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 1,135 records · Page 63
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A universal and constant rate of gene content change traces pangenome flux to LUCA

Abstract Prokaryotic genomes constantly undergo gene flux via lateral gene transfer, generating a pangenome structure consisting of a conserved core genome surrounded by a more variable accessory genome shell. Over time, flux generates change in genome content. Here, we measure and compare the rate of genome flux for 5655 prokaryotic genomes as a function of amino acid sequence divergence in 36 universally distributed proteins of the informational core (IC). We find a clock of gene content change. The long-term average rate of gene content flux is remarkably constant across all higher prokaryotic taxa sampled, whereby the size of the accessory genome—the proportion of the genome harboring gene content difference for genome pairs—varies across taxa. The proportion of species-level accessory genes per genome, varies from 0% (Chlamydia) to 30%–33% (Alphaproteobacteria, Gammaproteobacteria, and Clostridia). A clock-like rate of gene content change across all prokaryotic taxa sampled suggest that pangenome structure is a general feature of prokaryotic genomes and that it has been in existence since the divergence of bacteria and archaea.

Microbiology↗
DOE OSTI2024

A small secreted protein serves as a plant-derived effector mediating symbiosis between Populus and Laccaria bicolor

Beneficial symbiotic fungi colonize plant tissues, delivering crucial ecosystem services such as carbon sequestration and plant fertilization. Specifically, trees that form a nutrient-acquiring symbiosis with mutualistic ectomycorrhizal (ECM) fungi gain advantages from these associations by experiencing enhanced growth rates and increased resilience to both biotic and abiotic stresses. Despite the vital role ECM fungi play in the nutrition and well-being of trees, identifying key regulators participating in the molecular communication between plant and fungal cells is still in its early stages. The mutualistic relationship between Laccaria bicolor and Populus spp. has been utilized as a model system for investigating ECM symbiosis at the molecular level. It has been demonstrated that the fungus L. bicolor secretes Mycorrhiza-induced Small Secreted Proteins (MiSSPs) required for ECM development. Meanwhile, we have previously shown that P. trichocarpa small, secreted proteins (PtSSPs) are highly induced during mutualistic symbiosis and some of them can enter, via in-vitro feeding, L. bicolor hyphae affecting their growth and morphology. However, the exact role and mode of action of PtSSPs in mutualistic symbiosis remain unknown. Because previous study showed that PtSSP1 is taking up by fungal cell and then localize in fungal cells, we decide to dig further on its putative role in fungal cells and also by overexpressing it in poplar because it is not technically possible yet to overexpress poplar protein in Laccaria bicolor hyphae. Here, we further characterized the function of PtSSP1(Potri.009G063200) in ectomycorrhization, which accumulates in the nucleus of L. bicolor in an in-vitro feeding experiment. Our results provide new knowledge for the genetic engineering of plants to control associated microbes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genetic underpinnings of arthropod community distributions in Populus trichocarpa

Community genetics seeks to understand the mechanisms by which natural genetic variation in heritable host phenotypes can encompass assemblages of organisms such as bacteria, fungi, and many animals including arthropods. Prior studies that focused on plant genotypes have been unable to identify genes controlling community composition, a necessary step to predict ecosystem structure and function as underlying genes shift within plant populations. Here, we surveyed arthropods within an association population of Populus trichocarpa in three common gardens to discover plant genes that contributed to arthropod community composition. We analyzed our surveys with traditional single-trait genome-wide association analysis (GWAS), multitrait GWAS, and functional networks built from a diverse set of plant phenotypes. Plant genotype was influential in structuring arthropod community composition among several garden sites. Candidate genes important for higher level organization of arthropod communities had broadly applicable functions, such as terpenoid biosynthesis and production of dsRNA binding proteins and protein kinases, which may be capable of targeting multiple arthropod species. We have demonstrated the ability to detect, in an uncontrolled environment, individual genes that are associated with the community assemblage of arthropods on a host plant, further enhancing our understanding of genetic mechanisms that impact ecosystem structure.

Community genetics↗
DOE OSTI2022

A chromosome-level genome assembly of the Chinese cork oak (Quercus variabilis)

Quercus variabilis (Fagaceae) is an ecologically and economically important deciduous broadleaved tree species native to and widespread in East Asia. It is a valuable woody species and an indicator of local forest health, and occupies a dominant position in forest ecosystems in East Asia. However, genomic resources from Q. variabilis are still lacking. Here, we present a high-quality Q. variabilis genome generated by PacBio HiFi and Hi-C sequencing. The assembled genome size is 787 Mb, with a contig N50 of 26.04 Mb and scaffold N50 of 64.86 Mb, comprising 12 pseudo-chromosomes. The repetitive sequences constitute 67.6% of the genome, of which the majority are long terminal repeats, accounting for 46.62% of the genome. We used ab initio , RNA sequence-based and homology-based predictions to identify protein-coding genes. A total of 32,466 protein-coding genes were identified, of which 95.11% could be functionally annotated. Evolutionary analysis showed that Q. variabilis was more closely related to Q. suber than to Q. lobata or Q. robur. We found no evidence for species-specific whole genome duplications in Quercus after the species had diverged. This study provides the first genome assembly and the first gene annotation data for Q. variabilis. These resources will inform the design of further breeding strategies, and will be valuable in the study of genome editing and comparative genomics in oak species.

Han, Biao↗
DOE OSTI2026

The anaerobic fungus Caecomyces churrovis produces H 2 via a non-bifurcating NADH-dependent enzyme complex

ABSTRACT Hydrogenosomes are mitochondria-derived organelles that produce ATP and H 2 to support energy metabolism in anaerobic eukaryotes. H 2 production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H 2 production in anaerobic eukaryotes remains incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P) + and link electron transfer to H 2 formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H 2 production in the AF strain Caecomyces churrovis . Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H 2 :NAD + oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H 2 formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE, NuoF) in C. churrovis , and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H 2 directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H 2 production and altering fungal metabolism. IMPORTANCE H 2 production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H 2 production from AF was first reported, the molecular mechanism for hydrogenosomal H 2 production and redox balance remains unclear. We demonstrate that AF produce H 2 from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H 2 production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H 2 -producing systems.

Zhang, Bo [Department of Chemical Engineering, Uni↗
NASA NTRS1978

Design of polymeric immunomicrospheres for cell labelling and cell separation

Synthesis of several classes of hydrophylic microspheres applied to cell labeling and cell separation is described. Five classes of cross-linked microspheres with functional groups such as carboxyl, hydroxyl, amide and/or pyridine groups were synthesized. These functional groups were used to bind covalently antibodies and other proteins to the surface of the microspheres. To optimize the derivatisation technique, polyglutaraldehyde immunomicrospheres were prepared and utilized. Specific populations of human and murine lymphocytes were labelled with microspheres synthesized by the emulsion of the ionizing radiation technique. The labelling of the cells by means of microspheres containing an iron core produced successful separation of B from T lymphocytes by means of a magnetic field.

Rembaum, A.↗
NASA NTRS1995

STS-67 Post Flight Presentation

This video is the post-flight presentation by the astronauts of the STS-67 Space Shuttle Mission. The astronauts were: Steve Oswald (Mission Commander), Bill Gregory (Shuttle Pilot), John Grunsfeld (Mission Specialist), Sam Durrance (Payload Specialist), Ron Parise (Payload Specialist), and Tammy Jernigan (Payload Commander). Footage includes: pre-launch suitup and launch (liftoff), the deployment of the telescope package payload (Hopkins UV telescope, Wisconsin UV polarimeter, and Astrostar Tracker) for their astronomical observations of different stellar objects, inside Shuttle shots of data collection stations, protein crystal growth experiments, medical BSO of head and eye functions in microgravity environment, storm activity over the United States and other Earth observation shots, Mid-deck Act Control Experiments, school-Shuttle direct radio communication, and descent and landing footage. This launch was a night launch and the flight was a 17 day flight (extended two days from original flight plan).

Source record↗
NASA NTRS1995

Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Non-NASA Center↗
NASA NTRS2012

The Origins of Transmembrane Ion Channels

Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

Pohorille, Andrew↗
NASA NTRS2021

PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high‐throughput sequencing

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell‐free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE‐based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)‐peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry‐over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti‐FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 10(exp 12) random sequences, a round‐by‐round high‐throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr(Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE‐based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

cDNA display, FLAG epitope, mRNA display, peptide ↗
DOE OSTI2021

Predictive Models of Genetic Redundancy in Arabidopsis thaliana

Abstract Genetic redundancy refers to a situation where an individual with a loss-of-function mutation in one gene (single mutant) does not show an apparent phenotype until one or more paralogs are also knocked out (double/higher-order mutant). Previous studies have identified some characteristics common among redundant gene pairs, but a predictive model of genetic redundancy incorporating a wide variety of features derived from accumulating omics and mutant phenotype data is yet to be established. In addition, the relative importance of these features for genetic redundancy remains largely unclear. Here, we establish machine learning models for predicting whether a gene pair is likely redundant or not in the model plant Arabidopsis thaliana based on six feature categories: functional annotations, evolutionary conservation including duplication patterns and mechanisms, epigenetic marks, protein properties including posttranslational modifications, gene expression, and gene network properties. The definition of redundancy, data transformations, feature subsets, and machine learning algorithms used significantly affected model performance based on holdout, testing phenotype data. Among the most important features in predicting gene pairs as redundant were having a paralog(s) from recent duplication events, annotation as a transcription factor, downregulation during stress conditions, and having similar expression patterns under stress conditions. We also explored the potential reasons underlying mispredictions and limitations of our studies. This genetic redundancy model sheds light on characteristics that may contribute to long-term maintenance of paralogs, and will ultimately allow for more targeted generation of functionally informative double mutants, advancing functional genomic studies.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2019

De novo design of symmetric ferredoxins that shuttle electrons in vivo

A symmetric origin for bacterial ferredoxins was first proposed over 50 y ago, yet, to date, no functional symmetric molecule has been constructed. It is hypothesized that extant proteins have drifted from their symmetric roots via gene duplication followed by mutations. Phylogenetic analyses of extant ferredoxins support the independent evolution of N- and C-terminal sequences, thereby allowing consensus-based design of symmetric 4Fe-4S molecules. All designs bind two [4Fe-4S] clusters and exhibit strongly reducing midpoint potentials ranging from −405 to −515 mV. One of these constructs efficiently shuttles electrons through a designed metabolic pathway in Escherichia coli. These finding establish that ferredoxins consisting of a symmetric core can be used as a platform to design novel electron transfer carriers for in vivo applications. Outer-shell asymmetry increases sequence space without compromising electron transfer functionality.

Andrew C. Mutter↗
DOE OSTI2023

Inhibition of polyamine biosynthesis preserves β cell function in type 1 diabetes

In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing β cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with β cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during β cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125–1,000 mg/m 2 ) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve β cell function in T1D through islet cell-autonomous effects.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Combining chemistry and protein engineering for new-to-nature biocatalysis

Biocatalysis, the application of enzymes to solve synthetic problems of human import, has blossomed into a powerful technology for chemical innovation. In the past decade, a threefold partnership, where nature provides blueprints for enzymatic catalysis, chemists introduce innovative activity modes with abiological substrates, and protein engineers develop new tools and algorithms to tune and improve enzymatic function, has unveiled the frontier of new-to-nature enzyme catalysis. In this Perspective, we highlight examples of interdisciplinary studies, which have helped to expand the scope of biocatalysis, including concepts of enzymatic versatility explored through the lens of biomimicry, to achieve activities and selectivities beyond those currently possible with chemocatalysis. We indicate how modern tools, such as directed evolution, computational protein design and machine learning-based protein engineering methods, have already impacted and will continue to influence enzyme engineering for new abiological transformations. As a result, a sustained collaborative effort across disciplines is anticipated to spur further advances in biocatalysis in the coming years.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1999

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

Non-NASA Center↗
DOE OSTI2024

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family

Proteins from the small multidrug resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in the metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid-supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologs. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologs, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homolog, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homolog Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2002

XCTK1: A Xenopus C-terminal Kinesin-like Protein

XCTK1 is 97kDa kinesin-like protein homologous to FKIF2 and KIFC3. XCTK1 is present at picomolar levels in eggs, embryos and cultured cells in a soluble high-molecular weight complex that is not associated with membranes. XCKT1 localizes to centrosomes in Xenopus A6 cells. Anti-XCTK1 antibodies also localize to spindle poles when injected into A6 cells or when added to extracts during in vitro spindle assembly reactions. XCTK1 is associated with the center of taxol-induced microtubule asters in extracts. Therefore its localization to poles is dependent on microtubule minus-ends and not on centrosomes per se. Overexpression of XCTK1 leads to centrosome destruction in cultured cells. XCTK1 was tagged at either the N- or C-terminus and transfected into Xenopus A6 cells At low expression levels, XCTK1 associated with centrosomes. At higher levels, the protein localized to insoluble cytoplasmic structures. Gamma-tubulin staining was dramatically decreased from centrosomes or altogether absent. The centrosomal SPJ antigen colocalized with XCTK1-containing structures. Upon nocodozole treatment, microtubules failed to regrow from the centrosomes indicating that overexpression of XCTK1 severely compromises centrosomal function. Current studies are aimed at determining whether XCTK1 interacts directly with centrosomal proteins and to determine the effects of XCTK1 depletion on oocyte maturation and embryogenesis.

Winfree, Seth↗