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bioRxiv2018

Selective Permeability of Carboxysome Shell Pores to Anionic Molecules

Carboxysomes are closed polyhedral cellular microcompartments that increase the efficiency of carbon fixation in autotrophic bacteria. Carboxysome shells consist of small proteins that form hexameric units with semi-permeable central pores containing binding sites for anions. This feature is thought to selectively allow access to RuBisCO enzymes inside the carboxysome by [Formula] (the dominant form of CO2 in the aqueous solution at pH 7.4) but not O2, which leads to a non-productive reaction. To test this hypothesis, here we use molecular dynamics simulations to characterize the energetics and permeability of CO2, O2, and [Formula] through the central pores of two different shell proteins, namely, CsoS1A of -carboxysome and CcmK4 of {beta}-carboxysome shells. We find that the central pores are in fact selectively permeable to anions such as [Formula], as predicted by the model.

biochemistry↗

bioRxiv2018

Saccharomyces cerevisiae adapted to grow in the presence of low-dose rapamycin exhibit altered amino acid metabolism

Rapamycin is a potent inhibitor of the highly conserved TOR kinase, the nutrient-sensitive controller of growth and aging. It has been utilised as a chemotherapeutic agent due to its anti-proliferative properties and as an immunosuppressive drug, and is also known to extend lifespan in a range of eukaryotes from yeast to mammals. However, the mechanisms through which eukaryotic cells adapt to sustained exposure to rapamycin have not yet been thoroughly investigated. Here, S. cerevisiae response to long-term rapamycin exposure was investigated by identifying the physiological, transcriptomic and metabolic differences observed for yeast populations inoculated into low-dose rapamycin-containing environment. The effect of oxygen availability and acidity of extracellular environment on this response was further deliberated by controlling or monitoring the dissolved oxygen level and pH of the culture. Yeast populations grown in the presence of rapamycin reached higher cell densities complemented by an increase in their chronological lifespan, and these physiological adaptations were associated with a rewiring of the amino acid metabolism, particularly that of arginine. The ability to synthesise amino acids emerges as the key factor leading to the major mechanistic differences between mammalian and microbial TOR signalling pathways in relation to nutrient recognition. Furthermore, oxygen levels and extracellular acidity of the culture were observed to conjointly affect yeast populations, virtually acting as coupled physiological effectors; cells were best adapted when maximal oxygenation of the culture was maintained in slightly acidic pH, any deviation necessitated more extensive readjustment to additional stress factors.

biochemistry↗

bioRxiv2018

N-terminal BRCT domains of the DNA damage checkpoint proteins TOPBP1/Rad4 display distinct specificities for phosphopeptide ligands

TOPBP1 and its fission yeast homologue Rad4, are critical players in a range of DNA replication, repair and damage signalling processes. They are composed of multiple BRCT domains, some of which have the capacity to bind phosphorylated motifs in other proteins. They thus act as multi-point adaptors bringing proteins together into functional combinations, dependent on post-translational modifications downstream of cell cycle and DNA damage signals. We have now structurally and/or biochemically characterised a sufficient number of high-affinity complexes for the conserved N-terminal region of TOPBP1 and Rad4 in complex with diverse phospho-ligands - which include human RAD9 and Treslin, as well as S.pombe Crb2 and Sld3 - to define the key determinants of BRCT domain specificity. We use this information to identify and characterise previously unknown phosphorylation-dependent TOPBP1/Rad4-binding motifs in human RHNO1 and the fission yeast homologue of MDC1, Mdb1. These results provide important insights into how multiple BRCT domains within TOPBP1/Rad4 achieve selective and combinatorial binding of their multiple partner proteins.

biochemistry↗

bioRxiv2018

How molecular mechanisms of resistance affect resistance evolution

Combatting antibiotic resistance will require both new antibiotics and strategies to preserve the effectiveness of existing drugs. Both approaches would benefit from predicting optimal dosing of antibiotics based on drug-target binding parameters that can be measured early in drug development and that can change when bacteria become resistant. This would avoid the currently frequently employed trial-and-error approaches and might reduce the number of antibiotic candidates that fail late in drug development.\n\nHere, we describe a computational model (COMBAT-COmputational Model of Bacterial Antibiotic Target-binding) that leverages accessible biochemical parameters to quantitatively predict antibiotic dose-response relationships. We validate our model with MICs of a range of quinolone antibiotics in clinical isolates demonstrating that antibiotic efficacy can be predicted from drug-target binding (R2 > 0.9). To further challenge our approach, we do not only predict antibiotic efficacy from biochemical parameters, but also do the reverse: estimate the magnitude of changes in drug-target binding based on antibiotic dose-response curves. We experimentally demonstrate that changes in drug-target binding can be predicted from antibiotic dose-response curves with 92-94 % accuracy by exposing bacteria overexpressing target molecules to ciprofloxacin. To test the generality of COMBAT, we apply it to a different antibiotic class, the beta-lactam ampicillin, and can again predict binding parameters from dose-response curves with 90 % accuracy. We then apply COMBAT to predict antibiotic concentrations that can select for resistance due to novel resistance mutations.\n\nOur goal here is dual: First, we address a fundamental biological question and demonstrate that drug-target binding determines bacterial response to antibiotics, although antibiotic action involves many additional effects downstream of drug-target binding. Second, we create a tool that can help accelerate drug development by predicting optimal dosing and preserve the efficacy of existing antibiotics by predicting optimal treatment for possible resistant mutants.

biochemistry↗

bioRxiv2018

Lipids are the preferred substrate of the protist Naegleria gruberi, relative of a human brain pathogen

Naegleria gruberi is a free-living non-pathogenic amoeboflagellate and relative of Naegleria fowleri, a deadly pathogen causing primary amoebic meningoencephalitis (PAM). A genomic analysis of N. gruberi exists, but physiological evidence for its core energy metabolism or in vivo growth substrates is lacking. Here we show that N. gruberi trophozoites need oxygen for normal functioning and growth and that they furthermore shun both glucose and amino acids as growth substrates. Trophozoite growth depends mainly upon lipid oxidation via a mitochondrial branched respiratory chain, both ends of which require oxygen as final electron acceptor. Growing N. gruberi trophozoites thus have a strictly aerobic energy metabolism with a marked substrate preference for the oxidation of fatty acids. Analyses of N. fowleri genome data and comparison with those of N. gruberi indicate that N. fowleri has the same type of metabolism. Specialization to oxygen-dependent lipid breakdown represents a hitherto unprecedented metabolic strategy in protists.

biochemistry↗

bioRxiv2018

Global Adoption of High-Sensitivity Cardiac Troponins and the Universal Definition of Myocardial Infarction

ImportanceThe third Universal Definition of Myocardial Infarction aimed to standardize the approach to the diagnosis and management of myocardial infarction. High-sensitivity cardiac troponin testing was recommended, as these assays have improved precision at low concentrations, but concerns over specificity may have limited implementation.\n\nObjectiveTo determine the global adoption of high-sensitivity cardiac troponin assays and key recommendations from the Universal Definition.\n\nDesign, Setting and ParticipantsGlobal survey of 1,902 medical centers across 23 countries evenly distributed across all five continents. Included respondents were involved in the diagnosis and management of patients with suspected acute coronary syndrome at their institutions.\n\nMain Outcomes and MeasuresStructured questionnaire detailing the primary biomarker used for myocardial infarction, diagnostic thresholds and critical elements of clinical pathways for comparison to the third Universal Definition recommendations.\n\nResultsCardiac troponin was the primary diagnostic biomarker for myocardial infarction at 96% of all sites surveyed. Only 41% of centers had adopted high-sensitivity cardiac troponin assays, with wide variation from 7% in North America to 60% in Europe. Sites using high-sensitivity assays more frequently employed serial sampling pathways (91% vs. 78%) and the 99th percentile diagnostic threshold (74% vs. 66%) when compared to sites using the previous generation of troponin assays. Furthermore, sites using high-sensitivity assays more often used earlier serial sampling ([≤]3 hours) and accelerated diagnostic pathways. However, fewer than 1 in 5 sites using high-sensitivity assays had adopted sex-specific thresholds (18%).\n\nConclusions and RelevanceProgress has been made in adopting the recommendations of the Universal Definition of Myocardial Infarction, particularly in the use of the 99th percentile diagnostic threshold and serial sampling. However, high-sensitivity assays are used in a minority of sites and sex-specific thresholds in even fewer. These findings highlight regions where additional efforts are required to improve the risk stratification and diagnosis of patients with myocardial infarction.

biochemistry↗

bioRxiv2018

Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light resulting in dissociation into monomers, which are considered to be the active state and comprise a {beta}-propeller core domain and intrinsically disordered N- and C-terminal tails. The C-terminus is required for functional binding to signalling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photo-activation. We show that, whilst truncated UVR8 photo-converts from a single conformation of dimers to a single monomer conformation, the full-length protein exist in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C-terminus is primed for activation. In the monomer the extended C-terminus destabilises the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.\n\nTOC Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC=\"FIGDIR/small/371658_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (60K):\norg.highwire.dtl.DTLVardef@177c6f9org.highwire.dtl.DTLVardef@a845c9org.highwire.dtl.DTLVardef@17db14forg.highwire.dtl.DTLVardef@103d4f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2018

Requirements for efficient cotranscriptional regulatory switching in designed variants of the Bacillus subtilis pbuE adenine-responsive riboswitch

Riboswitches, generally located in the 5-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch informing the transcriptional or translational machinery. While the structure and function of riboswitch effector-binding (aptamer) domains have been intensely studied, only recently have the requirements for efficient linkage between small molecule binding and the structural switch in the cellular and cotranscriptional context begun to be actively explored. To address this, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches containing a secondary structural switch. Using a cell-based fluorescent protein reporter assay to assess ligand-dependent regulatory activity in E. coli, these studies revealed previously unrecognized features of the riboswitch. Most importantly, it was found that local and long-range conformational dynamics in two regions of the aptamer domain have a significant effect upon efficient regulatory switching. Further, sequence features of the expression platform including the pre-aptamer leader sequence, a nucleation helix and a putative programmed pause have clear affects upon ligand-dependent regulation. Together, these data point to sequence and structural features distributed throughout the riboswitch required to strike a balance between rates of ligand binding, transcription and secondary structural switching via a strand exchange mechanism.

biochemistry↗

bioRxiv2018

A Reconsideration of the Effect of Procyanidin on the Assembly of Collagen Type I

In order to elucidating the exact effect mechanism of polyphenols on the assembly of collagen, the assembled architectures of collagen treated with different amounts of procyanidin (PA) were investigated in details. The assembled morphologies of collagen were greatly influenced by the content of PA according to atomic force microcopy (AFM) images. When the content of PA was more than 20% (w/w), the fibrillar morphologies were substituted by globular aggregates, which were driven by the intense hydrogen bonding action originating from PA. While the formation of the non-fibrous aggregates was due to the coiling and entangling of flexible collagen molecules rather than their gelatinization based on the appearance of typical adsorption peaks at 222nm and 197nm on circular dichroism (CD) spectra. After being crosslinked by glutaraldehyde (GA), not only the diameters but also the lengths of fibrils increased. Unfortunately, the fibrillogenesis was still inhibited when the collagen suffered from 20% PA firstly and then 4% GA. Conversely, the fibrous morphologies of the fibrils stabilized by 4% GA and then underwent 20% PA maintained well, in spite of accompanying with grievous intertwining. This difference was derived from the change of flexibilities of collagen before and after being crosslinked by GA. Additionally, the differential scanning calorimeter (DSC) analysis confirmed the PA had no positive effect on the improvement of thermal stability of hydrous collagen, whereas the denaturation temperature of hydrated collagen stabilized by 4% GA increased from 40 {degrees}C to 80 {degrees}C.

biochemistry↗

bioRxiv2018

Q-FADD: A mechanistic approach for modeling the accumulation of proteins at sites of DNA damage by free diffusion

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring of the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing this process quantitatively. Here we introduce a free diffusion model that explicitly accounts for the unique topology of individual nuclei and quantitatively describes the accumulation of two test proteins, poly-ADP-ribose polymerases 1 and 2. Application of our model to other proteins will yield novel insights into the timing and mechanism of DNA repair.

biochemistry↗

bioRxiv2018

Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides. Facklamia ignava NrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone of F. ignava NrdB promotes formation of tetramers that are unable to form enzymatically competent complexes with F. ignava NrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP. F. ignava NrdB contains the recently identified radical factor Mn2III/IV. We show that NrdA from the firmicute F. ignava can form a catalytically competent RNR with the Mn2III/IV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis.

biochemistry↗

bioRxiv2018

Kinase Driver Mutations in Protein-protein Structure may associate to Disease by Effecting Kinase Stability

Activating mutations are significant drug targets of diseases. Statistical analysis on the mutated amino acids, the mutation characteristic and the related disease information of activating mutations is of great significance for the diagnosis and treatment of diseases. Study on the protein stability by predicting Gibbs free energy ({Delta}{Delta}G) change after mutation is very helpful for understanding the relationship between protein structure and function. By combining mCSM and disease datasets, this paper studies the effect of mutation on the protein stability and disease. The results show that the mutations in protein may be the cause of disease for PPI structures, which statistically afford significant information for disease related research and medical diagnosis and treatment.

biochemistry↗

bioRxiv2018

Identification and characterization of yeast and human glycosphingolipid flippases

Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid flippases (P4-ATPases) transport lipids across the membrane bilayer, and are involved in signal transduction, cell division, and vesicular transport. Mutations in flippase genes cause or contribute to a host of diseases such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disease. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis; and ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids are strong contributors to metabolic disease, little is known about how GlcCer is transported across cell membranes. We have identified an evolutionarily conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer. Further, we establish the structural determinants necessary for the specific recognition of this sphingolipid substrate. Our molecular observations clarify the relationship between these flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.

biochemistry↗

bioRxiv2018

Coordinative Metabolism of Glutamine Carbon and Nitrogen in proliferating Cancer Cells Under Hypoxia

Under hypoxia, most of glucose is converted to secretory lactate, which leads to the lack of carbon source from glucose and thus the overuse of glutamine-carbon. However, under such a condition how glutamine nitrogen is disposed to avoid releasing potentially toxic ammonia remains to be determined. Here we identify a metabolic flux of glutamine to secretory dihydroorotate under hypoxia. We found that glutamine nitrogen is indispensable to nucleotide biosynthesis, but enriched in dihyroorotate and orotate rather than processing to its downstream uridine monophosphate under hypoxia. Dihyroorotate, not orotate, is then secreted out of cells. The specific metabolic pathway occurs in vivo and is required for tumor growth. Such a metabolic pathway renders glutamine mainly to acetyl coenzyme A for lipogenesis, with the rest carbon and nitrogen being safely removed. Our results reveal how glutamine carbon and nitrogen are coordinatively metabolized under hypoxia, and provide a comprehensive understanding on glutamine metabolism.\n\nSignificanceTumor cells often addict to glutamine, and particularly utilize its carbon for lipogenesis under hypoxia. We reveal that tumor cells package the excessive glutamine-nitrogen into secretory dihydroorotate, instead of toxic ammonia. This specifically reprogrammed pathway supports in vivo tumor growth, and could offer diagnostic markers and therapeutic targets for cancers.

biochemistry↗

bioRxiv2018

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered that cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine motif traffic readily to the cytosol and nucleus with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using a broad spectrum of techniques, including an RNA interference (RNAi) screen, fluorescence correlation spectroscopy (FCS), and confocal imaging, we identify VPS39--a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex--as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. Subsequent multi-color confocal imaging studies identify CPMPs within the endosomal lumen, particularly within the intraluminal vesicles (ILVs) of Rab7+ and Lamp1+ endosomes that are the products of HOPS-mediated fusion. These results suggest that CPMPs require HOPS to reach ILVs--an environment that serves as a prerequisite for efficient endosomal escape.

biochemistry↗

bioRxiv2018

A conserved mechanism for meiotic chromosome organization through self-assembly of a filamentous chromosome axis core

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that \"axis core proteins\" from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify motifs in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core complexes form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture and role in axis assembly and recombination control. We propose that the meiotic chromosome axis self-assembles through cooperative interactions between dynamic DNA loop-extruding cohesin complexes and the filamentous axis core, then serves as a platform for chromosome organization, recombination, and synaptonemal complex assembly.

biochemistry↗

bioRxiv2018

Inhibition of proteasomal degradation rescues a pathogenic variant of mitochondrial Respiratory chain assembly 1 factor

Nuclear and mitochondrial genome mutations lead to various mitochondrial diseases, many of which affect the mitochondrial respiratory chain. The proteome of the intermembrane space (IMS) of mitochondria consists of several important assembly factors that participate in the biogenesis of mitochondrial respiratory chain complexes. The present study comprehensively analyzed a recently identified IMS protein, RESpiratory chain Assembly 1 (RESA1) factor, or cytochrome c oxidase assembly factor 7 (COA7) that is associated with a rare form of mitochondrial leukoencephalopathy and complex IV deficiency. We found that RESA1 requires the mitochondrial IMS import and assembly (MIA) pathway for efficient accumulation in the IMS. We also found that pathogenic mutant versions of RESA1 are imported slower than the wild type protein, and mislocalized mutant proteins are degraded in the cytosol by proteasome machinery. Interestingly, proteasome inhibition rescued both the mitochondrial localization of mutant RESA1 and complex IV activity in patient-derived fibroblasts. We propose that proteasome inhibition is a novel therapeutic approach for a broad range of mitochondrial pathologies that are associated with the excessive degradation of mitochondrial proteins that is caused by genetic mutations or biogenesis defects.

biochemistry↗

bioRxiv2018

A Ubiquitin-Binding Domain that Does Not Bind Ubiquitin

Ubiquitylation, the post-translational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function exclusively by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. We show that the SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. A structural model of the minimal KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography analysis is presented. Remarkably, the CUE1 domain, which resembles a canonical CUE domain, recognizes 2 clusters of exposed hydrophobic residues on KAP1, but these are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data challenge the well-established dogma that CUE domains exclusively recognize the ubiquitin-fold.

biochemistry↗