susceptible for atherosclerotic progression from other diabetic patient population thus circumventing undesirable drug exposure or conversely inadequate treatment. Research Design and MethodsThe study was designed to assess and compare the biochemical parameters along with lipoprotein ratios assessed in structural and functional aspects between the probable risk factor groups for CAD(diabetics with no symptoms for CAD) and untreated CAD patients (on admission for CAD) in a total of 593 participants. Anthropometric measurements were made and serum fasting glucose, lipid peroxide levels, lipid and lipoprotein ratio assessments were carried out. In addition to these basic parameters, oxidized LDL (Ox-LDL) and serum paraoxonase (PON) activity were also measured in this study. To ascertain the diagnostic accuracy of the individual markers, receiver operating characteristic curve analysis was carried out. ResultsSerum levels of blood lipids, lipid peroxides were higher in the diabetic and the cardiovascular patients with a concomitant decrease in the levels of HDL. Increased median levels of Ox-LDL and decreased PON activity was observed in the cardiovascular patients both with and without diabetes. ConclusionThis study proposes Ox-LDL/PON assessment would be more functional than the traditional LDL-c/HDL-c to categorize the risk group of patients into prospective atherosclerotic predisposed and non-predisposed population, thus enabling the former to get treated with appropriate medication.

biochemistry↗

bioRxiv2019

Interaction of NPC2 protein with Lysobisphosphatidic Acid is required for normal endolysosomal cholesterol trafficking

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates by the LE/LY phospholipid lysobisphosphatidic acid (LBPA) and in these studies sought to determine their functional relationship in normal LE/LY cholesterol egress. Here we demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Using its precursor phosphatidylglycerol (PG), we show that PG-induced LBPA enrichment results in clearance of accumulated cholesterol from NPC1-deficient cells but is ineffective in cells lacking functional NPC2. Together these studies reveal a heretofore unknown aspect of intracellular cholesterol trafficking, in which NPC2 and LBPA function together in an obligate step of sterol egress from the LE/LY compartment, which appears to be independent of NPC1.

biochemistry↗