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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 577 records · Page 32
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Cristae formation is a mechanical buckling event controlled by the inner mitochondrial membrane lipidome

Cristae are high–curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae–shaping proteins have been defined, analogous lipid–based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi–scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein–mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low–oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

Extremophiles in astrobiology: per Ardua ad Astra

As we consider the possibilities of finding life on other planets, it behooves us to evaluate what we know about the limits for life on planet Earth. In our continued exploration of Earth, we are finding microbes in a variety of unexpected habitats. In geothermal hot springs, we have discovered organisms thriving at temperatures near the boiling point of water and at pH values down to 0.5; in the deepest parts of the oceans, those that grow optimally at pressures above 1000 bars and die at pressures below 500 bars; and at the poles, those that grow below the freezing point of water and die at temperatures above 10 degrees C. All of these organisms are living proof that the biochemical "machinery" of life can be adapted to conditions that, from our anthropocentric perspective, appear to be extreme. By studying the molecular adaptations of extremophiles, we begin to identify the critical cellular components that expand the envelope for life. As an example, I will discuss what we have learned about the role of the proteins we call "heat shock proteins" in pushing the upper temperature limit of life and how our studies have provided a new perspective on the function of these proteins.

Chaperonin 60/physiology↗
DOE OSTI2024

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16 + NK cell activation

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16 + NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16 + NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1995

The statolith compartment in Chara rhizoids contains carbohydrate and protein

In contrast to higher plants, the alga Chara has rhizoids with single membrane-bound compartments that function as statoliths in gravity perception. Previous work has demonstrated that these statoliths contain barium sulfate crystals. In this study, we show that statoliths in Chara rhizoids react with a Coomassie Brilliant Blue cytochemical stain for proteins. While statoliths did not react with silver methenamine carbohydrate cytochemistry, the monoclonal antibody CCRC-M2, which is against a carbohydrate (sycamore-maple rhamnogalacturonan I), labeled the statolith compartment. These results demonstrate that in addition to barium sulfate, statoliths in Chara rhizoids have an organic matrix that consists of protein and carbohydrate moieties. Since the statoliths were silver methenamine negative, the carbohydrate in this compartment could be a 3-linked polysaccharide. CCRC-M2 also labeled Golgi cisternae, Golgi-associated vesicles, apical vesicles, and cell walls in the rhizoids. The specificity of CCRC-M2 immunolabeling was verified by several control experiments, including the demonstration that labeling was abolished when the antibody was preabsorbed with its antigen. Since in this and a previous study (John Z. Kiss and L. Andrew Staehelin, American Journal of Botany 80: 273-282, 1993) antibodies against higher plant carbohydrates crossreacted with cell walls of Chara in a specific manner, Characean algae may be a useful model system in biochemical and molecular studies of cell walls.

NASA Discipline Plant Biology↗
DOE OSTI2020

Atypical chemoreceptor arrays accommodate high membrane curvature

The prokaryotic chemotaxis system is arguably the best-understood signaling pathway in biology. In all previously described species, chemoreceptors organize into a hexagonal (P6 symmetry) extended array. Here, we report an alternative symmetry (P2) of the chemotaxis apparatus that emerges from a strict linear organization of the histidine kinase CheA in Treponema denticola cells, which possesses arrays with the highest native curvature investigated thus far. Using cryo-ET, we reveal that Td chemoreceptor arrays assume an unusual arrangement of the supra-molecular protein assembly that has likely evolved to accommodate the high membrane curvature. The arrays have several atypical features, such as an extended dimerization domain of CheA and a variant CheW-CheR-like fusion protein that is critical for maintaining an ordered chemosensory apparatus. Furthermore, the previously characterized Td oxygen sensor ODP influences CheA ordering. These results suggest a greater diversity of the chemotaxis signaling system than previously thought.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

The Molecular Basis of Hyperthermophily: The Role of HSP60/Chaperonins In Vivo

In this study, we aim to understand how S. shibatae copes with high temperatures. In particular, we investigated the role of the 60 kDa heat shock protein (HSP60 or chaperonin) with the hypothesis that chaperonin stabilizes the cell membrane under stressful conditions. To prove the hypothesis, this year two questions were addressed: (1) Is the chaperonin localized in the cytoplasm or on the cell membrane? (2) Does the chaperonin show affinity to lipid in vivo? In addition to those, we intensively studied newly discovered chaperonin-related protein, gamma, to understand how it influenced the function of the other components of chaperonin and how their combined activities contributed to hyperthermophily.

Kagawa, Hiromi↗
NASA NTRS1998

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Non-NASA Center↗
DOE OSTI2020

Engineered bacterial microcompartments: apps for programming metabolism

Bacterial Microcompartments (BMCs) are used by diverse bacteria to compartmentalize enzymatic reactions, functioning analogously to the organelles of eukaryotes. The bounding membrane and encapsulated components are composed entirely of protein, which makes them ideal targets for modification by genetic engineering. In contrast to viruses, in which generally only one protein forms the capsid, the shells of BMCs consist of a variety of shell proteins, each a potential unit of selection. Despite their differences in permeability, the shell proteins are surprisingly interchangeable. Recent developments have shown that they are also highly amenable to engineered modifications which poise them for a variety of biotechnological applications. Additionally, given their modular structure, with a module defined as a semi-autonomous functional unit, BMCs can be considered apps for programming metabolism that can be de-bugged by adaptive evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Mechanism of tethered agonist-mediated signaling by polycystin-1

Mutations of polycystin-1 (PC1) are the major cause (85% of cases) of autosomal dominant polycystic kidney disease (ADPKD), which is the fourth leading cause of kidney failure. PC1 is thought to function as an atypical G protein-coupled receptor, yet the mechanism by which PC1 regulates G-protein signaling remains poorly understood. A significant portion of ADPKD mutations of PC1 encode a protein with defects in maturation or reduced function that may be amenable to functional rescue. In this work, we have combined complementary biochemical and cellular assay experiments and accelerated molecular simulations, which revealed an allosteric transduction pathway in activation of the PC1 C-terminal fragment. Our findings will facilitate future rational drug design efforts targeting the PC1 signaling function. Polycystin-1 (PC1) is an important unusual G protein-coupled receptor (GPCR) with 11 transmembrane domains, and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal, and membrane-embedded, C-terminal fragments (CTF), and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless, and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain, and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074–S3585 and R3848–E4078 predicted from the GaMD simulations were validated by mutagenesis experiments. Together, these complementary analyses have provided insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Leading the way out: how a plant myosin facilitates vesicle tethering during exocytosis

Exocytosis is a process by which molecules, such as lipids, proteins, and polysaccharides, are delivered to the plasma membrane (PM) and the extracellular space via vesicular transport. Precise orchestration of this process ensures the spatiotemporal specificity of cargo delivery and therefore plays a vital role in cell growth, function, and response to environmental cues (reviewed in Wu and Guo, 2015; Ravikumar et al., 2017). Lastly, while sophisticated mechanisms for exocytosis have been described in animal and yeast cells, the extent of conservation of these mechanisms in plant systems is far less clear.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

RAS Protein Molecular Dynamics Simulations

This dataset contains 6401 files that describe parameters and other inputs for running molecular dynamics computer simulations of Ras proteins associated with a lipid-based cell membrane model in water, as well as resulting time-trajectories of atomic motion in these simulations. Specifically, the content includes: (a) 21 files containing generalized force field parameters, and (b) 22 files of similar format and content for each of 290 separate molecular dynamics simulations (22 ´ 290 = 6,380). The total size of this data set is 1.0 TB.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1970

Cell fine structure and function - Past and present

Electron microscopic studies of nerve membrane fine structure, discussing cell membrane multienzyme and macromolecular energy and information transduction, protein synthesis and nucleic acids interrelations

Fernandez-Moran, H.↗
NASA NTRS1996

Transmembrane transport of peptide type compounds: prospects for oral delivery

Synthesis and delivery of potential therapeutic peptides and peptidomimetic compounds has been the focus of intense research over the last 10 years. While it is widely recognized that numerous limitations apply to oral delivery of peptides, some of the limiting factors have been addressed and their mechanisms elucidated, which has lead to promising strategies. This article will briefly summarize the challenges, results and current approaches of oral peptide delivery and give some insight on future strategies. The barriers determining peptide bioavailability after oral administration are intestinal membrane permability, size limitations, intestinal and hepatic metabolism and in some cases solubility limitations. Poor membrane permeabilities of hydrophilic peptides might be overcome by structurally modifying the compounds, thus increasing their membrane partition characteristics and/or their affinity to carrier proteins. Another approach is the site-specific delivery of the peptide to the most permeable parts of the intestine. The current view on size limitation for oral drug delivery has neglected partition considerations. Recent studies suggest that compounds with a molecular weight up to 4000 might be significantly absorbed, assuming appropriate partition behavior and stability. Metabolism, probably the most significant factor in the absorption fate of peptides, might be controlled by coadministration of competitive enzyme inhibitors, structural modifications and administration of the compound as a well absorbed prodrug that is converted into the therapeutically active agent after its absorption. For some peptides poor solubility might present a limitation to oral absorption, an issue that has been addressed by mechanistically defining and therefore improving formulation parameters. Effective oral peptide delivery requires further development in understanding these complex mechanisms in order to maximize the therapeutic potential of this class of compounds.

NASA Discipline Regulatory Physiology↗
DOE OSTI2020

A Sec14 domain protein is required for photoautotrophic growth and chloroplast vesicle formation in Arabidopsis thaliana

In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans -Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Impact of PrsA on membrane lipid composition during daptomycin-resistance-mediated β-lactam sensitization in clinical MRSA strains

Abstract Background The cyclic anionic lipopeptide daptomycin is used in the treatment of severe infections caused by Gram-positive pathogens, including MRSA. Daptomycin resistance, although rare, often results in treatment failure. Paradoxically, in MRSA, daptomycin resistance is usually accompanied by a concomitant decrease in β-lactam resistance in what is known as the ‘see-saw effect’. This resensitization is extensively used for the treatment of MRSA infections, by combining daptomycin and a β-lactam antibiotic, such as oxacillin. Objectives We aimed: (i) to investigate the combined effects of daptomycin and oxacillin on the lipid composition of the cellular membrane of both daptomycin-resistant and -susceptible MRSA strains; and (ii) to assess the involvement of the post-translocational protein PrsA, which plays an important role in oxacillin resistance in MRSA, in membrane lipid composition and remodelling during daptomycin resistance/β-lactam sensitization. Results The combination of microbiological and biochemical studies, with fluorescence microscopy using lipid probes, showed that the lipid composition and surface charge of the daptomycin-resistant cells exposed to daptomycin/oxacillin were dependent on antibiotic concentration and directly associated with PrsA, which influenced cardiolipin remodelling/relocation. Conclusions Our findings show that PrsA, in addition to its post-transcriptional role in the maturation of PBP 2a, is a key mediator of cell membrane remodelling connected to the see-saw effect and may have a key role in the resensitization of daptomycin-resistant strains to β-lactams, such as oxacillin.

de Carvalho, Carla C. C. R.↗