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At least 793 records · Page 44

Salt Stress Induces Paramylon Accumulation and Fine-Tuning of the Macro-Organization of Thylakoid Membranes in Euglena gracilis Cells

The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR), and energy transfer between the photosystems did not change considerably in the salt-grown cells. We have observed notable increases in the carotenoid-to-Chl ratio and the accumulation of paramylon in the salt-treated cells. We propose that the accumulation of storage polysaccharides and changes in the pigment composition and thylakoid membrane organization help the adaptation of E. gracilis cells to salt stress and contribute to the maintenance of cellular processes under stress conditions.

59 BASIC BIOLOGICAL SCIENCES↗

Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.

59 BASIC BIOLOGICAL SCIENCES↗

Regioselectivity mechanism of the Thunbergia alata Δ 6 -16:0-acyl carrier protein desaturase

Plant plastidial acyl–acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ 9 cis )-ACP. Several variants expressing distinct regioselectivity have been described including a Δ 6 -16:0-ACP desaturase from black-eyed Susan vine ( Thunbergia alata ). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ 6 desaturase to Δ 9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. Furthermore, to validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ 6 to Δ 9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ 9 to Δ 6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ 6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications.

59 BASIC BIOLOGICAL SCIENCES↗

Physiological and metabolic responses of Zymomonas mobilis to lignocellulosic hydrolysate

Zymomonas mobilis is a promising biocatalyst for the sustainable conversion of lignocellulosic sugars into biofuels and bioproducts, yet its response to lignocellulosic hydrolysates remains poorly understood. Here, we investigate the physiological response of Z. mobilis to ammonia fiber expansion (AFEX)-pretreated switchgrass hydrolysate using a systems-level approach integrating LC–MS/MS-based lipidomics and shotgun proteomics. Growth on hydrolysate induced substantial shifts in fatty acid and membrane phospholipid composition, alongside broad proteomic remodeling. Notably, Z. mobilis exhibited a stress response characterized by the upregulation of heat shock proteins and efflux transporters and the downregulation of cell motility proteins. Unexpectedly, hydrolysate exposure also led to a robust upregulation of the Entner–Doudoroff pathway, the ethanol fermentation pathway, and other central carbon metabolism enzymes, indicating a substantial cellular investment potentially driven by additional nutrient availability in hydrolysate. These findings provide new insights into the metabolic adaptations of Z. mobilis to lignocellulosic hydrolysates, informing strategies to enhance its biofuel production capabilities.

Zymomonas mobilis↗

Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

Grant, Michael A.↗

Identification of sucrose synthase as an actin-binding protein

Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

Non-NASA Center↗

The Importance of Sample Return in Establishing Chemical Evidence for Life on Mars or Other Solar System Bodies

The search for evidence of life on Mars and elsewhere will continue to be one of the primary goals of NASA s robotic exploration program over the next decade. NASA and ESA are currently planning a series of robotic missions to Mars with the goal of understanding its climate, resources, and potential for harboring past or present life. One key goal will be the search for chemical biomarkers including complex organic compounds important in life on Earth. These include amino acids, the monomer building blocks of proteins and enzymes, nucleobases and sugars which form the backbone of DNA and RNA, and lipids, the structural components of cell membranes. Many of these organic compounds can also be formed abiotically as demonstrated by their prevalence in carbonaceous meteorites [1], though, their molecular characteristics may distinguish a biological source [2]. It is possible that in situ instruments may reveal such characteristics, however, return of the right sample (i.e. one with biosignatures or having a high probability of biosignatures) to Earth would allow for more intensive laboratory studies using a broad array of powerful instrumentation for bulk characterization, molecular detection, isotopic and enantiomeric compositions, and spatially resolved chemistry that may be required for confirmation of extant or extinct Martian life. Here we will discuss the current analytical capabilities and strategies for the detection of organics on the Mars Science Laboratory (MSL) using the Sample Analysis at Mars (SAM) instrument suite and how sample return missions from Mars and other targets of astrobiological interest will help advance our understanding of chemical biosignatures in the solar system.

Glavin, D. P.↗

EMC3 regulates trafficking and pulmonary toxicity of the SFTPC I73T mutation associated with interstitial lung disease

The most common mutation in surfactant protein C gene (SFTPC), SFTPC I73T , causes interstitial lung disease with few therapeutic options. We previously demonstrated that EMC3, an important component of the multiprotein endoplasmic reticulum membrane complex (EMC), is required for surfactant homeostasis in alveolar type 2 epithelial (AT2) cells at birth. In the present study, we investigated the role of EMC3 in the control of SFTPC I73T metabolism and its associated alveolar dysfunction. Using a knock-in mouse model phenocopying the I73T mutation, we demonstrated that conditional deletion of Emc3 in AT2 cells rescued alveolar remodeling/simplification defects in neonatal and adult mice. Proteomic analysis revealed that Emc3 depletion reversed the disruption of vesicle trafficking pathways and rescued the mitochondrial dysfunction associated with I73T mutation. Affinity mass spectrometry analysis identified potential EMC3 interacting proteins in lung AT2 cells, including Valosin Containing Protein (VCP) and its interactors. Treatment of Sftpc I73T knock-in mice and SFTPC I73T expressing iAT2 cells derived from SFTPC I73T patient-specific iPSCs with the specific VCP inhibitor CB5083 restored alveolar structure and SFTPC I73T trafficking respectively. Taken together, the present work identifies the EMC complex and VCP in the metabolism of the disease-associated SFTPC I73T mutant, providing novel therapeutical targets for SFTPC I73T -associated interstitial lung disease.

60 APPLIED LIFE SCIENCES↗

Elucidation of complex respiratory chains: a straightforward strategy to monitor electron transfer between cytochromes

Abstract Cytochromes are electron transfer (ET) proteins essential in various biological systems, playing crucial roles in the respiratory chains of bacteria. These proteins are particularly abundant in electrogenic microorganisms and are responsible for the efficient delivery of electrons to the cells’ exterior. The capability of sending electrons outside the cells open new avenues to be explored for emerging biotechnological applications in bioremediation, microbial electrosynthesis, and bioenergy fields. To develop these applications, it is critical to identify the different redox partners and to elucidate the stepwise ET along the respiratory paths. However, investigating direct ET events between proteins with identical features in nearly all spectroscopic techniques is extremely challenging. Nuclear magnetic resonance (NMR) spectroscopy offers the possibility to overcome this difficulty by analysing the alterations of the spectral signatures of each protein caused by electron exchange events. The uncrowded NMR spectral regions containing the heme resonances of the cytochromes display unique and distinct signatures in the reduced and oxidized states, which can be explored to monitor ET within the redox complex. In this study, we present a strategy for a fast and straightforward monitorization of ET between c-type cytochromes, using as model a triheme periplasmic cytochrome and a membrane-associated monoheme cytochrome from the electrogenic bacterium Geobacter sulfurreducens. The comparison between the 1D 1H NMR spectra obtained for samples containing the two cytochromes and for samples containing the individual proteins clearly demonstrated a unidirectional ET within the redox complex. This strategy provides a simple and straightforward means to elucidate complex biologic respiratory ET chains.

Morgado, Leonor (ORCID:0000000237605180)↗

Arabidopsis cytochrome b 5 proteins support fatty acid ω-3 but not ω-6 desaturation

Fatty acids are primary components of lipids, which serve as major energy sources in cells and play essential roles in membrane structure, signaling, and metabolic regulation (Shanklin and Cahoon 1998). The degree of fatty acid unsaturation critically influences lipid physicochemical properties, thereby affecting membrane fluidity and biological function (Nguyen et al. 2019). In Arabidopsis thaliana, fatty acid desaturation occurs via 2 parallel pathways: the “prokaryotic pathway” in plastids, involving glycosylglycerides, such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and phospholipid phosphatidylglycerol (PG); and the “eukaryotic pathway” in the endoplasmic reticulum (ER), involving phosphatidylcholine (PC) (Lou et al. 2014) (Supplementary Figure S1). Seven fatty acid desaturases (FADs) in Arabidopsis differentially desaturate each glycerolipid class in the plastid and ER (Nguyen et al. 2019). FAD2, an ER-resident ω-6 fatty acid desaturase, catalyzes the conversion of oleic acid (18:1) to linoleic acid (18:2), which can be further desaturated to α-linolenic acid (18:3) by FAD3, an ER-resident ω-3 fatty acid desaturase. In plastids, FAD6 catalyzes the desaturation of 18:1/16:1 to produce 18:2/16:2, while FAD7 and FAD8 redundantly convert 18:2/16:2 to 18:3/16:3 (Li-Beisson et al. 2013; Nguyen et al. 2019). Additionally, fatty acids synthesized in the ER can also be reimported into plastids to their site of de novo synthesis (Xu et al. 2010). All FADs require reducing power, in the form of 2 electrons, for catalysis, but the sources of the electrons vary between their subcellular localizations. In the ER, FAD2 and FAD3 receive electrons from a cytochrome b 5 (CB5)-based electron transfer chain comprising cytochrome b 5 reductase (CBR) and CB5. In contrast, ferredoxin serves as the electron donor for plastid-localized FAD6, FAD7, and FAD8 (Ohlrogge and Browse 1995; Andreu et al. 2007). While the relative contributions of the 2 pathways to total cellular desaturation products vary across tissues and species, most polyunsaturated FA biosynthesis in seeds occurs via ER-resident FAD2 and FAD3 (Miquel and Browse 1992; Ohlrogge and Browse 1995).

59 BASIC BIOLOGICAL SCIENCES↗

Single-Molecule Detection of the Encounter and Productive Electron Transfer Complexes of a Photosynthetic Reaction Center

Small, diffusible redox proteins play an essential role in electron transfer (ET) in respiration and photosynthesis, sustaining life on Earth by shuttling electrons between membrane-bound complexes via finely tuned and reversible interactions. Ensemble kinetic studies show transient ET complexes form in two distinct stages: an “encounter” complex largely mediated by electrostatic interactions, which subsequently, through subtle reorganization of the binding interface, forms a “productive” ET complex stabilized by additional hydrophobic interactions around the redox-active cofactors. Here, using single-molecule force spectroscopy (SMFS) we dissected the transient ET complexes formed between the photosynthetic reaction center-light harvesting complex 1 (RC-LH1) of Rhodobacter sphaeroides and its native electron donor cytochrome c 2 (cyt c 2 ). Importantly, SMFS resolves the distribution of interaction forces into low (~150 pN) and high (~330 pN) components, with the former more susceptible to salt concentration and to alteration of key charged residues on the RC. Thus, the low force component is suggested to reflect the contribution of electrostatic interactions in forming the initial encounter complex, whereas the high force component reflects the additional stabilization provided by hydrophobic interactions to the productive ET complex. Employing molecular dynamics simulations, we resolve five intermediate states that comprise the encounter, productive ET and leaving complexes, predicting a weak interaction between cyt c 2 and the LH1 ring near the RC-L subunit that could lie along the exit path for oxidized cyt c 2 . The multimodal nature of the interactions of ET complexes captured here may have wider implications for ET in all domains of life.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

The microbiome of a bacterivorous marine choanoflagellate contains a resource-demanding obligate bacterial associate

Abstract Microbial predators such as choanoflagellates are key players in ocean food webs. Choanoflagellates, which are the closest unicellular relatives of animals, consume bacteria and also exhibit marked biological transitions triggered by bacterial compounds, yet their native microbiomes remain uncharacterized. Here we report the discovery of a ubiquitous, uncultured bacterial lineage we name Candidatus Comchoanobacterales ord. nov., related to the human pathogen Coxiella and physically associated with the uncultured marine choanoflagellate Bicosta minor . We analyse complete ‘Comchoano’ genomes acquired after sorting single Bicosta cells, finding signatures of obligate host-dependence, including reduction of pathways encoding glycolysis, membrane components, amino acids and B-vitamins. Comchoano encode the necessary apparatus to import energy and other compounds from the host, proteins for host-cell associations and a type IV secretion system closest to Coxiella’s that is expressed in Pacific Ocean metatranscriptomes. Interactions between choanoflagellates and their microbiota could reshape the direction of energy and resource flow attributed to microbial predators, adding complexity and nuance to marine food webs.

59 BASIC BIOLOGICAL SCIENCES↗

A miniature CRISPR–Cas10 enzyme confers immunity by inhibitory signalling

Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signalling that share mechanistic features with human antiviral systems1. In these pathways, including cyclic oligonucleotide-based antiphage signalling systems (CBASSs) and type III CRISPR systems in bacteria and cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS–STING) in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response2, 3–4. Here, in an unexpected inversion of this process, we show that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that represses a toxic effector. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing the spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TMβ. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TMβ-mediated cell death due to inner membrane collapse. These findings unveil a powerful defence strategy against virus-mediated immune suppression, expanding our understanding of the role of oligonucleotides in immunity.

Doherty, Erin E↗

Early Diagnosis of Fibromyalgia Using Surface-Enhanced Raman Spectroscopy Combined with Chemometrics

Fibromyalgia (FM) is a chronic muscle pain disorder that shares several clinical features with other related rheumatologic disorders. This study investigates the feasibility of using surface-enhanced Raman spectroscopy (SERS) with gold nanoparticles (AuNPs) as a fingerprinting approach to diagnose FM and other rheumatic diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), osteoarthritis (OA), and chronic low back pain (CLBP). Blood samples were obtained on protein saver cards from FM (n = 83), non-FM (n = 54), and healthy (NC, n = 9) subjects. A semi-permeable membrane filtration method was used to obtain low-molecular-weight fraction (LMF) serum of the blood samples. SERS measurement conditions were standardized to enhance the LMF signal. An OPLS-DA algorithm created using the spectral region 750 to 1720 cm -1 enabled the classification of the spectra into their corresponding FM and non-FM classes (Rcv > 0.99) with 100% accuracy, sensitivity, and specificity. The OPLS-DA regression plot indicated that spectral regions associated with amino acids were responsible for discrimination patterns and can be potentially used as spectral biomarkers to differentiate FM and other rheumatic diseases. This exploratory work suggests that the AuNP SERS method in combination with OPLS-DA analysis has great potential for the label-free diagnosis of FM.

60 APPLIED LIFE SCIENCES↗

EphB2 guides axons at the midline and is necessary for normal vestibular function

Mice lacking the EphB2 receptor tyrosine kinase display a cell-autonomous, strain-specific circling behavior that is associated with vestibular phenotypes. In mutant embryos, the contralateral inner ear efferent growth cones exhibit inappropriate pathway selection at the midline, while in mutant adults, the endolymph-filled lumen of the semicircular canals is severely reduced. EphB2 is expressed in the endolymph-producing dark cells in the inner ear epithelium, and these cells show ultrastructural defects in the mutants. A molecular link to fluid regulation is provided by demonstrating that PDZ domain-containing proteins that bind the C termini of EphB2 and B-ephrins can also recognize the cytoplasmic tails of anion exchangers and aquaporins. This suggests EphB2 may regulate ionic homeostasis and endolymph fluid production through macromolecular associations with membrane channels that transport chloride, bicarbonate, and water.

Non-NASA Center↗

Using Domain Insertion to Create Sulfite Reductases That Present Chemical-Dependent Activities

Domain insertion can be used to create oxidoreductases whose activities are dependent upon analyte binding. To date, most domain insertion studies have targeted relatively small oxidoreductases of known structure, so it remains unclear how to apply this protein engineering approach to large hetero-oligomeric proteins that require dynamic conformational changes for catalysis. To address this question, we studied the effects of peptide and domain insertions on the activity of NADPH-dependent sulfite reductase (SiR) from Escherichia coli, a dodecameric oxidoreductase containing four hemoprotein and eight flavoprotein subunits. SiR mutational tolerance was first evaluated using systematic octapeptide insertion and a cellular selection, which identified regions across the hemoprotein structure that retain parent-like activity following insertion. When a ligand-binding domain was inserted at backbone locations tolerant to peptide insertion, including sites proximal and distal from the intersubunit interfaces, ∼90% retained catalytic activity, and >50% presented activity that is regulated by an endocrine disruptor. With one domain insertion variant, the conditional production of sulfide could be monitored electrochemically from cells using a bioelectrochemical reactor. These results show how systematic peptide insertion can be used to inform domain insertion in a large heterooligomeric protein complex, and they illustrate how SiR can be engineered to convert chemical information in the environment into a redox-active metabolite that diffuses across the cell membrane.

bacteria↗

Lipid sponge droplets as programmable synthetic organelles

Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here, we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and nonionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the colocalization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems.

59 BASIC BIOLOGICAL SCIENCES↗

Stromules, functional extensions of plastids within the plant cell

We report stromules are thin tubular extensions of the plastid compartment surrounded by the envelope membrane. A myriad of functions have been proposed for them, and they likely have multiple roles. Recent work has illuminated aspects of their formation, especially the important of microtubules in their movement and microfilaments in anchoring. A variety of biotic and abiotic stresses result in induction of stromule formation, and in recent years, stromule formation has been strongly implicated as part of the innate immune response. Both stromules and chloroplasts relocate to surround the nucleus when pathogens are sensed, possibly to supply signaling molecules such as reactive oxygen species. In addition to the nucleus, stromules have been observed in close proximity to other compartments such as mitochondria, endoplasmic reticulum, and the plasma membrane, potentially facilitating exchange of substrates and products to carry out important biosynthetic pathways. Much remains to be learned about the identity of proteins and other molecules released from chloroplasts and stromules and how they function in plant development and defense.

60 APPLIED LIFE SCIENCES↗