Duyệt theo Kiểu tài liệu "Periodicals (Báo – Tạp chí)"
- Ấn phẩm13C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding ‘Alcove’(Biochemical Journal, 2020) James, Christopher D; Wile, Seth; Ragsdale, Stephen WEPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and distal Nid. An alcove seen in the ACS crystal-structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO and is suggested to ‘cradle’ this CO. Previously, we only knew WT ACS bound a single CO in the Ared-CO intermediate, here seen as forming Nip(I)-CO with CO on-axis of the dz2 odd-electron orbital (g⊥>g‖∼2). The two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope now reveals a second CO bound in the dz2 orbital’s equatorial plane. This WT A-cluster conformer dominates the nearly-conservative F229W variant, but 13C-ENDOR reveals a minority “A” conformation with (g‖>g⊥∼2) characteristic of a ‘cloverleaf’ (eg. dx2-y2) odd-electron orbital, and with Nip binding two, apparently ‘in-plane’ CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni(I) ∼tenfold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the “A” conformation, but with ∼20% each of the WT conformer and an “O” state in which dz2 Nip(I) (g⊥>g‖∼2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.
- Ấn phẩmA clinically-relevant polymorphism in the Na+/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position(Biochemical Journal, 2020) Ruggiero, Melissa J; Malhotra, ShipraConventionally, most amino acid substitutions at important protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of non-conserved positions for which various substitutions produced progressive functional changes; we consider these evolutionary “rheostats”. Here, we report a strong rheostat position in the integral membrane protein, Na+/taurocholate cotransporting polypeptide (NTCP), at the site of a pharmacologically-relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions (“S267X”) with three substrates (taurocholate, estrone-3-sulfate and rosuvastatin). The S267X set showed strong rheostatic effects on overall transport, and individual substitutions showed varied effects on transport kinetics (Km and Vmax). However, the outcomes were substrate dependent, indicating altered specificity. To assess protein stability, we measured surface expression and used the Rosetta software suite to model structure and stability changes of S267X. Although buried near the substrate binding site, S267X substitutions were easily accommodated in the NTCP structure model. Across the modest range of changes, calculated stabilities correlated with surface-expression differences, but neither parameter correlated with altered transport. Thus, substitutions at rheostat position 267 had wide-ranging effects on the phenotype of this integral membrane protein. We further propose that polymorphic positions in other proteins might be locations of rheostat positions.
- Ấn phẩmA clinically-relevant polymorphism in the Na+/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position(Biochemical Journal, 2020) Strzelecka, Dominika; Smietanski, MiroslawChemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5’,3’-phosphodiester bonds in the mRNA body and polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases—SP6, T7 and polyA polymerase. To verify the efficiency of incorporation of ATP analogues in the presence of ATP, we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5’-mononucleotides.
- Ấn phẩmA Live-Cell Assay for the Detection of pre-microRNA-Protein Interactions(Biochemical Journal, 2020) Rosenblum, Sydney L; Lorenz, Daniel A; Garner, Amanda LRecent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of both coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we have utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.
- Ấn phẩmA molecular mechanism for LINC complex branching by structurally diverse SUN-KASH 6:6 assemblies(Biochemical Journal, 2020) Gurusaran, Manickam; Davies, Owen RThe LINC complex mechanically couples cytoskeletal and nuclear components across the nuclear envelope to fulfil a myriad of cellular functions, including nuclear shape and positioning, hearing and meiotic chromosome movements. The canonical model of the LINC complex is of individual linear nucleocytoskeletal linkages provided by 3:3 interactions between SUN and KASH proteins. Here, we provide crystallographic and biophysical evidence that SUN-KASH is a constitutive 6:6 complex in which two SUN trimers interact back-to-back. A common SUN-KASH topology is achieved through structurally diverse 6:6 interaction mechanisms by distinct KASH proteins, including zinc-coordination by Nesprin-4. The SUN-KASH 6:6 complex is incompatible with the current model of a linear LINC complex and instead suggests the formation of a branched LINC complex network. In this model, SUN-KASH 6:6 complexes act as nodes for force distribution and integration between adjacent SUN and KASH molecules, enabling the coordinated transduction of large forces across the nuclear envelope.
- Ấn phẩmA nanobody suite for yeast scaffold nucleoporins provides details of the Nuclear Pore Complex structure(Biochemical Journal, 2020) Nordeen, Sarah A; Andersen, Kasper; Knockenhauer, Kevin ENuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 or inner ring complex. Working in S. cerevisiae, and to study the assembly of these two essential subcomplexes, we developed a set of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. These nanobodies all bind specifically and with high affinity. We present structures of several nup-nanobody complexes, revealing their binding sites. Additionally, constitutive expression of the nanobody suite in S. cerevisiae revealed accessible and obstructed surfaces of the Y complex and Nic96 within the NPC. Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.
- Ấn phẩmA novel in vitro Caenorhabditis elegans transcription system(Biochemical Journal, 2020) Wibisono, Phillip; Liu, Yiyong; Sun, JingruCaenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for biochemical studies of C. elegans is the high difficulty of preparing functionally active nuclear extract due to its thick surrounding cuticle. By employing Balch homogenization, we have achieved effective disruption of larval and adult worms and have obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, two non-radioactive detection methods, PCR and qRT-PCR, have been adapted into our system to qualitatively and quantitatively detect transcription, respectively. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection increased transcription activity. Our in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulations. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.
- Ấn phẩmA rod conformation of the Pyrococcus furiosus Rad50 coiled coil(Biochemical Journal, 2020) Min Soh, Young; Gruber, StephanThe Rad50-Mre11 nuclease complex plays a vital role in DNA repair in all domains of life. It recognizes and processes DNA double-strand breaks. Rad50 proteins fold into an extended structure with a ~20-60 nm long coiled coil connecting a globular ABC ATPase domain with a zinc hook dimerization domain. A published structure of an archaeal Rad50 zinc hook shows coiled coils pointing away from each other. Here we present the crystal structure of an alternate conformation displaying co-aligned coiled coils. Archaeal Rad50 may thus switch between rod-shaped and ring-like conformations as recently proposed for a bacterial homolog.
- Ấn phẩmA survey of pairwise epistasis supports an outside-in hierarchy of clade-specifying and function-defining residues in PSD95 PDZ3(Biochemical Journal, 2020) Nedrud, David; Maestas, Willow Coyote; Schmidt, DanielDeep mutational scanning enables data-driven models of protein structure and function. Here, we adapted Saturated Programmable Insertion Engineering as an economical and programmable deep mutational scanning technique. We validate this approach with an existing single mutant dataset in the PSD95 PDZ3 domain, and further characterize most pairwise double mutants to study how a mutation’s phenotype depends on mutations at other sites, a phenomenon called epistasis. We observe wide-spread proximal negative epistasis, which we attribute to mutations affecting thermodynamic stability, and strong long-range positive epistasis, which is enriched in an evolutionarily conserved and function-defining network of ‘sector’ and clade-specifying residues. Conditional neutrality of mutations in clade-specifying residues compensates for deleterious mutations in sector positions. This suggests an outside-in hierarchy of interactions through which positive epistasis between clade-specifying residues and the PDZ sector facilitated the evolutionary expansion and specialization of PDZ domains.
- Ấn phẩmA temperature-sensitive Mycobacterium smegmatis glgE mutation leads to a loss of GlgE enzyme activity and thermostability and the accumulation of -maltose-1-phosphate(Biochimica et Biophysica Acta (BBA), 2020) Syson, KarlTo reconstruct and examine the temperature-sensitive mutant and characterise the mutated GlgE enzyme. Results- The mutant strain accumulated the substrate for GlgE, -maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with -maltose-1-phosphate. The accumulation of -maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 ºC. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. Conclusions- We have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. General Significance- This study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria.
- Ấn phẩmABPP-HT - high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context(Frontiers in Chemistry, 2020) Jones, HannahThe potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes (ABPs) are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.
- Ấn phẩmAbsence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation(Biochemical Journal, 2020) Liu, Liu; Dumbrepatil, Arti B; Fleischhacker, Angela SHeme oxygenase-2 (HO2) and −1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 level by heme is elucidated. Under heme deficient conditions, HO2 is destabilized and targeted for degradation. In HO2, three heme binding sites are potential targets of heme-dependent regulation: one at its catalytic site; the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. However, cellular heme overload does not affect HO2 stability. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2’s role in maintaining heme homeostasis, paving the way for future investigation into HO2’s pathophysiological role in heme deficiency response.
- Ấn phẩmActivity-specificity trade-off gives PI5P4Kβ a nucleotide preference to function as a GTP-sensing kinase(Biochemical Journal, 2020) Takeuchi, Koh; Ikeda, Yoshiki; Harada, AyakaMost kinases function with ATP. However, contrary to the prevailing dogma, phosphatidylinositol 5-phosphate 4-kinase β (PI5P4Kβ) utilizes GTP as a primary phosphate donor with a unique binding mode for GTP. Although PI5P4Kβ is evolved from a primordial ATP-utilizing enzyme, PI4P5K, how PI5P4Kβ evolutionarily acquired the GTP preference to function as a cellular GTP sensor remains unclear
- Ấn phẩmAcute radiofrequency electromagnetic radiation exposures cause neuronal DNA damage and impair neurogenesis in the young adolescent rat brain(2020) Singh, Kumari VandanaMobile phone is now a commonly used communication device in all age groups. Young adolescents use it for longer duration and effect of its radiofrequency electromagnetic radiation (RF-EMR) on their brain structure and function need detailed investigation. In the present study, we investigated the effect of RF-EMR emitted from mobile phones, on young adolescent rat brain. Wistar rats (5 weeks, male) were exposed to RF-EMR signal (2,115 MHz) from a mobile phone at a whole body averaged specific absorption rate (SAR) of 1.15 W/kg continuously for 8 h. Higher level of lipid peroxidation, carbon centered lipid radicals and DNA damage were observed in the brain of rat exposed to RF-EMR. Number of neural progenitor cells (NPCs) in dentate gyrus (DG) were found to be relatively low in RF-EMR exposed rats that may be due to reduced neurogenesis. Acute exposure to RF-EMR induced neuronal degeneration in DG region with insignificant variation in CA3, CA1 and cerebral cortex sub regions of hippocampus. Findings of this study, indicate that acute exposure of high frequency RF-EMR at relatively higher SAR may adversely impact the neurogenesis and function of adolescent rat brain. Generation of carbon centered lipid radicals, and nuclear DNA damage might be playing critical role in reduced neurogenesis and higher neuronal degeneration in the cortex and hippocampus of brain. Detailed understanding of RF-EMR induced alteration in brain function will be useful to develop appropriate interventions for reducing the impact caused by RF-EMR damage.
- Ấn phẩmAir and surface measurements of SARS-CoV-2 inside a bus during normal operation(Biochemical Journal, 2020) Di Carlo, Piero; Chiacchiaretta, Piero; Sinjari, BrunaTransmission pathways of SARS-CoV-2 are through aerosol, droplet and touching infected material. Indoor locations are more likely environments for the diffusion of the virus contagion among people, but direct detection of SARS-CoV-2 in air or on surfaces is quite sparse, especially regarding public transport. In fact, an important demand is to know how and if it is safe to use them. To understand the possible spreading of COVID-19 inside a city bus during normal operation and the effectiveness of the protective measures adopted for transportation, we analysed the air and the surfaces most usually touched by passengers. The measurements were carried out across the last week of the lockdown and the first week when gradually all the travel restrictions were removed.
- Ấn phẩmAlcohol-Induced Increase in the Content of CYP2E1 in Human Liver Microsomes Causes a Multifold Activation of CYP3A4 and Attenuates its Cooperativity(Biochemical Journal, 2020) Bikash, Dang; Nadzehda Y, DavydovaHere we investigate the effect of alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. In these studies we used a model that implements enrichment of HLM samples with CYP2E1 through membrane incorporation of the purified protein. Enrichment of HLM with CYP2E1 considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. Incorporation of CYP2E1 also eliminates the activating effect of α-Naphthoflavone (ANF) on BQ metabolism seen in some untreated HLM samples. To probe the physiological relevance of these effects we compared three pooled preparations of HLM from normal donors (HLM-N) with a preparation obtained from heavy alcohol consumers (HLM-A). The composition of the P450 pool in all four samples was characterized with mass-spectrometric determination of 11 cytochrome P450 species. The molar content of CYP2E1 in HLM-A was from 2.5 to 3.3 times higher than that found in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all four HLM samples. Despite of that, HLM-A exhibited much higher rate of metabolism and lower degree of homotropic cooperativity with BQ, similar to that observed in CYP2E1-enriched HLM-N. In order to substantiate the hypothesis on the involvement of physical interactions between CYP2E1 and CYP3A4 in the observed effects we probed hetero-association of these proteins in Supersomes™ containing recombinant CYP3A4 with a technique based on homo-FRET and employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments demonstrated high affinity interactions between the two enzymes and revealed an inhibitory effect of ANF on their hetero-association. Our results demonstrate that the catalytic activity and allosteric properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and imply a profound impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.
- Ấn phẩmAltered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity(Biochemical Journal, 2020) Fellner, MatthiasMutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with exchange between conformational states proposed to regulate changes in function. Here we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. We find that three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with increased overall flexibility of cytochrome c, particularly movement of the Ω loops. This suggests that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an “induced fit” mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics suggesting that the mechanism by which the variants alter peroxidase activity is not related to the conformation dynamics of the hexacoordinate heme Fe state of cytochrome c analyzed in the simulations. Recent suggestions that conformational mobility of specific regions of cytochrome c underpins changes in reduction potential and the alkaline transition pK were not supported. These data highlight that conformational dynamics of cytochrome c drives some but not all of its properties and activities.
- Ấn phẩmAmphipathic Fluorescent Dyes for Sensitive and Long-Term Monitoring of Plasma Membranes(Science Signaling, 2020) Daly, LeonardAdaption of cells to low oxygen environments is an essential process mediated in part by the Hypoxia Inducible Factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs, and consequently he hypoxic response, are regulated by post-translational modification (PTM) and changes in biomolecular interactions. However, our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies, with validation typically having been conducted by in cellulo fragment expression and hypoxia mimicking drugs. Consequently, we still lack an understanding of true oxygen deprivation signaling via HIFα. Using an immunoprecipitation-based, mass spectrometry approach, we characterize the regulation of in cellulo expressed full-length HIF-1α and HIF-2α, in terms of both PTM and binding partners, in response to normoxia (21% oxygen) and hypoxia (1% oxygen). These studies revealed that a change in oxygen tension significantly alters the complexity and composition of HIF-α protein interaction networks, with HIF-2α in particular having an extended hypoxia-induced interactome, most notably with mitochondrial-associated proteins. Both HIFα isoforms are heavily covalently modified: we define ~40 different sites of PTM on each of HIF-1α and HIF-2α, comprising 13 different PTM types, including multiple cysteine modifications and a highly unusual phosphocysteine. Over 80% of the PTMs identified are novel, and pproximately half exhibit oxygen-dependency under these conditions.
- Ấn phẩmAnalysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product(Biochemical Journal, 2020) Zacchi, Lucia F; Recinos, Dinora Roche; Pegg, Cassandra LCoagulation factor IX (FIX) is a highly complex post-translationally modified human serum glycoprotein and a high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Changes in bioreactor operating conditions can impact rFIX production and occupancy and structure of rFIX post-translational modifications (PTMs). We hypothesized that monitoring the bioreactor cell culture supernatant with Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics would allow us to predict product yield and quality after purification. With the goal of optimizing rFIX production, we developed a suite of MS proteomics analytical methods and used these to investigate changes in rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. Our methods provided a detailed overview of the dynamics of site-specific PTM occupancy and abundance on rFIX during production, which accurately predicted the efficiency of purification and the quality of the purified product from different culture conditions. In addition, we identified new PTMs in rFIX, some of which were near the GLA domain and could impact rFIX GLA-dependent purification efficiency and protein function. The workflows presented here are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.
- Ấn phẩmApoC-III helical structure determines its ability to bind plasma lipoproteins and inhibit Lipoprotein Lipase-mediated triglyceride lipolysis(Biochemical Journal, 2020) Vitali1, Cecilia; Stankov, Sylvia; Sumeet A, KhetarpalIn humans, apolipoprotein C-III (apoC-III) plasma levels have been associated with increased risk of cardiovascular disease. This association is in part explained by the effects of apoC-III on triglyceride (TG) metabolism; apoC-III raises plasma TG by increasing very low density lipoprotein (VLDL) secretion, inhibiting lipoprotein lipase (LPL)-mediated TG lipolysis, and impairing the removal of triglyceride-rich lipoprotein (TRL) remnants from the circulation. In this study, we explored the structure-function relationship the interaction of apoC-III with plasma lipoproteins and its ultimate impact on LPL activity. The structural and functional properties of wild-type (WT) apoC-III were compared with two missense variants previously associated with lower (A23T) and higher (Q38K) plasma TG. ApoC-III in the lipid-free state is unstructured but its helix content and stability increases when bound to lipid. Lipid-bound apoC-III contains two alpha helices spanning residues amino acids 11 - 38 (helix 1) and 44 – 64 (helix 2). Investigation of the structural and functional consequences of the A23T and Q38K variants showed that these amino acid substitutions within helix 1 do not significantly alter the stability of the helical structure but affect its hydrophilic-lipophilic properties. The A23T substitution impairs lipoprotein binding capacity, reduces LPL inhibition, and ultimately leads to lower plasma TG levels. Conversely, the Q to K substitution at position 38 enhances the lipid affinity of helix 1, increases TRL binding capacity and LPL inhibition, and is associated with hypertriglyceridemia. This study indicates that structural modifications that perturb the hydrophilic/lipophilic properties of the alpha helices can modulate the hypertriglyceridemic effects of apoC-III.
- Ấn phẩmATP2, the essential P4-ATPase of malaria parasites, catalyzes lipid-dependent ATP hydrolysis in complex with a Cdc50 β-subunit(Biochemical Journal, 2020) Lamy, Anaïs; Bruzaferro, Ewerton Macarini; Marín, Alex PerálvarezEfficient mechanisms of lipid transport are indispensable for the Plasmodium malaria parasite along the different stages of its intracellular life-cycle. Gene targeting approaches have recently revealed the irreplaceable role of the Plasmodium-encoded type 4 P-type ATPases (P4-ATPases or lipid flippases), ATP2, together with its potential involvement as antimalarial drug target. In eukaryotic membranes, P4-ATPases assure their asymmetric phospholipid distribution by translocating phospholipids from the outer to the inner leaflet. As ATP2 is a yet putative transporter, in this work we have used a recombinantly-produced P. chabaudi ATP2, PcATP2, to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 heterodimerizes with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A, indispensable partners for most P4-ATPases. Moreover, the purified PcATP2/PcCdc50B complex catalyses ATP hydrolysis in the presence of phospholipids containing either phosphatidylserine, phosphatidylethanolamine or phosphatidylcholine head groups, and that this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work provides the first study of the function and quaternary organization of ATP2, a promising antimalarial drug target candidate.
- Ấn phẩmCharacterization of redox sensitive algal mannitol-1-phosphatases of the haloacid dehalogenase superfamily of proteins(2020) Le Strat, Yoran; Tonon, Thierry; Leblanc, CatherineMacroalgae (or seaweeds) are the dominant primary producers in marine vegetated coastal habitats and largely contribute to global ocean carbon fluxes. They also represent attractive renewable production platforms for biofuels, food, feed, and bioactives, notably due to their diverse and peculiar polysaccharides and carbohydrates. Among seaweeds, brown algae produce alginates and sulfated fucans as constituents of their cell wall, and the photoassimilates laminarin and mannitol for carbon storage. Availability of brown algal genomes, including those of the kelp Saccharina japonica and the filamentous Ectocarpus sp., has paved the way for biochemical characterization of recombinant enzymes involved in their polysaccharide and carbohydrates synthesis, notably mannitol. Biosynthesis of mannitol in brown algae starts from fructose-6-phospate, which is converted into mannitol-1-phosphate (M1P), and this intermediate is then hydrolysed by a haloacid dehalogenase type M1P phosphatase (M1Pase) to produce mannitol. We report here the biochemical characterization of a second M1Pase in Ectocarpus sp after heterologous expression in Escherichia coli. (EsM1Pase1). Our results show that both Ectocarpus M1Pases were redox sensitive, with EsM1Pase1 being active only in presence of reducing agent. Such catalytic properties have not been observed for any of the M1Pase characterized so far. EsM1Pases were specific to mannitol, in contrast to S. japonica M1Pases that can use other phosphorylated sugars as substrates. Finally, brown algal M1Pases grouped into two well-supported clades, with potential different subcellular localization and physiological role(s) under diverse environmental conditions and/or stages of life cycle.
- Ấn phẩmClosing coronavirus spike glycoproteins by structure-guided design(Biochemical Journal, 2020) McCallum, Matthew; Walls, Alexandra C; Corti, DavideThe recent spillover of SARS-CoV-2 in the human population resulted in the ongoing COVID-19 pandemic which has already caused 4.9 million infections and more than 326,000 fatalities. To initiate infection the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface, determining host and tissue tropism, and fusion of the viral and host membranes. Although SARS-CoV- 2 S is the main target of neutralizing antibodies and the focus of vaccine design, its stability and conformational dynamics are limiting factors for developing countermeasures against this virus. We report here the design of a prefusion SARS-CoV-2 S ectodomain trimer construct covalently stabilized in the closed conformation. Structural and antigenicity analysis showed we successfully shut S in the closed state without otherwise altering its architecture. Finally, we show that this engineering strategy is applicable to other β-coronavirus S glycoproteins and might become an important tool for vaccine design, structural biology, serology and immunology studies.
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