MP
Re-evaluating TRP channel mechanosensitivity.
Cox CD, Poole K, Martinac B.
Trends Biochem Sci. 2024 Aug;49(8):693-702.
doi: 10.1016/j.tibs.2024.05.004. Epub 2024 Jun 8.
PMID: 38851904.
Transient receptor potential (TRP) channels are implicated in a wide array of mechanotransduction processes. However, a question remains whether TRP channels directly sense mechanical force, thus acting as primary mechanotransducers. We use several recent examples to demonstrate the difficulty in definitively ascribing mechanosensitivity to TRP channel subfamilies. Ultimately, despite being implicated in an ever-growing list of mechanosignalling events in most cases limited robust or reproducible evidence supports the contention that TRP channels act as primary transducers of mechanical forces. They either (i) possess unique and as yet unspecified structural or local requirements for mechanosensitivity; or (ii) act as mechanoamplifiers responding downstream of the activation of a primary mechanotransducer that could include Ca2+-permeable mechanosensitive (MS) channels or other potentially unidentified mechanosensors.
Cobalamin decyanation by the membrane transporter BtuM.
Martínez Felices JM, Barreto YB, Thangaratnarajah C, Whittaker JJ, Alencar AM, Guskov A, Slotboom DJ.
Structure. 2024 Aug 8;32(8):1165-1173.e3.
doi: 10.1016/j.str.2024.04.014. Epub 2024 May 10.
PMID: 38733996.
BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the β-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the β-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (KD = 4.0 μM), followed by the formation of a covalent bond (rate constant of 0.163 s-1), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.
Structures of the dopamine transporter point to ways to target addiction and disease.
Sitte HH.
Nature. 2024 Aug;632(8025):509-511.
doi: 10.1038/d41586-024-02435-0.
PMID: 39112574.
Three studies of the human dopamine transporter reveal how it binds to molecules such as dopamine and cocaine. Dopamine imbalances underlie some brain conditions and these data will aid targeted drug design.
- Structure of the human dopamine transporter and mechanisms of inhibition. Srivastava DK, Navratna V, Tosh DK, Chinn A, Sk MF, Tajkhorshid E, Jacobson KA, Gouaux E. Nature. 2024 Aug;632(8025):672-677. doi: 10.1038/s41586-024-07739-9. Epub 2024 Aug 7. PMID: 39112705.
- Structure of the human dopamine transporter in complex with cocaine. Nielsen JC, Salomon K, Kalenderoglou IE, Bargmeyer S, Pape T, Shahsavar A, Loland CJ. Nature. 2024 Aug;632(8025):678-685. doi: 10.1038/s41586-024-07804-3. Epub 2024 Aug 7. PMID: 39112703.
- Dopamine reuptake and inhibitory mechanisms in human dopamine transporter. Li Y, Wang X, Meng Y, Hu T, Zhao J, Li R, Bai Q, Yuan P, Han J, Hao K, Wei Y, Qiu Y, Li N, Zhao Y. Nature. 2024 Aug;632(8025):686-694. doi: 10.1038/s41586-024-07796-0. Epub 2024 Aug 7. PMID: 39112701.
The role of FoxA, FiuA, and FpvB in iron acquisition via hydroxamate-type siderophores in Pseudomonas aeruginosa.
Will V, Frey C, Normant V, Kuhn L, Chicher J, Volck F, Schalk IJ.
Sci Rep. 2024 Aug 13;14(1):18795.
doi: 10.1038/s41598-024-69152-6.
PMID: 39138320.
Siderophores are specialized molecules produced by bacteria and fungi to scavenge iron, a crucial nutrient for growth and metabolism. Catecholate-type siderophores are mainly produced by bacteria, while hydroxamates are mostly from fungi. This study investigates the capacity of nine hydroxamate-type siderophores from fungi and Streptomyces to facilitate iron acquisition by the human pathogen Pseudomonas aeruginosa. Growth assays under iron limitation and 55Fe incorporation tests showed that all nine siderophores promoted bacterial growth and iron transport. The study also aimed to identify the TonB-dependent transporters (TBDTs) involved in iron import by these siderophores. Using mutant strains lacking specific TBDT genes, it was found that iron is imported into P. aeruginosa cells by FpvB for coprogen, triacetylfusarinine, fusigen, ferrirhodin, and ferrirubin. Iron complexed by desferioxamine G is transported by FpvB and FoxA, ferricrocin-Fe and ferrichrycin-Fe by FpvB and FiuA, and rhodotoluric acid-Fe by FpvB, FiuA, and another unidentified TBDT. These findings highlight the effectiveness of hydroxamate-type siderophores in iron transport into P. aeruginosa and provide insights into the complex molecular mechanisms involved, which are important for understanding microbial interactions and ecological balance.
Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging.
Hirschi S, Lemmin T, Ayoub N, Kalbermatter D, Pellegata D, Ucurum Z, Gertsch J, Fotiadis D.
Nat Commun. 2024 Aug 13;15(1):6950.
doi: 10.1038/s41467-024-50960-3.
PMID: 39138159.
Microbial ion-pumping rhodopsins (MRs) are extensively studied retinal-binding membrane proteins. However, their biogenesis, including oligomerisation and retinal incorporation, remains poorly understood. The bacterial green-light absorbing proton pump proteorhodopsin (GPR) has emerged as a model protein for MRs and is used here to address these open questions using cryo-electron microscopy (cryo-EM) and molecular dynamics (MD) simulations. Specifically, conflicting studies regarding GPR stoichiometry reported pentamer and hexamer mixtures without providing possible assembly mechanisms. We report the pentameric and hexameric cryo-EM structures of a GPR mutant, uncovering the role of the unprocessed N-terminal signal peptide in the assembly of hexameric GPR. Furthermore, certain proteorhodopsin-expressing bacteria lack retinal biosynthesis pathways, suggesting that they scavenge the cofactor from their environment. We shed light on this hypothesis by solving the cryo-EM structure of retinal-free proteoopsin, which together with mass spectrometry and MD simulations suggests that decanoate serves as a temporary placeholder for retinal in the chromophore binding pocket. Further MD simulations elucidate possible pathways for the exchange of decanoate and retinal, offering a mechanism for retinal scavenging. Collectively, our findings provide insights into the biogenesis of MRs, including their oligomeric assembly, variations in protomer stoichiometry and retinal incorporation through a potential cofactor scavenging mechanism.
The ‘dispanins’ and related proteins in physiology and neurological disease.
Deuis JR, Klasfauseweh T, Walker L, Vetter I.
Trends Neurosci. 2024 Aug;47(8):622-634.
doi: 10.1016/j.tins.2024.06.004. Epub 2024 Jul 17.
PMID: 39025729.
The dispanins are a family of 15 transmembrane proteins that have diverse and often unclear physiological functions. Many dispanins, including synapse differentiation induced gene 1 (SynDIG1), proline-rich transmembrane protein 1 (PRRT1)/SynDIG4, and PRRT2, are expressed in the central nervous system (CNS), where they are involved in the development of synapses, regulation of neurotransmitter release, and interactions with ion channels, including AMPA receptors (AMPARs). Others, including transmembrane protein 233 (TMEM233) and trafficking regulator of GLUT4-1 (TRARG1), are expressed in the peripheral nervous system (PNS); however, the function of these dispanins is less clear. Recently, a family of neurotoxins isolated from the giant Australian stinging tree was shown to target TMEM233 to modulate the function of voltage-gated sodium (NaV) channels, suggesting that the dispanins are inherently druggable. Here, we review current knowledge about the structure and function of the dispanins, in particular TMEM233 and its two most closely related homologs PRRT2 and TRARG1, which may be drug targets involved in neurological disease.
Structure and mechanism of the osmoregulated choline transporter BetT.
Yang T, Nian Y, Lin H, Li J, Lin X, Li T, Wang R, Wang L, Beattie GA, Zhang J, Fan M.
Sci Adv. 2024 Aug 16;10(33):eado6229.
doi: 10.1126/sciadv.ado6229. Epub 2024 Aug 14.
PMID: 39141726.
The choline-glycine betaine pathway plays an important role in bacterial survival in hyperosmotic environments. Osmotic activation of the choline transporter BetT promotes the uptake of external choline for synthesizing the osmoprotective glycine betaine. Here, we report the cryo-electron microscopy structures of Pseudomonas syringae BetT in the apo and choline-bound states. Our structure shows that BetT forms a domain-swapped trimer with the C-terminal domain (CTD) of one protomer interacting with the transmembrane domain (TMD) of a neighboring protomer. The substrate choline is bound within a tryptophan prism at the central part of TMD. Together with functional characterization, our results suggest that in Pseudomonas species, including the plant pathogen P. syringae and the human pathogen Pseudomonas aeruginosa, BetT is locked at a low-activity state through CTD-mediated autoinhibition in the absence of osmotic stress, and its hyperosmotic activation involves the release of this autoinhibition.
Cryo-EM structure of the human glucose transporter GLUT7
Sang Soo Lee, Subin Kim, Mi Sun Jin,
Biochemical and Biophysical Research Communications, 2024,150544,
https://doi.org/10.1016/j.bbrc.2024.150544.
GLUT7 is a Class II glucose transporter predominantly expressed at the apical membrane of enterocytes in the small intestine. Here, we report the cryo-EM structure of nanodisc-reconstituted human GLUT7 in the apo state at 3.3 Å resolution. Our atomic model reveals a typical major facilitator superfamily fold, with the substrate-binding site open to the extracellular side of the membrane. Despite the nearly identical conformation to its closest family member, rat GLUT5, our structure unveils distinct features of the substrate-binding cavity that may influence substrate specificity and binding mode. A homology model of the inward-open human GLUT7 indicates that similar to other members of the GLUT family, it may undergo a global rocker-switch-like reorientation of the transmembrane bundles to facilitate substrate translocation across the membrane. Our work enhances the current structural understanding of the GLUT family, and lays a foundation for rational design of regulators of GLUTs and other sugar transporters.
Membranes
A pH-sensitive motif in an outer membrane protein activates bacterial membrane vesicle production.
Dehinwal R, Gopinath T, Smith RD, Ernst RK, Schifferli DM, Waldor MK, Marassi FM.
Nat Commun. 2024 Aug 13;15(1):6958.
doi: 10.1038/s41467-024-51364-z.
PMID: 39138228.
Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins.
Prominin 1 and Tweety Homology 1 both induce extracellular vesicle formation.
Bell TA, Luce BE, Hakim P, Ananda VY, Dardari H, Nguyen TH, Monshizadeh A, Chao LH.
Elife. 2024 Aug 13;13:e100061.
doi: 10.7554/eLife.100061. Epub ahead of print.
PMID: 39136554.
Prominin-1 (Prom1) is a five-transmembrane-pass integral membrane protein that associates with curved regions of the plasma membrane. Prom1 interacts with membrane cholesterol and actively remodels the plasma membrane. Membrane bending activity is particularly evident in photoreceptors, where Prom1 loss-of-function mutations cause failure of outer segment homeostasis, leading to cone-rod retinal dystrophy (CRRD). The Tweety Homology (Ttyh) protein family has been proposed to be homologous to Prominin, but it is not known whether Ttyh proteins have an analogous membrane-bending function. Here, we characterize the membrane-bending activity of human Prom1 and Ttyh1 in native bilayer membranes. We find that Prom1 and Ttyh1 both induce formation of extracellular vesicles (EVs) in cultured mammalian cells and that the EVs produced are physically similar. Ttyh1 is more abundant in EV membranes than Prom1 and produces EVs with membranes that are more tubulated than Prom1 EVs. We further show that Prom1 interacts more stably with membrane cholesterol than Ttyh1 and that this may contribute to membrane bending inhibition in Prom1 EVs. Intriguingly, a loss-of-function mutation in Prom1 associated with CRRD induces particularly stable cholesterol binding. These experiments provide mechanistic insight into Prominin function in CRRD and suggest that Prom and Ttyh belong to a single family of functionally related membrane-bending, EV-generating proteins.
Engineering Outer Membrane Vesicles to Carry Enzymes: Encapsulation, Isolation, Characterization, and Modification.
Thakur M, Dean SN.
Methods Mol Biol. 2024;2843:177-194.
doi: 10.1007/978-1-0716-4055-5_12.
PMID: 39141301.
Outer membrane vesicles (OMVs) are small, spherical, nanoscale proteoliposomes released from Gram-negative bacteria that play an important role in cellular defense, pathogenesis, and signaling, among other functions. The functionality of OMVs can be enhanced by engineering developed for biomedical and biochemical applications. Here, we describe methods for directed packaging of enzymes into bacterial OMVs of E. coli using engineered molecular systems, such as localizing proteins to the inner or outer surface of the vesicle. Additionally, we detail some modification strategies for OMVs such as lyophilization and surfactant conjugation that enable the protection of activity of the packaged enzyme when exposed to non-physiological conditions such as elevated temperature, organic solvents, and repeated freeze/thaw that otherwise lead to a substantial loss in the activity of the free enzyme.
Nanoscale lipid domains determine the dynamic molecular portraits of mixed DOPC/DOPS bilayers in a fluid phase: A computational insight.
Veretenenko II, Trofimov YA, Krylov NA, Efremov RG.
Biochim Biophys Acta Biomembr. 2024 Aug 5;1866(7):184376.
doi: 10.1016/j.bbamem.2024.184376. Epub ahead of print.
PMID: 39111381.
Lateral heterogeneity, or mosaicity, is a fundamental property inherent to cell membranes that is crucial for their functioning. While microscopic inhomogeneities (e.g. rafts) are easily detected experimentally, lipid domains with nanoscale dimensions (nanoclusters of nanodomains, NDs) resist reliable characterization by instrumental methods. In such a case, important insight can be gained via computer modeling. Here, NDs composed of lipid’s head groups in the mixed zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylserine (DOPS) bilayers were studied by molecular dynamics. A new algorithm has been developed to identify NDs. Unlike most similar methods, it implicitly considers the heterogeneous distribution of lipid head atomic density and does not require subjectively chosen parameters. In DOPS-rich membranes, lipids form more compact and stable NDs due to strong interlipid interactions. In DOPC-rich systems, NDs arise due to the “packing” effect of weakly bound lipid heads. The clustering picture is related to the physical properties of the bilayer surface: DOPS-rich systems show more pronounced surface heterogeneity of hydrophilic/hydrophobic regions compared to DOPC-rich ones. The results obtained are important for the effective quantitative characterization of the “dynamic molecular portrait” of a membrane surface – its “fingerprint” characterizing dynamical distribution of its physicochemical properties.
Involvement of mammalian SoLute Carriers (SLC) in the traffic of polyamines.
Pochini L.
Front Mol Biosci. 2024 Jul 25;11:1452184.
doi: 10.3389/fmolb.2024.1452184. PMID: 39130372;
PMC11310933.
Polyamines interact with different molecular targets to regulate a vast range of cellular processes. A network of enzymes and transport systems is crucial for the maintenance of polyamine homeostasis. Indeed, polyamines after synthesis must be distributed to the various tissues and some intracellular organelles. Differently from the well characterized enzymes devoted to polyamine synthesis, the transport systems are not unequivocally identified or characterized. Besides some ATPases which have been identified as polyamine transporters, much less is known about solute carriers (SLC) involved in the transport of these compounds. Only two SLCs have been unequivocally identified as polyamine transporters: SLC18B1 (VPAT) and SLC22A4 (OCTN1). Transport studies have been performed with cells transfected with the cDNAs encoding the two and other SLCs or, in the case of OCTN1, also by in vitro assay using proteoliposomes harboring the recombinant human protein. According to the role proposed for OCTN1, polyamines have been associated with prolonged and quality of life. This review provides an update on the most recent findings concerning the polyamine transporters or the prediction of the putative ones.
Determining the Role of Surfactant on the Cytosolic Delivery of DNA Cross-Linked Micelles.
Fuente IF, Sawant SS, Kho KW, Sarangi NK, Canete RC, Pal S, Liang LH, Keyes TE, Rouge JL.
ACS Appl Mater Interfaces. 2024 Aug 12.
doi: 10.1021/acsami.4c09894. Epub ahead of print.
PMID: 39132807.
Nucleic Acid Nanocapsules (NANs) are nucleic acid nanostructures that radially display oligonucleotides on the surface of cross-linked surfactant micelles. Their chemical makeup affords the stimuli-responsive release of therapeutically active DNA-surfactant conjugates into the cells. While NANs have so far demonstrated the effective cytosolic delivery of their nucleic acid cargo, as seen indirectly by their gene regulation capabilities, there remain gaps in the molecular understanding of how this process happens. Herein, we examine the enzymatic degradation of NANs and confirm the identity of the DNA-surfactant conjugates formed by using mass spectrometry (MS). With surface enhanced (resonance) Raman spectroscopy (SE(R)RS), we also provide evidence that the energy-independent translocation of such DNA-surfactant conjugates is contingent upon their release from the NAN structure, which, when intact, otherwise buries the hydrophobic surfactant tail in its interior. Such information suggests a critical role of the surfactant in the lipid disruption capability of the DNA surfactant conjugates generated from degradation of the NANs. Using NANs made with different tail lengths (C12 and C10), we show that this mechanism likely holds true despite significant differences in the physical properties (i.e., critical micelle concentration (CMC), surfactants per micelle, Nagg) of the resultant particles (C12 and C10 NANs). While the total cellular uptake efficiencies of C12 and C10 NANs are similar, there were differences observed in their cellular distribution and localized trafficking, even after ensuring that the total concentration of DNA was the same for both particles. Ultimately, C10 NANs appeared less diffuse within cells and colocalized less with lysosomes over time, achieving more significant knockdown of the target gene investigated, suggesting more effective endosomal escape. These differences indicate that the surfactant assembly and disassembly properties, including the number of surfactants per particle and the CMC can have important implications for the cellular delivery efficacy of DNA micelles and surfactant conjugates.
Bound Water Enhances the Ion Selectivity of Highly Charged Polymer Membranes.
Espinoza C, Díaz JC, Kitto D, Kim HK, Kamcev J.
ACS Appl Mater Interfaces. 2024 Aug 13.
doi: 10.1021/acsami.4c07516. Epub ahead of print.
PMID: 39136307.
Electrochemical technologies for water treatment, resource recovery, energy generation, and energy storage rely on charged polymer membranes to selectively transport ions. With the rise of applications involving hypersaline brines, such as management of desalination brine or the recovery of ions from brines, there is an urgent need for membranes that can sustain high conductivity and selectivity under such challenging conditions. Current membranes are constrained by an inherent trade-off between conductivity and selectivity, alongside concerns regarding their high costs. Moreover, a gap in the fundamental understanding of ion transport within charged membranes at high salinities prevents the development of membranes that could meet these stringent requirements efficiently. Here, we present the synthesis of scalable, highly charged membranes that demonstrate high conductivity and selectivity while contacting 1 and 5 molal NaCl solutions. A detailed analysis of the membrane transport properties reveals that the high proportion of bound water in the membranes, enabled by the high charge content and hydrophilic structure of the polymers, enhances both the ion partitioning and diffusion selectivities of the membranes. These structure/property relationships derived from this study offer valuable guidance for designing next-generation membranes that simultaneously achieve exceptional conductivity and selectivity in high-salinity conditions.
Molecules
Illuminating the monoamine transporters: Fluorescently labelled ligands to study dopamine, serotonin and norepinephrine transporters.
Camacho-Hernandez GA, Jahan K, Newman AH.
Basic Clin Pharmacol Toxicol. 2023 Nov;133(5):473-484.
doi: 10.1111/bcpt.13827. Epub 2023 Jan 7.
PMID: 36527444.
Fluorescence microscopy has revolutionized the visualization of physiological processes in live-cell systems. With the recent innovations in super resolution microscopy, these events can be examined with high precision and accuracy. The development of fluorescently labelled small molecules has provided a significant advance in understanding the physiological relevance of targeted proteins that can now be visualized at the cellular level. One set of physiologically important target proteins are the monoamine transporters (MATs) that play an instrumental role in maintaining monoamine signalling homeostasis. Understanding the mechanisms underlying their regulation and dysregulation is fundamental to treating several neuropsychiatric conditions such as attention deficit hyperactivity disorder (ADHD), anxiety, depression and substance use disorders. Herein, we describe the rationale behind the small molecule design of fluorescently labelled ligands (FLL) either as MAT substrates or inhibitors as well as their applications to advance our understanding of this class of transporters in health and disease.
Pyrazolone-Protein Interaction Enables Long-Term Retention Staining and Facile Artificial Biorecognition on Cell Membranes.
Xiong T, Chen Y, Peng Q, Li M, Lu S, Chen X, Fan J, Wang L, Peng X.
J Am Chem Soc. 2024 Aug 13.
doi: 10.1021/jacs.4c08987. Epub ahead of print.
PMID: 39138141.
Cell membrane genetic engineering has been utilized to confer cell membranes with functionalities for diagnostic and therapeutic purposes but concerns over cost and variable modification results. Although nongenetic chemical modification and phospholipid insertion strategies are more convenient, they still face bottlenecks in either biosafety or stability of the modifications. Herein, we show that pyrazolone-bearing molecules can bind to proteins with high stability, which is mainly contributed to by the multiple interactions between pyrazolone and basic amino acids. This new binding model offers a simple and versatile noncovalent approach for cell membrane functionalization. By binding to cell membrane proteins, pyrazolone-bearing dyes enabled precise cell tracking in vitro (>96 h) and in vivo (>21 days) without interfering with the protein function or causing cell death. Furthermore, the convenient anchor of pyrazolone-bearing biotin on cell membranes rendered the biorecognition to avidin, showing the potential for artificially creating cell targetability.
Methods
Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids.
Jann C, Giofré S, Bhattacharjee R, Lemke EA.
Chem Rev. 2024 Aug 9.
doi: 10.1021/acs.chemrev.3c00878. Epub ahead of print.
PMID: 39120726.
Over 500 natural and synthetic amino acids have been genetically encoded in the last two decades. Incorporating these noncanonical amino acids into proteins enables many powerful applications, ranging from basic research to biotechnology, materials science, and medicine. However, major challenges remain to unleash the full potential of genetic code expansion across disciplines. Here, we provide an overview of diverse genetic code expansion methodologies and systems and their final applications in prokaryotes and eukaryotes, represented by Escherichia coli and mammalian cells as the main workhorse model systems. We highlight the power of how new technologies can be first established in simple and then transferred to more complex systems. For example, whole-genome engineering provides an excellent platform in bacteria for enabling transcript-specific genetic code expansion without off-targets in the transcriptome. In contrast, the complexity of a eukaryotic cell poses challenges that require entirely new approaches, such as striving toward establishing novel base pairs or generating orthogonally translating organelles within living cells. We connect the milestones in expanding the genetic code of living cells for encoding novel chemical functionalities to the most recent scientific discoveries, from optimizing the physicochemical properties of noncanonical amino acids to the technological advancements for their in vivo incorporation. This journey offers a glimpse into the promising developments in the years to come.
Reconstitution of the Melibiose Permease of Salmonella enterica serovar Typhimurium (MelBSt) into Lipid Nanodiscs.
Hariharan P, Guan L.
Bio Protoc. 2024 Aug 5;14(15):e5045.
doi: 10.21769/BioProtoc.5045.
PMID: 39131193.
Membrane proteins play critical roles in cell physiology and pathology. The conventional way to study membrane proteins at protein levels is to use optimal detergents to extract proteins from membranes. Identification of the optimal detergent is tedious , and in some cases, the protein functions are compromised. While this detergent-based approach has produced meaningful results in membrane protein research, a lipid environment should be more suitable to recapture the protein’s native folding and functions. This protocol describes how to prepare amphipathic membrane scaffold-proteins (MSPs)-based nanodiscs of a cation-coupled melibiose symporter of Salmonella enterica serovar Typhimurium (MelBSt), a member of the major facilitator superfamily. MSPs generate nano-assemblies containing membrane proteins surrounded by a patch of native lipids to better preserve their native conformations and functions. This protocol requires purified membrane protein in detergents, purified MSPs in solution, and detergent-destabilized phospholipids. The mixture of all three components at specific ratios is incubated in the presence of Bio-Beads SM-2 resins, which absorb all detergent molecules, allowing the membrane protein to associate with lipids surrounded by the MSPs. By reconstituting the purified membrane proteins back into their native-like lipid environment, these nanodisc-like particles can be directly used in cryo-EM single-particle analysis for structure determination and other biophysical analyses. It is noted that nanodiscs may potentially limit the dynamics of membrane proteins due to suboptimal nanodisc size compared to the native lipid bilayer. Key features • This protocol was built based on the method originally developed by Sligar et al. [1] and modified for a specific major facilitator superfamily transporter • This protocol is robust and reproducible • Lipid nanodiscs can increase membrane protein stability, and reconstituted transporters in lipid nanodiscs can regain function if their function is compromised using detergents • The reconstituted lipids nanodisc can be used for cryo-EM single-particle analysis.
Microbio
Investigating bottom phase extraction from aqueous two-phase systems for detecting bacteria using the lateral-flow immunoassay.
Luu AP, Rao SS, Malik HY, Shi RB, Toubian AA, Kamei DT.
Anal Biochem. 2024 Jul 31;694:115634.
doi: 10.1016/j.ab.2024.115634. Epub ahead of print.
PMID: 39094782.
Lateral-flow immunoassays (LFAs) can be used to diagnose urinary tract infections caused by Escherichia coli (E. coli) at the point of care. Unfortunately, urine samples containing dilute concentrations of E. coli can yield false negative results on LFAs. Our laboratory was first to implement aqueous two-phase systems (ATPSs) to preconcentrate samples into smaller volumes prior to their application on LFAs. This is achieved by manipulating the ratio of the volume of the top phase to that of the bottom phase (volume ratio; VR) and concentrating biomarkers in the bottom phase which, when applied to LFAs in fixed volumes, leads to corresponding improvements in sensitivity. This work is the first demonstration that the same LOD can be achieved irrespective of the VR when the entire bottom phase is added to LFAs. A custom 3D-printed device was also developed to decrease liquid handling steps. Across different VRs expected from patient urine variability, this diagnostic workflow successfully detected E. coli concentrations down to 2 × 105 colony-forming units (cfu) mL-1 in synthetic urine, demonstrating consistent 10-fold improvements in sensitivity compared to trials conducted without ATPS preconcentration. This method successfully addresses the variability of patient samples while remaining easy to use at the point of care.
In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli.
Srivastava R, González-Prieto C, Lynch JP, Muscolo M, Lin CY, Brown MA, Lemos L, Shrestha A, Osburne MS, Leong JM, Lesser CF.
bioRxiv [Preprint]. 2024 Jul 30:2024.07.30.605899.
doi: 10.1101/2024.07.30.605899.
PMID: 39131305.
Engineered smart microbes that secrete therapeutics are emerging as treatment modalities, particularly for gut-based diseases. With the growing threat of multidrug-resistant infection, non-antibiotic treatments are urgently needed. The gastrointestinal pathogen enterohemorrhagic E coli (EHEC) can cause the potentially lethal hemolytic uremic syndrome, a toxin-driven disease. Given concerns that antibiotics increase toxin release, treatment is largely limited to supportive care. Here, we show that pre-treatment with a commensal E. coli (HS-PROT 3 EcT) engineered to secrete an antibody that blocks an essential EHEC virulence factor delays the establishment of an EHEC-like infection in mice. This study strongly suggests that smart microbes that deliver payloads that block colonization factors of gut pathogens can be developed as critically needed alternatives to antibiotics for fighting bacterial infections.
Miscellaneous
From Vikings to Beethoven: what your DNA says about your ancient relatives.
Callaway E.
Nature. 2024 Aug;632(8024):246-249.
doi: 10.1038/d41586-024-02536-w.
PMID: 39112623.
Scientists are using consumer-genomics databases to link living people to ancestors from the recent and not-so-recent past. But the meaning of these connections isn’t always clear.
Scientists are falling victim to deepfake AI video scams – here’s how to fight back.
Nordling L.
Nature. 2024 Aug 7.
doi: 10.1038/d41586-024-02521-3. Epub ahead of print.
PMID: 39112581.
Bad actors are snatching videos of researchers and creating deepfakes that hawk fake drugs using their likenesses. These AI-created videos are becoming easier to make and pose significant reputational risks to scientists, not to mention the potential harm to impressionable viewers. “To spread misinformation, you want to manipulate what people think are the trusted sources,” says educational policy expert Christopher Doss. Scientists affected should contact the platform hosting the deepfake and tell their employers and professional societies. They can also consider legal action or police involvement, although prosecuting perpetrators has proved challenging.
Slow productivity worked for Marie Curie — here’s why you should adopt it, too.
Gulland A.
Nature. 2024 Aug;632(8024):461-463.
doi: 10.1038/d41586-024-02540-0. PMID: 39103528.
“Do fewer things. Work at a natural pace. Obsess over quality.”
In his new book Slow Productivity, computer scientist and time-management guru Cal Newport urges us to learn from scientists such as Marie Curie — whose work straddled decades and involved periods of rest and relaxation.
Your microwave oven has its own microbiome.
Soliman A.
Nature. 2024 Aug 8.
doi: 10.1038/d41586-024-02553-9. Epub ahead of print.
PMID: 39112590.
Your microwave oven probably has its own microbiome — challenging the common misconception that microwaves make food safe by wiping out bacteria. Microbiologists swabbed 30 microwave ovens, and discovered 101 bacterial strains. Most were bacteria found on human skin, but some were associated with food-borne illnesses. In laboratory microwave ovens, researchers found ‘extremophiles’ that can withstand high radiation and high temperatures. The team suggests that these strains might have been ‘selected’ evolutionarily by surviving repeated rounds of radiation, and could turn out to be useful — for the bioremediation of toxic waste, for example.
Stonehenge’s enigmatic centre stone was hauled 800 kilometres from Scotland.
Gaind N, Smith R.
Nature. 2024 Aug;632(8025):484-485.
doi: 10.1038/d41586-024-02584-2.
PMID: 39143344.
Chemical analysis of tiny crystals in Stonehenge’s central Altar Stone suggest that it came from Scotland — and not from Wales as previously thought. The fingerprint of zircon, rutile and apatite crystals in the stone allowed it to be traced to the Orcadian Basin, which includes parts of northeastern Scotland and the Orkney Islands. Whether the six-tonne monolith was transported by land or by sea is hotly debated.
One-quarter of unresponsive people with brain injuries are conscious.
Nowogrodzki J.
Nature. 2024 Aug 14.
doi: 10.1038/d41586-024-02614-z. Epub ahead of print.
PMID: 39147810.
A study of around 240 people with brain injuries who are physically unresponsive found that one quarter were actually conscious. People were given a brain scan and asked to imagine themselves playing tennis, or opening and closing their hand — 25% showed brain activity that indicated they could consistently respond to commands mentally. The responsive group tended to be younger, to have injuries that were from physical trauma and to have been living with their injuries for longer than the others. The actual proportion of people experiencing this state of ‘cognitive motor dissociation’ is likely to be even higher than measured by the test, says neurologist and study leader Nicholas Schiff. “I’ve been in the MRI, and I’ve done this experiment, and it’s hard.”
The Taliban said women could study – three years on they still can’t.
Naddaf M.
Nature. 2024 Aug 14.
doi: 10.1038/d41586-024-02626-9. Epub ahead of print.
PMID: 39147811.
Three years ago, just days after their takeover of Afghanistan, a Taliban spokesman said: “We are going to allow women to work and study…. Women will be very active in society, but within the framework of Islam.” Yet since December 2022, girls over the age of 12 and women in Afghanistan have been banned from schools and universities, and they are unlikely to return anytime soon. “Three years are a lot in education — it’s almost a bachelor’s degree,” says one Afghan computer scientist, who now lives in Europe.”
Chatbots in science: What can ChatGPT do for you?
Pividori M.
Nature. 2024 Aug 14.
doi: 10.1038/d41586-024-02630-z. Epub ahead of print.
PMID: 39147814.
Biomedical-informatics researcher Milton Pividori’s laboratory spent 18 months studying how best to incorporate ChatGPT into scientific work. This is what they learnt: Engineer your prompt — there are ‘cheat sheets’ to help with this. Target tasks that are more mechanical — and usually, boring — and keep creative work for humans. Using a chatbot to write is less risky than asking it to read: it’s easier to catch ‘hallucinations’ in text you’re editing than to know when a summary contains inaccuracies. A key point, especially for trainees. “If you don’t know how to do something, I strongly discourage you from using a chatbot to do it for you,” writes Pividori.
A.L.S. Stole His Voice. A.I. Retrieved it.
A version of this article appears in print on Aug. 15, 2024, Section A, Page 16 of the New York edition with the headline: An Insidious Disease Stole His Voice, but A.I. Helped to Retrieve It.
https://www.nytimes.com/2024/08/14/health/als-ai-brain-implants.html?smid=url-share
“What good is that?” One of the first sentences read aloud by an AI-powered brain implant system for Casey Harrell — an experience that reduced him to tears. Harrell’s speech has been affected by amyotrophic lateral sclerosis, and the system interprets his vocalizations and brain activity, and talks with his own voice.