MP
Light-driven Anion-Pumping Rhodopsin with Unique Cytoplasmic Anion-release Mechanism.
Ishizuka T, Suzuki K, Konno M, Shibata K, Kawasaki Y, Akiyama H, Murata T, Inoue K.
J Biol Chem. 2024 Sep 19:107797.
doi: 10.1016/j.jbc.2024.107797. Epub ahead of print.
PMID: 39305959.
Microbial rhodopsins are photoreceptive membrane proteins found in microorganisms with an all-trans-retinal chromophore. The function of many microbial rhodopsins is determined by three residues in the third transmembrane helix called motif residues. Here, we report a group of microbial rhodopsins with a novel Thr-Thr-Gly (TTG) motif. The ion-transport assay revealed that they function as light-driven inward anion pumps similar to halorhodopsins previously found in archaea and bacteria. Based on the characteristic glycine residue in their motif and light-driven anion-pumping function, these new rhodopsins are called glycylhalorhodopsins (GHRs). X-ray crystallographic analysis found large cavities on the cytoplasmic side, which are produced by the small side-chain volume of the glycine residue in the motif. The opened structure of GHR on the cytoplasmic side is related to the anion releasing process to the cytoplasm during the photoreaction compared to canonical halorhodopsin from Natronomonas pharaonis (NpHR). GHR also transports SO42- and the extracellular glutamate residue plays an essential role in extracellular SO42- uptake. In summary, we have identified TTG motif-containing microbial rhodopsins that display an anion-releasing mechanism.
Substrate binding and inhibition mechanism of norepinephrine transporter.
Ji W, Miao A, Liang K, Liu J, Qi Y, Zhou Y, Duan X, Sun J, Lai L, Wu JX.
Nature. 2024 Sep;633(8029):473-479.
doi: 10.1038/s41586-024-07810-5. Epub 2024 Aug 14.
PMID: 39143211.
Norepinephrine transporter (NET; encoded by SLC6A2) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic noradrenaline level. Genetic mutations and dysregulation of NET are associated with a spectrum of neurological conditions in humans, making NET an important therapeutic target. However, the structure and mechanism of NET remain unclear. Here we provide cryogenic electron microscopy structures of the human NET (hNET) in three functional states-the apo state, and in states bound to the substrate meta-iodobenzylguanidine (MIBG) or the orthosteric inhibitor radafaxine. These structures were captured in an inward-facing conformation, with a tightly sealed extracellular gate and an open intracellular gate. The substrate MIBG binds at the centre of hNET. Radafaxine also occupies the substrate-binding site and might block the structural transition of hNET for inhibition. These structures provide insights into the mechanism of substrate recognition and orthosteric inhibition of hNET.
Structural Models of Human Norepinephrine Transporter Ensemble Reveal the Allosteric Sites and Ligand-Binding Mechanism.
Luo D, Zhang Y, Li Y, Liu Z, Wu H, Xue W.
J Phys Chem B. 2024 Sep 12;128(36):8651-8661.
doi: 10.1021/acs.jpcb.4c03731. Epub 2024 Aug 29.
PMID: 39207306.
The norepinephrine transporter (NET) plays a pivotal role in recycling norepinephrine (NE) from the synaptic cleft. However, the structures referring to the conformational heterogeneity of NET during the transport cycle remain poorly understood. Here, three structural models of NE bound to the orthosteric site of NET in outward-open (OOholo), outward-occluded (OCholo), and inward-open (IOholo) conformations were first obtained using the multistate structures of serotonin transporter as templates and further characterized through Gaussian-accelerated molecular dynamics and free energy reweighting. Analysis of the structures revealed eight potential allosteric sites on the functional-specific states of NET. One of the pharmacologically relevant pockets located at the extracellular vestibule was further verified by simulating the binding behaviors of a clinical trial drug χ-MrIA that is allosterically regulating NET. These structural and energetic insights into NET advanced our understanding of NE reuptake and paved the way for discovering novel molecules targeting the allosteric sites.
Positive allosteric modulation of a GPCR ternary complex.
Burger WAC, Draper-Joyce CJ, Valant C, Christopoulos A, Thal DM.
Sci Adv. 2024 Sep 13;10(37):eadp7040.
doi: 10.1126/sciadv.adp7040. Epub 2024 Sep 11.
PMID: 39259792.
The activation of a G protein-coupled receptor (GPCR) leads to the formation of a ternary complex between agonist, receptor, and G protein that is characterized by high-affinity binding. Allosteric modulators bind to a distinct binding site from the orthosteric agonist and can modulate both the affinity and the efficacy of orthosteric agonists. The influence allosteric modulators have on the high-affinity active state of the GPCR-G protein ternary complex is unknown due to limitations on attempting to characterize this interaction in recombinant whole cell or membrane-based assays. Here, we use the purified M2 muscarinic acetylcholine receptor reconstituted into nanodiscs to show that, once the agonist-bound high-affinity state is promoted by the G protein, positive allosteric modulators stabilize the ternary complex that, in the presence of nucleotides, leads to an enhanced initial rate of signaling. Our results enhance our understanding of how allosteric modulators influence orthosteric ligand signaling and will aid the design of allosteric therapeutics.
Structural insights into CXCR4 modulation and oligomerization.
Saotome K, McGoldrick LL, Ho JH, Ramlall TF, Shah S, Moore MJ, Kim JH, Leidich R, Olson WC, Franklin MC.
Nat Struct Mol Biol. 2024 Sep 23.
doi: 10.1038/s41594-024-01397-1. Epub ahead of print.
PMID: 39313635.
Activation of the chemokine receptor CXCR4 by its chemokine ligand CXCL12 regulates diverse cellular processes. Previously reported crystal structures of CXCR4 revealed the architecture of an inactive, homodimeric receptor. However, many structural aspects of CXCR4 remain poorly understood. Here, we use cryo-electron microscopy to investigate various modes of human CXCR4 regulation. CXCL12 activates CXCR4 by inserting its N terminus deep into the CXCR4 orthosteric pocket. The binding of US Food and Drug Administration-approved antagonist AMD3100 is stabilized by electrostatic interactions with acidic residues in the seven-transmembrane-helix bundle. A potent antibody blocker, REGN7663, binds across the extracellular face of CXCR4 and inserts its complementarity-determining region H3 loop into the orthosteric pocket. Trimeric and tetrameric structures of CXCR4 reveal modes of G-protein-coupled receptor oligomerization. We show that CXCR4 adopts distinct subunit conformations in trimeric and tetrameric assemblies, highlighting how oligomerization could allosterically regulate chemokine receptor function.
Structural insights into the human P2X1 receptor and ligand interactions.
Bennetts FM, Venugopal H, Glukhova A, Mobbs JI, Ventura S, Thal DM.
Nat Commun. 2024 Sep 28;15(1):8418.
doi: 10.1038/s41467-024-52776-7.
PMID: 39341830.
The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions, offering the potential for new drug treatments. However, progress in this area has been hindered by limited structural information and a lack of well-characterised tool compounds. In this study, we employ cryogenic electron microscopy (cryo-EM) to elucidate the structures of the P2X1 receptor in an ATP-bound desensitised state and an NF449-bound closed state. NF449, a potent P2X1 receptor antagonist, engages the receptor distinctively, while ATP, the endogenous ligand, binds in a manner consistent with other P2X receptors. To explore the molecular basis of receptor inhibition, activation, and ligand interactions, key residues involved in ligand and metal ion binding were mutated. Radioligand binding assays with [3H]-α,β-methylene ATP and intracellular calcium ion influx assays were used to evaluate the effects of these mutations. These experiments validate key ligand-receptor interactions and identify conserved and non-conserved residues critical for ligand binding or receptor modulation. This research expands our understanding of the P2X1 receptor structure at a molecular level and opens new avenues for in silico drug design targeting the P2X1 receptor.
Function and firing of the Streptomyces coelicolor contractile injection system requires the membrane protein CisA
Bastien Casu, Joseph W. Sallmen, Peter E. Haas, Govind Chandra, Pavel Afanasyev, Jingwei Xu, Susan Schlimpert, and Martin Pilhofer
bioRxiv posted 1 October 2024
doi:10.1101/2024.06.25.600559
Bacterial contractile injection systems (CIS) are phage tail-like macromolecular complexes that mediate cell-cell interactions by injecting effector proteins into target cells. CIS from Streptomyces coelicolor (CISSc) are localized in the cytoplasm. Under stress, they induce cell death and impact the bacteria′s life cycle. It remains unknown whether CISSc require accessory proteins to directly interact with the cytoplasmic membrane and function. Here, we characterize the putative membrane adaptor CisA, a conserved factor in CIS gene clusters across Streptomyces species. We show by cryo-electron tomography imaging and in vivo assays that CISSc contraction and function depend on CisA. Using single-particle cryo-electron microscopy, we provide an atomic model of the extended CISSc apparatus; however, CisA is not part of the complex. Instead, our findings show that CisA is a membrane protein with a cytoplasmic N-terminus predicted to interact with CISSc components, thereby providing a possible mechanism for mediating CISSc recruitment to the membrane and subsequent firing. Our work shows that CIS function in multicellular bacteria is distinct from Type 6 Secretion Systems and extracellular CIS, and possibly evolved due to the role CISSc play in regulated cell death.
Hybrid Exb/Mot stators require substitutions distant from the chimeric pore to power flagellar rotation.
Ridone P, Baker MAB.
J Bacteriol. 2024 Sep 16:e0014024.
doi: 10.1128/jb.00140-24. Epub ahead of print.
PMID: 39283106.
Ion-powered rotary motors (IRMs) underpin the rotation of one of nature’s oldest wheels, the flagellar motor. Recent structures show that this complex appears to be a fundamental molecular module with diverse biological utility where electrical energy is coupled to torque. Here, we attempted to rationally design chimeric IRMs to explore the cross-compatibility of these ancient motors. We succeeded in making one working chimera of a flagellar motor and a non-flagellar transport system protein. This had only a short hybrid stretch in the ion-conducting channel, and function was subsequently improved through additional substitutions at sites distant from this hybrid pore region. Our goal was to test the cross-compatibility of these homologous systems and highlight challenges arising when engineering new rotary motors.
Membranes
Extreme makeover: the incredible cell membrane adaptations of extremophiles to harsh environments.
Maiti A, Erimban S, Daschakraborty S.
Chem Commun (Camb). 2024 Sep 16;60(75):10280-10294.
doi: 10.1039/d4cc03114h.
PMID: 39190300.
The existence of life beyond Earth has long captivated humanity, and the study of extremophiles-organisms surviving and thriving in extreme environments-provides crucial insights into this possibility. Extremophiles overcome severe challenges such as enzyme inactivity, protein denaturation, and damage of the cell membrane by adopting several strategies. This feature article focuses on the molecular strategies extremophiles use to maintain the cell membrane’s structure and fluidity under external stress. Key strategies include homeoviscous adaptation (HVA), involving the regulation of lipid composition, and osmolyte-mediated adaptation (OMA), where small organic molecules protect the lipid membrane under stress. Proteins also have direct and indirect roles in protecting the lipid membrane. Examining the survival strategies of extremophiles provides scientists with crucial insights into how life can adapt and persist in harsh conditions, shedding light on the origins of life. This article examines HVA and OMA and their mechanisms in maintaining membrane stability, emphasizing our contributions to this field. It also provides a brief overview of the roles of proteins and concludes with recommendations for future research directions.
The role of oleosins and phosphatidylcholines on the membrane mechanics of oleosomes.
Yang J, Plankensteiner L, de Groot A, Hennebelle M, Sagis LMC, Nikiforidis CV.
J Colloid Interface Sci. 2024 Sep 19;678(Pt C):1001-1011.
doi: 10.1016/j.jcis.2024.09.171. Epub ahead of print.
PMID: 39326161.
Hypothesis: Oilseeds use triacylglycerides as main energy source, and pack them into highly stable droplets (oleosomes) to facilitate the triacylglycerides long-term storage in the aqueous cytosol. To prevent the coalescence of oleosomes, they are stabilized by a phospholipid monolayer and unique surfactant-shaped proteins, called oleosins. In this study, we use state-of-the-art interfacial techniques to reveal the function of each component at the oleosome interface.
Experiments: We created model oil-water interfaces with pure oleosins, phosphatidylcholines, or mixtures of both components (ratios of 3:1, 1:1, 1:3), and applied large oscillatory dilatational deformations (LAOD). The obtained rheological response was analyzed with general stress decomposition (GSD) to get insights into the role of phospholipids and oleosins on the mechanics of the interface.
Findings: Oleosins formed viscoelastic solid interfacial films due to network formation via in-plane interactions. Between adsorbed phosphatidylcholines, weak interactions were observed, suggesting the surface stress response upon dilatational deformations was dominated by density changes. In mixtures with 3:1 and 1:1 oleosin-to-phosphatidylcholine ratios, oleosins dominated the interfacial mechanics and formed a network, while phosphatidylcholines contributed to interfacial tension reduction. At higher phosphatidylcholine concentrations (1:3 oleosin-to-phosphatidylcholine), phosphatidylcholine dominated the interface, and no network formation occurred. Our findings improve the understanding of both components role for oleosomes.
Establishment of the Meyer-Overton correlation in an artificial membrane without protein.
Matsumoto A, Uesono Y.
Biochim Biophys Acta Gen Subj. 2024 Sep 27:130717.
doi: 10.1016/j.bbagen.2024.130717. Epub ahead of print.
PMID: 39343251.
Background: The potency of anesthetics with various structures increases exponentially with lipophilicity, which is the Meyer-Overton (MO) correlation discovered over 120 years ago. The MO correlation was also observed with various biological effects and chemicals, including alcohols; thus, the correlation represents a fundamental relationship between chemicals and organisms. The MO correlation was explained by the lipid and protein theories, although the principle remains unknown because these are still debating.
Methods: The gentle hydration method was used to form giant unilamellar vesicles (GUVs) consisting of high- and low-melting phospholipids and cholesterol in the presence of n-alcohols (C2-C12). Confocal fluorescence microscopy was used to determine the percentage of GUVs with domains in relation to the n-alcohol concentrations.
Results: n-Alcohols inhibited the domain formation of GUVs, and the half inhibitory concentration (IC50) in the aqueous phase (Cw) decreased exponentially with increasing chain length (lipophilicity). In contrast, the membrane concentrations (Cm) of alcohols for the inhibition, which is a product of the membrane-water partition coefficient and the IC50 values, remained constant irrespective of the chain length.
Conclusions: The MO correlation is established in GUVs, which supports the lipid theory. When alcohols reach the same critical concentration in the membrane, similar biological effects appear irrespective of the chain length, which is the principle underlying the MO correlation.
General significance: The protein theory states that a highly lipophilic compound targets minor membrane proteins due to the low Cw. However, our lipid theory states that the compound targets various membrane proteins due to the high Cm.
Shape transformation of vesicles induced by orientational arrangement of membrane proteins
Menglong Feng, Kunhao Dong, Yuansheng Cao, RUI MA
bioRxiv 2024.09.28.615559;
doi: https://doi.org/10.1101/2024.09.28.615559
Vesicles of lipid bilayer can adopt a variety of shapes due to different coating proteins. The ability of proteins to reshape membrane is typically characterized by inducing spontaneous curvature of the membrane at the coated area. BAR family proteins are known to have a crescent shape and can induce membrane curvature along its concaved body axis but not in the perpendicular direction. We model this type of proteins as a rod-shaped molecule with an orientation and induce normal curvature along its orientation in the tangential plane of the membrane surface. We show how a ring of these proteins reshape an axisymmetric vesicle when the protein curvature or orientation is varied. A discontinuous shape transformation from a protrusion shape without a neck to a one with a neck is found. Increasing the rigidity of the protein ring is able to smooth out the transition. Furthermore, we show that varying the protein orientation is able to induce an hourglass-shaped neck, which is significantly narrower than the reciprocal of the protein curvature. Our results offer a new angle to rationalize the helical structure formed by many proteins that carry out membrane fission functions.
A minimalist model lipid system mimicking the biophysical properties of Escherichia coli membrane
Nicolo Tormena, Teuta Pilizota, and Kislon Voitchovsky
bioRxiv posted 1 October 2024
doi:10.1101/2024.09.29.615671
Biological membrane are highly complex systems that are of fundamental importance to the development and survival of organisms. Native membranes typically comprise different types of lipids, biomolecules and proteins assembled around a lipid bilayer structure. This complexity can render investigations challenging, with many studies relying on model membranes such as artificial vesicles and supported lipid bilayers (SLBs). The purpose of a model system is to capture the desired dominant features of the native context while remaining uniquely defined and simpler. Here, we search for a minimal lipid-only model system of the Escherichia coli inner membrane. We aim to retain the main lipidomic components in their native ratio while mimicking the membrane thermal and mechanical properties. We design a collection of candidate model systems reflecting the main aspects of the known native lipidomic composition and narrow down our selection based on the systems phase transition temperature. We further test our candidate model systems by independently measuring their elastic properties. We identify 3 ternary model systems able to form stable bilayers that closely mimic E. coli inner membrane lipid composition and mechanical properties. These model systems are made of commercially available synthetic 16:0-18:1 phosphatidylethanolamine (POPE), 16:0-18:1 phosphatidylglycerol (POPG), and 16:0-18:1 Cardiolipin (CL). We anticipate our results to be of interest for future studies making use of E. coli models, for example investigating membrane proteins function or macromolecule-membrane interactions.
Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets.
Waeterschoot J, Barniol-Xicota M, Verhelst S, Baatsen P, Koos E, Lammertyn J, Casadevall I Solvas X.
Heliyon. 2024 Sep 18;10(18):e37915.
doi: 10.1016/j.heliyon.2024.e37915.
PMID: 39347415.
Understanding the intricate functions of membrane proteins is pivotal in cell biology and drug discovery. The composition of the cell membrane is highly complex, with different types of membrane proteins and lipid species. Hence, studying cellular membranes in a complexity-reduced context is important to enhance our understanding of the roles of these different elements. However, reconstitution of membrane proteins in an environment that closely mimics the cell, like giant unilamellar vesicles (GUVs), remains challenging, often requiring detergents that compromise protein function. To address this challenge, we present a novel strategy to manufacture GUVs from styrene maleic acid lipid particles (SMALPs) that utilises surfactant-stabilised droplets as a template. As a first step towards the incorporation of membrane proteins, this work focusses on the conversion of pure lipid SMALPs in GUVs. To evaluate the method, we produced a new form of SMA linked to fluorescein, referred to as FSMA. We demonstrate the assembly of SMALPs at the surfactant-stabilised droplet interface, resulting in the formation of GUVs when released upon addition of a demulsifying agent. The released vesicles appear similar to electroformed vesicles imaged with confocal light microscopy, but a fluorescein leakage assay and cryo-TEM imaging reveal their porous nature, potentially as a result of residual interactions of SMA with the lipid bilayer. Our study represents a significant step towards opening new avenues for comprehensive protein research in a complexity-reduced, yet biologically relevant, setting.
Molecules
Stimulation of cytochrome c oxidase activity by detergents.
Smirnova I, Wu F, Brzezinski P.
Biochim Biophys Acta Bioenerg. 2024 Sep 7;1866(1):149509.
doi: 10.1016/j.bbabio.2024.149509. Epub ahead of print.
PMID: 39251013.
Cytochrome c oxidase (CytcO) is an integral membrane protein, which catalyzes four-electron reduction of oxygen linked to proton uptake and pumping. Amphipathic molecules bind in sites near the so-called K proton pathway of CytcO to reversibly modulate its activity. However, purification of CytcO for mechanistic studies typically involves the use of detergents, which may interfere with binding of these regulatory molecules. Here, we investigated the CytcO enzymatic activity as well as intramolecular electron transfer linked to proton transfer upon addition of different detergents to bovine heart mitoplasts. The CytcO activity increased upon addition of alkyl glucosides (DDM and DM) and the steroid analog GDN. The maximum stimulating effect was observed for DDM and DM, and the half-stimulating effect correlated with their CMC values. With GDN the stimulation effect was smaller and occurred at a concentration higher than CMC. A kinetic analysis suggests that the stimulation of activity is due to removal of a ligand bound near the K proton pathway, which indicates that in the native membrane this site is occupied to yield a lower than maximal possible CytcO activity. Possible functional consequences are discussed.
Methods
Chemiosmotic nutrient transport in synthetic cells powered by electrogenic antiport coupled to decarboxylation.
Patiño-Ruiz MF, Anshari ZR, Gaastra B, Slotboom DJ, Poolman B.
Nat Commun. 2024 Sep 12;15(1):7976.
doi: 10.1038/s41467-024-52085-z.
PMID: 39266519.
Cellular homeostasis depends on the supply of metabolic energy in the form of ATP and electrochemical ion gradients. The construction of synthetic cells requires a constant supply of energy to drive membrane transport and metabolism. Here, we provide synthetic cells with long-lasting metabolic energy in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution with the transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. We couple the accumulation of lactose to a metabolic network for the generation of intermediates of the glycolytic and pentose phosphate pathways. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, paving the way for the development of more complex synthetic systems.
Protocol for evaluating S-acylated protein membrane affinity using protein-lipid conjugates.
Xie C, Luo D, Liu L, Huang X, Zhang SL, Yang A.
STAR Protoc. 2024 Sep 20;5(3):103182.
doi: 10.1016/j.xpro.2024.103182. Epub 2024 Aug 1.
PMID: 39093703.
S-acylation of proteins allows their association with membranes. Here, we present a protocol for establishing a platform for membrane affinity evaluation of S-acylated proteins in vitro. We describe steps for preparing lipid-maleimide compounds, mCherry-p62 recombinant proteins, and total cellular membranes. We then detail procedures for synthesizing protein-lipid conjugates using lipid-maleimide compounds and recombinant proteins and evaluating the membrane affinity of protein-lipid conjugates.
Microbio
Inosine reverses multidrug resistance in Gram-negative bacteria carrying mobilized RND-type efflux pump gene cluster tmexCD-toprJ.
Li F, Xu T, Fang D, Wang Z, Liu Y.
mSystems. 2024 Sep 10:e0079724.
doi: 10.1128/msystems.00797-24. Epub ahead of print.
PMID: 39254032.
TMexCD1-TOprJ1, a mobilized resistance-nodulation-division-type efflux pump, confers phenotypic resistance to multiple classes of antibiotics. Nowadays, tmexCD-toprJ has disseminated among diverse species of clinical pathogens, exacerbating the need for novel anti-infective strategies. In this study, we report that tmexCD1-toprJ1-negative and -positive bacteria exhibit significantly different metabolic flux and characteristics, especially in purine metabolism. Intriguingly, the addition of inosine, a purine metabolite, effectively restores the antibacterial activity of tigecycline by promoting antibiotic uptake. Our findings highlight the correlation between bacterial mechanism and antibiotic resistance, and offer a distinct approach to overcome tmexCD-toprJ-mediated multidrug resistance.
Evaluating the Expression of Efflux Pumps in Pseudomonas aeruginosa in Exposure to Sodium Dodecyl Sulfate, Didecyldimethylammonium Chloride, and Octenidine Dihydrochloride.
Alsamhary K.
Microb Drug Resist. 2024 Sep;30(9):385-390.
doi: 10.1089/mdr.2024.0070. Epub 2024 Jul 31.
PMID: 39082183.
Emerging resistance of Gram-negative bacteria, including Pseudomonas aeruginosa, to commonly used detergents and disinfectant is encountering us with hazard. Inappropriate use of disinfectants has forced bacteria to gain resistance. The ability of bacteria to extrude substrates from the cellular interior to the external environment has enabled them to persist in exposure to toxic compounds, which is due to existence of transport proteins. Efflux pumps, in Gram-negative bacteria, are proteins responsible for exporting molecules outside of the cell, by crossing the two membranes. In this study, 40 P. aeruginosa strains from hospitals, clinics, and burn center laundries and 40 P. aeruginosa strains from urban laundries were collected. This study evaluated the minimum inhibitory concentration (MIC) level of sodium dodecyl sulfate (SDS), didecyldimethylammonium chloride (DDAC), and octenidine dihydrochloride (Od) in P. aeruginosa strains. The real-time PCR was carried out to evaluate the expression of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux system. The obtained results indicated a higher MIC level for SDS, DDAC, and Od in medical laundries. The sub-MIC level of DDAC and Od increased the expression level of MexAB-OprM, MexCD-OprJ, and MexXY-OprM in P. aeruginosa strains, suggesting that efflux pumps contribute to disinfectant resistance in P. aeruginosa.
A Metagenomic Study of Antibiotic Resistance Across Diverse Soil Types and Geographical Locations
Stephanie Pillay, Yasin Ilkagan Tepeli, Paul van Lent, and Thomas Abeel
bioRxiv posted 30 September 2024
doi:10.1101/2024.09.30.615846
Antimicrobial resistance in soil may be driven by anthropogenic activities and biogeographical factors, increasing the risk of bacteria developing resistance and leading to higher morbidity and mortality rates in humans and animals.
Miscellaneous
Combining systems and synthetic biology for in vivo enzymology.
Castaño-Cerezo S, Chamas A, Kulyk H, Treitz C, Bellvert F, Tholey A, Galéote V, Camarasa C, Heux S, Garcia-Alles LF, Millard P, Truan G.
EMBO J. 2024 Sep 25.
doi: 10.1038/s44318-024-00251-w. Epub ahead of print.
PMID: 39322757.
Enzymatic parameters are classically determined in vitro, under conditions that are far from those encountered in cells, casting doubt on their physiological relevance. We developed a generic approach combining tools from synthetic and systems biology to measure enzymatic parameters in vivo. In the context of a synthetic carotenoid pathway in Saccharomyces cerevisiae, we focused on a phytoene synthase and three phytoene desaturases, which are difficult to study in vitro. We designed, built, and analyzed a collection of yeast strains mimicking substantial variations in substrate concentration by strategically manipulating the expression of geranyl-geranyl pyrophosphate (GGPP) synthase. We successfully determined in vivo Michaelis-Menten parameters (KM, Vmax, and kcat) for GGPP-converting phytoene synthase from absolute metabolomics, fluxomics and proteomics data, highlighting differences between in vivo and in vitro parameters. Leveraging the versatility of the same set of strains, we then extracted enzymatic parameters for two of the three phytoene desaturases. Our approach demonstrates the feasibility of assessing enzymatic parameters directly in vivo, providing a novel perspective on the kinetic characteristics of enzymes in real cellular conditions.
‘Afraid to talk’: researchers fear the end for science in Venezuela.
Taylor L.
Nature. 2024 Sep 27.
doi: 10.1038/d41586-024-03144-4. Epub ahead of print.
PMID: 39327522.
A lack of funding and academic freedom amid a political crackdown leave scientists feeling hopeless and pondering an exodus from the country.
Computing the Human Interactome
Jing Zhang, Ian R Humphreys, Jimin Pei, Jinuk Kim, Chulwon Choi, Rongqing Yuan, Jesse Durham, Siqi Liu, Hee-Jung Choi, Minkyung Baek, David Baker, and Qian Cong
bioRxiv posted 1 October 2024
doi:10.1101/2024.10.01.615885
Protein-protein interactions (PPI) are essential for biological function. Recent advances in coevolutionary analysis and Deep Learning (DL) based protein structure prediction have enabled comprehensive PPI identification in bacterial and yeast proteomes, but these approaches have limited success to date for the more complex human proteome. Here, we overcome this challenge by 1) enhancing the coevolutionary signals with 7-fold deeper multiple sequence alignments harvested from 30 petabytes of unassembled genomic data, and 2) developing a new DL network trained on augmented datasets of domain-domain interactions from 200 million predicted protein structures. These advancements allow us to systematically screen through 200 million human protein pairs and predict 18,316 PPIs with an expected precision of 90%, among which 5,578 are novel predictions. 3D models of these predicted PPIs nearly triple the number of human PPIs with accurate structural information, providing numerous insights into protein function and mechanisms of human diseases.
Bridging structural biology and clinical research through in-tissue cryo-electron tomography.
Kixmoeller K, Creekmore BC, Lee EB, Chang YW.
EMBO J. 2024 Sep 16.
doi: 10.1038/s44318-024-00216-z. Epub ahead of print.
PMID: 39284913.
Structural biology is a field of research that investigates the structure and organization of macromolecules to decipher their functional mechanisms. Traditionally, it has adopted a reductionist approach out of necessity, focusing on the high-resolution analysis of macromolecules outside their natural cellular contexts. This contrasts sharply with clinical research, which examines human biology holistically through patient studies and cellular disease models, albeit with less detailed structural insights. Recent advancements are bridging this gap, enabling the study of macromolecular structures within more complex biological systems, from single cells to multicellular organisms and primary human tissues. These developments signal a pivotal shift within structural biology from traditional methodologies towards a more integrated understanding of biological complexity. Here, we highlight recent technological advancements that facilitate structural studies in tissue environments, showcasing the discoveries made possible with these approaches and the prospect of their applications in human clinical research.
The First ‘Zeta-Class’ Supercomputer Will Revolutionize Science in Just 6 Years
Work to develop a supercomputer that could out-pace the world’s current fastest by 1000 times is officially underway. Expected to cost the Japanese government around US$775 million the Fugaku Next machine should be online by 2030. The world’s fastest current supercomputer functions in the realm of one quintillion (1017) calculations per second or exaFLOPS. Fugaku Next is expected to operate in the realm of zetaFLOPS 1000 times that speed.
How cells swap instant messages
Cells in our bodies are passing delicate time-sensitive notes to each other. These notes come in the form of messenger RNA (mRNA) which cells have to neatly package in sacs called vesicles to send between cells. This year researchers showed that cells in all three domains of life – archaea bacteria and eukaryotes – can send these messages and even that they can be used as weapons between species. There are still questions to answer such as whether other molecules packed into vesicles are necessary for mRNA’s message to land but “it’s a fun challenge to unravel all of that” says biologist Amy Buck. “I’ve been in awe of what RNA can do.”
Twenty years of Addgene.
Remmel A.
Nature. 2024 Oct;634(8032):254-256.
doi: 10.1038/d41586-024-03152-4.
PMID: 39349632.
The non-profit plasmid repository Addgene is celebrating two decades of helping scientists to share and access these crucial life-science DNA tools. Researchers who develop a popular plasmid can offload the hassle of handling requests to Addgene for free. Meanwhile, scientists who order these reagents can count on receiving materials on time and up to standard. Addgene has accelerated the development of groundbreaking technologies such as CRISPR-Cas9 gene editing, prime editing (which uses RNA to guide Cas9 to a specific site in the genome) and tools for studying the SARS-CoV-2 virus.
Spring-loaded DNA origami arrays as energy-supplied hardware for modular nanorobots
Martina Pfeiffer, Fiona Cole, Dongfang Wang, Yonggang Ke, and Philip Tinnefeld
bioRxiv posted 1 October 2024
doi:10.1101/2024.09.30.615428