Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “protein function”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,639 records · Page 91
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Reactive oxygen species control protein degradation at the mitochondrial import gate

While reactive oxygen species (ROS) have long been known to drive aging and neurodegeneration, their persistent depletion below basal levels also disrupts organismal function. Cells counteract loss of basal ROS via the reductive stress response, but the identity and biochemical activity of ROS sensed by this pathway remain unknown. Here, we show that the central enzyme of the reductive stress response, the E3 ligase Cullin 2-FEM1 homolog B (CUL2 FEM1B ), specifically acts at mitochondrial TOM complexes, where it senses ROS produced by complex III of the electron transport chain (ETC). ROS depletion during times of low ETC activity triggers the localized degradation of CUL2 FEM1B substrates, which sustains mitochondrial import and ensures the biogenesis of the rate-limiting ETC complex IV. As complex III yields most ROS when the ETC outpaces metabolic demands or oxygen availability, basal ROS are sentinels of mitochondrial activity that help cells adjust their ETC to changing environments, as required for cell differentiation and survival.

FEM1B↗
DOE OSTI2020

Antibody Targeted Metal–Organic Frameworks for Bioimaging Applications

Here, we report on the availability and chemical utility of primary amines within metal–organic frameworks (MOFs) for cell targeting. Primary amine groups represent one of the most versatile chemical moieties for conjugation to biologically relevant molecules, including antibodies and enzymes. Specifically, we used two different chemical conjugations schemes, utilizing the amino functionality on the organic linker: first, carbodiimide chemistry was used to link the primary amine to available carboxyl groups on the protein neutravidin; second, sulfhydryl cross-linking chemistry was used via Traut’s reagent scheme. Importantly, this is the first report that documents this methodology implemented with MOF systems. Finally, the ability of the EpCAM antibody targeted MOFs to bind to a human epithelial cell line (A549), a common target for imaging studies, was confirmed with confocal microscopy.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme’s biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A’s function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Improvement of eukaryotic protein predictions from soil metagenomes

During the last decades, metagenomics has highlighted the diversity of microorganisms from environmental or host-associated samples. Most metagenomics public repositories use annotation pipelines tailored for prokaryotes regardless of the taxonomic origin of contigs. Consequently, eukaryotic contigs with intrinsically different gene features, are not optimally annotated. Using a bioinformatics pipeline, we have filtered 7.9 billion contigs from 6,872 soil metagenomes in the JGI’s IMG/M database to identify eukaryotic contigs. We have re-annotated genes using eukaryote-tailored methods, yielding 8 million eukaryotic proteins and over 300,000 orphan proteins lacking homology in public databases. Comparing the gene predictions we made with initial JGI ones on the same contigs, we confirmed our pipeline improves eukaryotic proteins completeness and contiguity in soil metagenomes. The improved quality of eukaryotic proteins combined with a more comprehensive assignment method yielded more reliable taxonomic annotation. This dataset of eukaryotic soil proteins with improved completeness, quality and taxonomic annotation reliability is of interest for any scientist aiming at studying the composition, biological functions and gene flux in soil communities involving eukaryotes.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain

Ca 2+ /calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

ATM–dependent phosphorylation of CHD7 regulates morphogenesis-coupled DSB stress response in fetal radiation exposure

Following radiation exposure, unrepaired DNA double-strand breaks (DSBs) persist to some extent in a subset of cells as residual damage; they can exert adverse effects, including late-onset diseases. In search of the factor(s) that characterize(s) cells bearing such damage, we discovered ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of the transcription factor chromodomain helicase DNA binding protein 7 (CHD7). CHD7 controls the morphogenesis of cell populations derived from neural crest cells during vertebrate early development. Indeed, malformations in various fetal bodies are attributable to CHD7 haploinsufficiency. Following radiation exposure, CHD7 becomes phosphorylated, ceases promoter/enhancer binding to target genes, and relocates to the DSB-repair protein complex, where it remains until the damage is repaired. Thus, ATM-dependent CHD7 phosphorylation appears to act as a functional switch. As such stress responses contribute to improved cell survival and canonical nonhomologous end joining, we conclude that CHD7 is involved in both morphogenetic and DSB-response functions. Thus, we propose that higher vertebrates have evolved intrinsic mechanisms underlying the morphogenesis-coupled DSB stress response. In fetal exposure, if the function of CHD7 becomes primarily shifted toward DNA repair, morphogenic activity is reduced, resulting in malformations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Crystal structure of MbnF: an NADPH-dependent flavin monooxygenase from Methylocystis strain SB2

Methanobactins (MBs) are ribosomally produced and post-translationally modified peptides (RiPPs) that are used by methanotrophs for copper acquisition. The signature post-translational modification of MBs is the formation of two heterocyclic groups, either an oxazolone, pyrazinedione or imidazolone group, with an associated thioamide from an X -Cys dipeptide. The precursor peptide (MbnA) for MB formation is found in a gene cluster of MB-associated genes. The exact biosynthetic pathway of MB formation is not yet fully understood, and there are still uncharacterized proteins in some MB gene clusters, particularly those that produce pyrazinedione or imidazolone rings. One such protein is MbnF, which is proposed to be a flavin monooxygenase (FMO) based on homology. To help to elucidate its possible function, MbnF from Methylocystis sp. strain SB2 was recombinantly produced in Escherichia coli and its X-ray crystal structure was resolved to 2.6 Å resolution. Based on its structural features, MbnF appears to be a type A FMO, most of which catalyze hydroxylation reactions. Preliminary functional characterization shows that MbnF preferentially oxidizes NADPH over NADH, supporting NAD(P)H-mediated flavin reduction, which is the initial step in the reaction cycle of several type A FMO enzymes. It is also shown that MbnF binds the precursor peptide for MB, with subsequent loss of the leader peptide sequence as well as the last three C-terminal amino acids, suggesting that MbnF might be needed for this process to occur. Finally, molecular-dynamics simulations revealed a channel in MbnF that is capable of accommodating the core MbnA fragment minus the three C-terminal amino acids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A Melanoma Brain Metastasis CTC Signature and CTC:B-cell Clusters Associate with Secondary Liver Metastasis: A Melanoma Brain–Liver Metastasis Axis

Melanoma brain metastasis is linked to dismal prognosis and low overall survival and is detected in up to 80% of patients at autopsy. Circulating tumor cells (CTC) are the smallest functional units of cancer and precursors of fatal metastasis. We previously used an unbiased multilevel approach to discover a unique ribosomal protein large/small subunit (RPL/RPS) CTC gene signature associated with melanoma brain metastasis. In this study, we hypothesized that CTC-driven melanoma brain metastasis secondary metastasis (“metastasis of metastasis” per clinical scenarios) has targeted organ specificity for the liver. We injected parallel cohorts of immunodeficient and newly developed humanized NBSGW (huNBSGW) mice with cells from CTC-derived melanoma brain metastasis to identify secondary metastatic patterns. We found the presence of a melanoma brain–liver metastasis axis in huNBSGW mice. Furthermore, RNA sequencing analysis of tissues showed a significant upregulation of the RPL/RPS CTC gene signature linked to metastatic spread to the liver. Additional RNA sequencing of CTCs from huNBSGW blood revealed extensive CTC clustering with human B cells in these mice. CTC:B-cell clusters were also upregulated in the blood of patients with primary melanoma and maintained either in CTC-driven melanoma brain metastasis or melanoma brain metastasis CTC–derived cells promoting liver metastasis. CTC-generated tumor tissues were interrogated at single-cell gene and protein expression levels (10x Genomics Xenium and HALO spatial biology platforms, respectively). Collectively, our findings suggest that heterotypic CTC:B-cell interactions can be critical at multiple stages of metastasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Comparing four heat-inducible promoters in stably transformed sugarcane regarding spatial and temporal control of transgene expression reveals candidates to drive stem-preferred transgene expression

Small heat shock protein (sHSP) promoters contain cis-regulatory elements that facilitate transcription in response to heat stress, making them valuable tools for functional studies through controlled gene expression and the precise regulation of gene-editing tools or morphogenic regulators. To evaluate their utility, GUS reporter gene expression driven by four plant-sourced HSP promoters (p GmHSP17.5 , p HvHSP17 , p ZmHSP17.7 , and p ZmHSP26 ) was compared across various tissues of stably transformed sugarcane before and after heat treatment. At 22°C, all promoters showed minimal activity in leaves and roots, although p ZmHSP17.7 and p HvHSP17 displayed moderate expression in stems. Following heat treatment, all promoters exhibited their highest activity in stems, followed by leaves and roots. In stem tissues, p GmHSP17.5 displayed heat-induced uidA expression comparable to the constitutive p ZmUbi promoter. Notably, heat-induced reporter gene activity in stem middle sections of single-copy transgenic lines containing p ZmHSP17.7 , p HvHSP17 , or p ZmHSP26 exceeded p ZmUbi -derived uidA activity by 9.7-fold, 3.8-fold, and 3.0-fold, respectively, with 346- to 3,672-fold induction compared to control conditions. Most promoters showed peak expression in the middle sections of the stem, while p HvHSP17 was the most active in the stem apices. Histochemical analysis revealed that p ZmHSP17.7 and p HvHSP17 were active in both parenchyma cells and vascular bundles within sugarcane stems. Among leaf tissues, mature leaves exhibited greater expression than senescing or immature leaves, while root activity remained consistently minimal across all promoters. Temperature-course experiments identified distinct activation thresholds: 34°C–36°C for p ZmHSP17.7 , 36°C for p ZmHSP26 , 36°C–38°C for p HvHSP17 , and 40°C–42°C for p GmHSP17.5 . Drought stress also induced reporter gene transcription in stems under HSP promoters, although with lower fold induction than heat treatment. These findings provide valuable tools for gene function studies and biotechnology applications, including heat stress tolerance research, controlled transgene expression in metabolic engineering, precision gene editing, and developmental biology studies.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1987

Gravitational haemodynamics and oedema prevention in the giraffe

The question of how giraffes avoid pooling of blood and tissue fluid (edema) in dependent tissues of their extremities is addressed. As monitored by radiotelemetry, the blood and tissue fluid pressures that govern transcapillary exchange vary greatly with exercise. These pressures, combined with a tight skin layer, move fluid upward against gravity. The skin thus functions like a natural antigravity suit. Other mechanisms that prevent edema include precapillary vasoconstriction and low permeability of capillaries to plasma proteins.

Hargens, Alan R.↗
NASA NTRS2002

Parathyroid hormone-dependent signaling pathways regulating genes in bone cells

Parathyroid hormone (PTH) is an 84-amino-acid polypeptide hormone functioning as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. PTH and PTH-related protein (PTHrP) indirectly activate osteoclasts resulting in increased bone resorption. During this process, PTH changes the phenotype of the osteoblast from a cell involved in bone formation to one directing bone resorption. In addition to these catabolic effects, PTH has been demonstrated to be an anabolic factor in skeletal tissue and in vitro. As a result, PTH has potential medical application to the treatment of osteoporosis, since intermittent administration of PTH stimulates bone formation. Activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix, production of growth factors, and stimulation and recruitment of osteoclasts. The ability of PTH to drive changes in gene expression is dependent upon activation of transcription factors such as the activator protein-1 family, RUNX2, and cAMP response element binding protein (CREB). Much of the regulation of these processes by PTH is protein kinase A (PKA)-dependent. However, while PKA is linked to many of the changes in gene expression directed by PTH, PKA activation has been shown to inhibit mitogen-activated protein kinase (MAPK) and proliferation of osteoblasts. It is now known that stimulation of MAPK and proliferation by PTH at low concentrations is protein kinase C (PKC)-dependent in both osteoblastic and kidney cells. Furthermore, PTH has been demonstrated to regulate components of the cell cycle. However, whether this regulation requires PKC and/or extracellular signal-regulated kinases or whether PTH is able to stimulate other components of the cell cycle is unknown. It is possible that stimulation of this signaling pathway by PTH mediates a unique pattern of gene expression resulting in proliferation in osteoblastic and kidney cells; however, specific examples of this are still unknown. This review will focus on what is known about PTH-mediated cell signaling, and discuss the established or putative PTH-regulated pattern of gene expression in osteoblastic cells following treatment with catabolic (high) or anabolic (low) concentrations of the hormone.

NASA Discipline Musculoskeletal↗
NASA NTRS1993

Climbing Darwin's ladder

The work of Bartel and Szostak, in which RNA molecules were selected to enhance the ability to catalyze a reaction similar to a step in protein-catalyzed RNA replication, is discussed. An important aspect of this experiment was the ability to reach a high level of functional organization in ten evolutionary steps. Further steps necessary to obtain an RNA enzyme with RNA replicase activity include performing the reaction with mononucleoside 5'-triphosphates, generalizing the reaction to include a variety of sequences without loss of template-dependent specificity, and overcoming template self-structure that could prevent some regions from being copied efficiently.

NASA Discipline Exobiology↗
NASA NTRS2011

Evaluation of the Combined Effects of Gamma Radiation and High Dietary Iron on Peripheral Leukocyte Distribution and Function

NASA is concerned with the health risks to astronauts, particularly those risks related to radiation exposure. Both radiation and increased iron stores can independently increase oxidative damage, resulting in protein, lipid and DNA oxidation. Oxidative stress increases the risk of many health problems including cancer, cataracts, and heart disease. This study, a subset of a larger interdisciplinary investigation of the combined effect of iron overload on sensitivity to radiation injury, monitored immune parameters in the peripheral blood of rats subjected to gamma radiation, high dietary iron or both. Specific immune measures consisted of (A) peripheral leukocyte distribution; (B) plasma cytokine levels; (C) cytokine production profiles following whole blood stimulation of either T cells or monocytes.

Crucian, Brian E.↗
DOE OSTI2019

Liquid-like and rigid-body motions in molecular-dynamics simulations of a crystalline protein

To gain insight into crystalline protein dynamics, we performed molecular-dynamics (MD) simulations of a periodic 2 × 2 × 2 supercell of staphylococcal nuclease. We used the resulting MD trajectories to simulate X-ray diffraction and to study collective motions. The agreement of simulated X-ray diffraction with the data is comparable to previous MD simulation studies. We studied collective motions by analyzing statistically the covariance of alpha-carbon position displacements. The covariance decreases exponentially with the distance between atoms, which is consistent with a liquidlike motions (LLM) model, in which the protein behaves like a soft material. To gain finer insight into the collective motions, we examined the covariance behavior within a protein molecule (intraprotein) and between different protein molecules (interprotein). The interprotein atom pairs, which dominate the overall statistics, exhibit LLM behavior; however, the intraprotein pairs exhibit behavior that is consistent with a superposition of LLM and rigid-body motions (RBM). Our results indicate that LLM behavior of global dynamics is present in MD simulations of a protein crystal. They also show that RBM behavior is detectable in the simulations but that it is subsumed by the LLM behavior. Finally, the results provide clues about how correlated motions of atom pairs both within and across proteins might manifest in diffraction data. Overall, our findings increase our understanding of the connection between molecular motions and diffraction data and therefore advance efforts to extract information about functionally important motions from crystallography experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching

Nanoarchitectural control of matter is crucial for next-generation technologies. DNA origami templates are harnessed to accurately position single molecules; however, direct single molecule evidence is lacking regarding how well DNA origami can control the orientation of such molecules in three-dimensional space, as well as the factors affecting control. Here, we present two strategies for controlling the polar (θ) and in-plane azimuthal (Φ) angular orientations of cyanine Cy5 single molecules tethered on rationally-designed DNA origami templates that are physically adsorbed (physisorbed) on glass substrates. By using dipolar imaging to evaluate Cy5's orientation and super-resolution microscopy, the absolute spatial orientation of Cy5 is calculated relative to the DNA template. The sequence-dependent partial intercalation of Cy5 is discovered and supported theoretically using density functional theory and molecular dynamics simulations, and it is harnessed as our first strategy to achieve θ control for a full revolution with dispersion as small as ±4.5°. In our second strategy, Φ control is achieved by mechanically stretching the Cy5 from its two tethers, being the dispersion ±10.3° for full stretching. These results can in principle be applied to any single molecule, expanding in this way the capabilities of DNA as a functional templating material for single-molecule orientation control. The experimental and modeling insights provided herein will help engineer similar self-assembling molecular systems based on polymers, such as RNA and proteins.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2026

Beyond Component Optimization: Systems Level Biodesign for Lanthanide Recovery

Global demand for lanthanides (Ln) is projected to rise sharply over the next decade, while geographically concentrated supply chains and the low concentrations and matrix complexity of secondary feedstocks limit the reach of conventional hydro- and pyrometallurgical separation. Engineered biological systems offer a selective, low-energy alternative, and component-level advances in Ln-binding proteins, AI-designed selective scaffolds, and cell-surface display platforms now rival synthetic chelators in affinity and selectivity. These components, however, remain functionally isolated. Currently, there are no engineered chassis coupling recognition, intracellular trafficking, accumulation, and controlled release into an end-to-end pipeline. Here, we outline how new biodesign strategies and chassis selection must move beyond bioleaching to encompass the full recovery pathway. Achieving this requires integrating AI/ML-guided design, genome-scale build tools, high-throughput phenotyping, and biophysical transport modeling within a Design–Build–Test–Learn cycle tuned to recognition, trafficking, accumulation, and release.

Biodesign↗