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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 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation

The many variants of human Ca2+/calmodulin-dependent protein kinase II (CaMKII) differ in the lengths and sequences of disordered linkers connecting the kinase domains to the oligomeric hubs of the holoenzyme. CaMKII activity depends on the balance between activating and inhibitory autophosphorylation (on Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-β, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers.

Life Sciences & Biomedicine - Other Topics↗
DOE OSTI2020

Charting the native architecture of Chlamydomonas thylakoid membranes with single-molecule precision

Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Previously, we used in situ cryo-electron tomography to reveal the native architecture of thylakoid membranes (Engel et al., 2015). Here, we leverage technical advances to resolve the individual protein complexes within these membranes. Combined with a new method to visualize membrane surface topology, we map the molecular landscapes of thylakoid membranes inside green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed membrane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli

Many photosynthetic organisms employ a CO 2 concentrating mechanism (CCM) to increase the rate of CO 2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an Escherichia coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO 2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO 2 assimilation in diverse organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evolution of pathogen tolerance and emerging infections: A missing experimental paradigm

Researchers worldwide are repeatedly warning us against future zoonotic diseases resulting from humankind’s insurgence into natural ecosystems. The same zoonotic pathogens that cause severe infections in a human host frequently fail to produce any disease outcome in their natural hosts. What precise features of the immune system enable natural reservoirs to carry these pathogens so efficiently? To understand these effects, we highlight the importance of tracing the evolutionary basis of pathogen tolerance in reservoir hosts, while drawing implications from their diverse physiological and life-history traits, and ecological contexts of host-pathogen interactions. Long-term co-evolution might allow reservoir hosts to modulate immunity and evolve tolerance to zoonotic pathogens, increasing their circulation and infectious period. Such processes can also create a genetically diverse pathogen pool by allowing more mutations and genetic exchanges between circulating strains, thereby harboring rare alive-on-arrival variants with extended infectivity to new hosts (i.e., spillover). Finally, we end by underscoring the indispensability of a large multidisciplinary empirical framework to explore the proposed link between evolved tolerance, pathogen prevalence, and spillover in the wild.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A novel bivalent chromatin associates with rapid induction of camalexin biosynthesis genes in response to a pathogen signal in Arabidopsis

Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana , camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, trimethylation of lysine 27 of histone 3 (H3K27me3) (repression) and acetylation of lysine 18 of histone 3 (H3K18ac) (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27me3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name as kairostat, plays an important role in controlling the timely induction of gene expression upon stress stimuli in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Partial connectomes of labeled dopaminergic circuits reveal non-synaptic communication and axonal remodeling after exposure to cocaine

Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change with drug exposure remains poorly understood. We leverage cell type-specific labeling with large volume serial electron microscopy to detail DA connections in the nucleus accumbens (NAc) of the mouse (Mus musculus) before and after exposure to cocaine. We find that individual DA axons contain different varicosity types based on their vesicle contents. Spatially ordering along individual axons further suggests that varicosity types are non-randomly organized. DA axon varicosities rarely make specific synapses (<2%, 6/410), but instead are more likely to form spinule-like structures (15%, 61/410) with neighboring neurons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls, formed blind-ended ‘bulbs’ filled with mitochondria, and were surrounded by elaborated glia. Finally, mitochondrial lengths increased by ~2.2 times relative to control only in DA axons and NAc spiny dendrites after cocaine exposure. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large-scale axonal re-arrangements with correlated changes in mitochondria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The molecular mechanism of load adaptation by branched actin networks

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes, including phagocytosis, endocytosis, and pseudopod protrusion. When confronted with opposing forces, the growth rate of these networks slows and their density increases, but the stoichiometry of key components does not change. The molecular mechanisms governing this force response are not well understood, so we used single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Although load forces are observed to increase the density of growing filaments, we find that they actually decrease the rate of filament nucleation due to inhibitory interactions between actin filament ends and nucleation promoting factors. The force-induced increase in network density turns out to result from an exponential drop in the rate constant that governs filament capping. The force dependence of filament capping matches that of filament elongation and can be explained by expanding Brownian Ratchet theory to cover both processes. We tested a key prediction of this expanded theory by measuring the force-dependent activity of engineered capping protein variants and found that increasing the size of the capping protein increases its sensitivity to applied forces. In summary, we find that Brownian Ratchets underlie not only the ability of growing actin filaments to generate force but also the ability of branched actin networks to adapt their architecture to changing loads.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A molecular mechanism for the generation of ligand-dependent differential outputs by the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity

In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4 PPH-4.1 phosphatase and ATR ATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4 PPH-4.1 , ATR ATL-1 , and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The Neurodata Without Borders ecosystem for neurophysiological data science

The neurophysiology of cells and tissues are monitored electrophysiologically and optically in diverse experiments and species, ranging from flies to humans. Understanding the brain requires integration of data across this diversity, and thus these data must be findable, accessible, interoperable, and reusable (FAIR). This requires a standard language for data and metadata that can coevolve with neuroscience. We describe design and implementation principles for a language for neurophysiology data. Our open-source software (Neurodata Without Borders, NWB) defines and modularizes the interdependent, yet separable, components of a data language. We demonstrate NWB’s impact through unified description of neurophysiology data across diverse modalities and species. NWB exists in an ecosystem, which includes data management, analysis, visualization, and archive tools. Thus, the NWB data language enables reproduction, interchange, and reuse of diverse neurophysiology data. More broadly, the design principles of NWB are generally applicable to enhance discovery across biology through data FAIRness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An auto-inhibited state of protein kinase G and implications for selective activation

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition

Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme–PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structure and flexibility of the yeast NuA4 histone acetyltransferase complex

The NuA4 protein complex acetylates histones H4 and H2A to activate both transcription and DNA repair. We report the 3.1-Å resolution cryo-electron microscopy structure of the central hub of NuA4, which flexibly tethers the histone acetyltransferase (HAT) and Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) modules. The hub contains the large Tra1 subunit and a core that includes Swc4, Arp4, Act1, Eaf1, and the C-terminal region of Epl1. Eaf1 stands out as the primary scaffolding factor that interacts with the Tra1, Swc4, and Epl1 subunits and contributes the conserved HSA helix to the Arp module. Using nucleosome-binding assays, we find that the HAT module, which is anchored to the core through Epl1, recognizes H3K4me3 nucleosomes with hyperacetylated H3 tails, while the TINTIN module, anchored to the core via Eaf1, recognizes nucleosomes that have hyperacetylated H2A and H4 tails. Together with the known interaction of Tra1 with site-specific transcription factors, our data suggest a model in which Tra1 recruits NuA4 to specific genomic sites then allowing the flexible HAT and TINTIN modules to select nearby nucleosomes for acetylation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR Cas systems. We report a cryo-EM (cryo-electron microscopy) structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact on RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Deep learning-driven insights into super protein complexes for outer membrane protein biogenesis in bacteria

To reach their final destinations, outer membrane proteins (OMPs) of gram-negative bacteria undertake an eventful journey beginning in the cytosol. Multiple molecular machines, chaperones, proteases, and other enzymes facilitate the translocation and assembly of OMPs. These helpers usually associate, often transiently, forming large protein assemblies. They are not well understood due to experimental challenges in capturing and characterizing protein-protein interactions (PPIs), especially transient ones. Using AF2Complex, we introduce a high-throughput, deep learning pipeline to identify PPIs within the Escherichia coli cell envelope and apply it to several proteins from an OMP biogenesis pathway. Among the top confident hits obtained from screening ~1500 envelope proteins, we find not only expected interactions but also unexpected ones with profound implications. Subsequently, we predict atomic structures for these protein complexes. These structures, typically of high confidence, explain experimental observations and lead to mechanistic hypotheses for how a chaperone assists a nascent, precursor OMP emerging from a translocon, how another chaperone prevents it from aggregating and docks to a β-barrel assembly port, and how a protease performs quality control. This work presents a general strategy for investigating biological pathways by using structural insights gained from deep learning-based predictions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Tyramine and its Amtyr1 receptor modulate attention in honey bees (Apis mellifera)

Animals must learn to ignore stimuli that are irrelevant to survival and attend to ones that enhance survival. When a stimulus regularly fails to be associated with an important consequence, subsequent excitatory learning about that stimulus can be delayed, which is a form of nonassociative conditioning called ‘latent inhibition’. Honey bees show latent inhibition toward an odor they have experienced without association with food reinforcement. Moreover, individual honey bees from the same colony differ in the degree to which they show latent inhibition, and these individual differences have a genetic basis. To investigate the mechanisms that underly individual differences in latent inhibition, we selected two honey bee lines for high and low latent inhibition, respectively. We crossed those lines and mapped a Quantitative Trait Locus for latent inhibition to a region of the genome that contains the tyramine receptor gene Amtyr1 [We use Amtyr1 to denote the gene and AmTYR1 the receptor throughout the text.]. We then show that disruption of Amtyr1 signaling either pharmacologically or through RNAi qualitatively changes the expression of latent inhibition but has little or slight effects on appetitive conditioning, and these results suggest that AmTYR1 modulates inhibitory processing in the CNS. Electrophysiological recordings from the brain during pharmacological blockade are consistent with a model that AmTYR1 indirectly regulates at inhibitory synapses in the CNS. Our results therefore identify a distinct Amtyr1 -based modulatory pathway for this type of nonassociative learning, and we propose a model for how Amtyr1 acts as a gain control to modulate hebbian plasticity at defined synapses in the CNS. We have shown elsewhere how this modulation also underlies potentially adaptive intracolonial learning differences among individuals that benefit colony survival. Finally, our neural model suggests a mechanism for the broad pleiotropy this gene has on several different behaviors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

B cell receptor-induced IL-10 production from neonatal mouse CD19 + CD43 - cells depends on STAT5-mediated IL-6 secretion

Newborns are unable to reach the adult-level humoral immune response partly due to the potent immunoregulatory role of IL-10. Increased IL-10 production by neonatal B cells has been attributed to the larger population of IL-10-producting CD43 + B-1 cells in neonates. Here, we show that neonatal mouse CD43 - non-B-1 cells also produce substantial amounts of IL-10 following B cell antigen receptor (BCR) activation. In neonatal mouse CD43 - non-B-1 cells, BCR engagement activated STAT5 under the control of phosphorylated forms of signaling molecules Syk, Btk, PKC, FAK, and Rac1. Neonatal STAT5 activation led to IL-6 production, which in turn was responsible for IL-10 production in an autocrine/paracrine fashion through the activation of STAT3. In addition to the increased IL-6 production in response to BCR stimulation, elevated expression of IL-6Rα expression in neonatal B cells rendered them highly susceptible to IL-6-mediated STAT3 phosphorylation and IL-10 production. Finally, IL-10 secreted from neonatal mouse CD43 - non-B-1 cells was sufficient to inhibit TNF-α secretion by macrophages. Our results unveil a distinct mechanism of IL-6-dependent IL-10 production in BCR-stimulated neonatal CD19 + CD43 - B cells.

59 BASIC BIOLOGICAL SCIENCES↗