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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 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Structure of the phosphoinositide 3-kinase (PI3K) p110γ-p101 complex reveals molecular mechanism of GPCR activation

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein–coupled receptors. Here, we report the cryo–electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ–adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101–specific signaling events in vivo.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Molecular mechanism of trehalose 6-phosphate inhibition of the plant metabolic sensor kinase SnRK1

SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), a central plant metabolic sensor kinase, phosphorylates its target proteins, triggering a global shift from anabolism to catabolism. Molecular modeling revealed that upon binding of KIN10 to GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1), KIN10’s activation T-loop reorients into GRIK1’s active site, enabling its phosphorylation and activation. Trehalose 6-phosphate (T6P) is a proxy for cellular sugar status and a potent inhibitor of SnRK1. T6P binds to KIN10, a SnRK1 catalytic subunit, weakening its affinity for GRIK1. Here, we investigate the molecular details of T6P inhibition of KIN10. Molecular dynamics simulations and in vitro phosphorylation assays identified and validated the T6P binding site on KIN10. Under high-sugar conditions, T6P binds to KIN10, blocking the reorientation of its activation loop and preventing its phosphorylation and activation by GRIK1. Under these conditions, SnRK1 maintains only basal activity levels, minimizing phosphorylation of its target proteins, thereby facilitating a general shift from catabolism to anabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Rhoptry kinase protein 39 (ROP39) is a novel factor that recruits host mitochondria to the parasitophorous vacuole of Toxoplasma gondii

ABSTRACT Most intracellular pathogens replicate in a vacuole to avoid the defense system of the host. A few pathogens recruit host mitochondria around those vacuoles, but the molecules responsible for mitochondrial recruitment remain unidentified. It is only in the apicomplexan parasite Toxoplasma gondii, that mitochondrial association factor 1b (MAF1b) has been identified as an association factor for host mitochondria. Here, we show that rhoptry kinase family protein 39 (ROP39) induces host mitochondrial recruitment in T. gondii. We found that the abundance of ROP39 was increased on host mitochondria extracted from human foreskin fibroblasts (HFFs) infected with T. gondii. ROP39 expressed exogenously in HFFs localized on host mitochondria, indicating that it has the potential to bind to host mitochondria without assistance from other parasite factors. Confocal microscopy revealed that ROP39 colocalized with host mitochondria on the membrane of parasitophorous vacuoles, in which the parasites reside. Moreover, we observed about a 10% reduction in the level of mitochondrial association in rop39-knockout parasites compared with a parental strain.

Fukumoto, Junpei↗
DOE OSTI2021

Mitogen-activated protein kinase 6 negatively regulates secondary wall biosynthesis by modulating MYB46 protein stability in Arabidopsis thaliana

The R2R3-MYB transcription factor MYB46 functions as a master switch for secondary cell wall biosynthesis, ensuring the exquisite expression of the secondary wall biosynthetic genes in the tissues where secondary walls are critical for growth and development. At the same time, suppression of its function is needed when/where formation of secondary walls is not desirable. Little is known about how this opposing control of secondary cell wall formation is achieved. We used both transient and transgenic expression of MYB46 and mitogen-activated protein kinase 6 (MPK6) to investigate the molecular mechanism of the post-translational regulation of MYB46. We show that MYB46 is phosphorylated by MPK6, leading to site specific phosphorylation-dependent degradation of MYB46 by the ubiquitin-mediated proteasome pathway. In addition, the MPK6-mediated MYB46 phosphorylation was found to regulate in planta secondary wall forming function of MYB46. Furthermore, we provide experimental evidences that MYB83, a paralog of MYB46, is not regulated by MPK6. The coupling of MPK signaling to MYB46 function provides insights into the tissue- and/or condition-specific activity of MYB46 for secondary wall biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Pyrolysis_Molecular_Beam_Mass_Spectrometry_Analysis_of_hybrid_cross_of_Populus_tremula_x_P_alba_717-1B4_and_overexpression_of_a_lectin_receptor-like_kinase_(PtLecRLK1)

Stem tissues from the hybrid poplarPopulus tremula × P. albaclone 717-1B4 and from lectin receptor-like kinase overexpression lines PP7 and PP19 were individually colonized with the ectomycorrhizal fungiLaccaria bicolorstrain S238N,Hyaloscypha finlandicastrain PMI746, orUmbelopsis vinaceastrain PMI3018, as well as with a mixed fungal inoculum; non-inoculated plants served as controls. Plants were grown in a greenhouse at Oak Ridge National Laboratory and harvested in January 2025. Stem samples were analyzed using Pyrolysis–Molecular Beam Mass Spectrometry (Py-MBMS). Stems were harvested, debarked, dried, milled, destarched and ethanol extracted prior to analysis. Py-MBMS analysis was conducted using approximately 4 mg of wood from biomass and each sample was analyzed in duplicate. A Frontier PY2020 unit pyrolyzed samples at 500°C for 30 s in 80 µL deactivated stainless steel cups. An Extrel Super-Sonic MBMS Model Max 1000 was used to collect mass spectral data fromm/z30 to 450 at 17 eV and processed using Merlin Automation software (V3). Spectral ion intensities were normalized to the total ion chromatogram signal for each sample for analysis of spectral variance. Lignin content (wt %) was estimated based on relative responses from standards of known Klason lignin content using mean-normalized ion intensities ofm/z120, 124 (G), 137 (G), 138 (G), 150 (G), 152, 154 (S), 164 (G), 167 (S), 168 (S), 178 (G), 180, 181, 182 (S), 194 (S), 208 (S) and 210 (S) where G indicates guaiacyl-derived ions, S indicates syringyl-derived ions, and other ions either derive from other lignin monomers or multiple sources. Ratios of S and G lignin monomer units (S/G) were obtained by dividing the sum of S-based ions by the sum of G-based ions using mean-normalized ion intensities.

CBI↗
DOE OSTI2024

Polyphosphate kinase deletion increases laboratory productivity in cyanobacteria

Identification and manipulation of cellular energy regulation mechanisms may be a strategy to increase productivity in photosynthetic organisms. This work tests the hypothesis that polyphosphate synthesis and degradation play a role in energy management by storing or dissipating energy in the form of ATP. A polyphosphate kinase ( ppk ) knock-out strain unable to synthesize polyphosphate was generated in the cyanobacterium Synechocystis sp. PCC 6803. This mutant strain demonstrated higher ATP levels and faster growth than the wildtype strain in high-carbon conditions and had a growth defect under multiple stress conditions. In a strain that combined ppk deletion with heterologous expression of ethylene-forming enzyme, higher ethylene productivity was observed than in the wildtype background. These results support the role of polyphosphate synthesis and degradation as an energy regulation mechanism and suggest that such mechanisms may be effective targets in biocontainment design.

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 OSTI2023

Development of Novel Small-Molecule Activators of Pyruvate Kinase Muscle Isozyme 2, PKM2, to Reduce Photoreceptor Apoptosis

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2’s efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye.

59 BASIC BIOLOGICAL SCIENCES↗