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At least 163 records · Page 9

All sources NASA NTRS DOE OSTI

DOE OSTI2017

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution

Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. Here, we mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of these pruned enzymes with a series of substrates. A substantial rate enhancement of ~1011-fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7 –10 8 -fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1996

Positive selection moments identify potential functional residues in human olfactory receptors

Correlated mutation analysis and molecular models of olfactory receptors have provided evidence that residues in the transmembrane domains form a binding pocket for odor ligands. As an independent test of these results, we have calculated positive selection moments for the alpha-helical sixth transmembrane domain (TM6) of human olfactory receptors. The moments can be used to identify residues that have been preferentially affected by positive selection and are thus likely to interact with odor ligands. The results suggest that residue 622, which is commonly a serine or threonine, could form critical H-bonds. In some receptors a dual-serine subsite, formed by residues 622 and 625, could bind hydroxyl determinants on odor ligands. The potential importance of these residues is further supported by site-directed mutagenesis in the beta-adrenergic receptor. The findings should be of practical value for future physiological studies, binding assays, and site-directed mutagenesis.

Non-NASA Center↗

DOE OSTI2018

Identification of ATIC as a Novel Target for Chemoradiosensitization

Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

NASA NTRS1999

Mutations in a new Arabidopsis cyclophilin disrupt its interaction with protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) is a component of multiple signaling pathways in eukaryotes. Disruption of PP2A activity in Arabidopsis is known to alter auxin transport and growth response pathways. We demonstrated that the regulatory subunit A of an Arabidopsis PP2A interacts with a novel cyclophilin, ROC7. The gene for this cyclophilin encodes a protein that contains a unique 30-amino acid extension at the N-terminus, which distinguishes the gene product from all previously identified Arabidopsis cyclophilins. Altered forms of ROC7 cyclophilin with mutations in the conserved DENFKL domain did not bind to PP2A. Unlike protein phosphatase 2B, PP2A activity in Arabidopsis extracts was not affected by the presence of the cyclophilin-binding molecule cyclosporin. The ROC7 transcript was expressed to high levels in all tissues tested. Expression of an ROC7 antisense transcript gave rise to increased root growth. These results indicate that cyclophilin may have a role in regulating PP2A activity, by a mechanism that differs from that employed for cyclophilin regulation of PP2B.

NASA Discipline Plant Biology↗

DOE OSTI2023

The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

Abstract Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans , which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2 , respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1 . In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Cyanobacterial photosystem II reaction center design in tobacco chloroplasts increases biomass in low light

Abstract The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Q) in the D1 polypeptide (NtLL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 μE m−2 s−1, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (NtHL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin-resistance gene.

Zhang, Yuan (ORCID:0000000291785207)↗

NASA NTRS1998

Radiation-induced instability and its relation to radiation carcinogenesis

PURPOSE: A model that identifies radiation-induced genetic instability as the earliest cellular event in the multi-step sequence leading to radiation-induced cancer was previously proposed. In this paper ongoing experiments are discussed which are designed to test this model and its predictions in mouse mammary epithelial cells. RESULTS: Several lines of evidence are presented that appear to support this model: first, the development of delayed mutations in p53 following irradiation in altered growth variants; secondly, the high frequencies for the induction of both instability and transformation following irradiation in mammary epithelial cells; and finally, the demonstration that susceptibility to the induction of cytogenetic instability is a heritable trait that correlates with susceptibility to transformation and radiation-induced mammary cancer. Mice resistant to transformation and mammary cancer development are also resistant to the development of instability after irradiation. In contrast, mice sensitive to transformation and cancer are also sensitive to the development of cytogenetic instability. CONCLUSIONS: Data from this laboratory and from the studies cited above suggest a specific, and perhaps unique, role for radiation-induced instability as a critical early event associated with initiation of the carcinogenic process.

Review↗

DOE OSTI2022

Trajectories for the evolution of bacterial CO 2 -concentrating mechanisms

Cyanobacteria rely on CO 2 -concentrating mechanisms (CCMs) to grow in today’s atmosphere (0.04% CO 2 ). These complex physiological adaptations require ≈15 genes to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+ nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air. If any plausible ancestral form—i.e., lacking a single gene—cannot grow, how did the CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was “catalyzed” by historically high CO 2 levels that decreased over geologic time. Using an E. coli reconstitution of a bacterial CCM, we constructed strains lacking one or more CCM components and evaluated their growth across CO 2 concentrations. We expected these experiments to demonstrate the importance of the carboxysome. Instead, we found that partial CCMs expressing CA or Ci uptake genes grew better than controls in intermediate CO 2 levels (≈1%) and observed similar phenotypes in two autotrophic bacteria, Halothiobacillus neapolitanus and Cupriavidus necator . To understand how CA and Ci uptake improve growth, we model autotrophy as colimited by CO 2 and HCO 3 − , as both are required to produce biomass. Our experiments and model delineated a viable trajectory for CCM evolution where decreasing atmospheric CO 2 induces an HCO 3 − deficiency that is alleviated by acquisition of CA or Ci uptake, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity.

Flamholz, Avi I.↗

NASA NTRS1992

Space Exposed Experiment Developed for Students (SEEDS) (P0004-2)

SEEDS, a cooperative endeavor of NASA Headquarters, the NASA Langley Research Center, and the George W. Park Seed Company, resulted in the distribution of approximately 132,000 SEEDS kits to 3.3 million students. Kits contained Rutger's tomato seeds that had flown on the Long Duration Exposure Facility (LDEF), as well as seeds that had been stored in a climate controlled warehouse for the same time period. Preliminary data indicates the germination rate for space exposed seeds was 73.8 percent while Earth based seeds germinated at a rate of 70.3 percent. Tests conducted within the first six months after retrieval indicated space exposed seeds germinated in an average of 8.0 days, while Earth based seeds' average germination time was 8.3 days. Some mutations (assumed to be radiation induced) include plants that added a leaf instead of the usual flower at the end of the flower frond. Also, fruit produced from a flower with a variegated calyx bore seeds producing albino plants, while fruit from a flower with a green calyx from the same plant bore seeds producing green plants.

Grigsby, Doris K.↗

DOE OSTI2018

SAPAP4 Deletion Causes Synaptic Dysfunction in the nucleus accumbens

Highlights: • SAPAP4 is a postsynaptic scaffolding protein at excitatory synapses. • SAPAP4−/− mice display decreased cocaine-induced locomotor activity. • The spine density of medium spiny neurons in the nucleus accumbens shell is lower in SAPAP4−/− mice than in wild-type mice. • SAPAP4 plays a critical role in the synaptic function of the nucleus accumbens. SAP90/PSD95-associated proteins (SAPAPs) are one type of scaffold protein in the postsynaptic density (PSD). Scaffold proteins play an important role in synaptic function. Recently, many studies have shown that mutations associated with scaffold proteins cause dysfunction in neuronal circuitry and in behavior. SAPAP4, as a protein in the SAPAP family, may have an impact on synaptic functions and on behaviors. To test this hypothesis, mice with a genetic deletion of SAPAP4 were used in our study. SAPAP4−/− mice displayed decreased cocaine sensitivity behavior after an acute injection of 20 mg/kg cocaine. We also found that the spine density of medium spiny neurons (MSNs) in the nucleus accumbens (NAc) shell was reduced in SAPAP4−/− mice. Furthermore, SAPAP4−/− mice displayed altered synaptic transmission and a decreased frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in the NAc. Our findings demonstrate that SAPAP4 plays a critical role in cocaine-related behavior and in the synaptic function of the NAc.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1991

Space Exposed Experiment Developed for Students (SEEDS) P-0004-2

This cooperative endeavor of NASA Headquarters, the NASA Langley Research Center, and the George W. Park Seed Company, resulted in the distribution, by the end of March, 1990, of approximately 132,000 space exposed experiment developed for students (SEEDS) kits to 64,000 teachers representing 40,000 classrooms and 3.3 million kindergarden through university students. Kits were sent to every state, as well as to 30 foreign countries. Preliminary radiation data indicates that layer A received 725 rads, while layer D received 350 rads. Germination rate was reported to be 73.8 percent for space exposed seeds and 70.3 percent for earth based control seeds. Tests conducted within the first six months after retrieval indicated space exposed seeds germinated in an average of 8.0 days, while earth based control seeds' average germination rate was 8.3 days. Some mutations (assumed to be radiation induced) reported by students and Park Seed include plants that added a leaf instead of the usual flower at the end of the flower front and fruit produced from a flower with a variegated calyx bore seeds producing albino plants, while fruit from a flower with a green calyx from the same plant bore seeds produced green plants.

Grigsby, Doris K.↗

DOE OSTI2021

Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury

The signaling of cells by scaffolds of synthetic molecules that mimic proteins is known to be effective in the regeneration of tissues. Here, we describe peptide amphiphile supramolecular polymers containing two distinct signals and test them in a mouse model of severe spinal cord injury. One signal activates the transmembrane receptor β1-integrin and a second one activates the basic fibroblast growth factor 2 receptor. By mutating the peptide sequence of the amphiphilic monomers in nonbioactive domains, we intensified the motions of molecules within scaffold fibrils. This resulted in notable differences in vascular growth, axonal regeneration, myelination, survival of motor neurons, reduced gliosis, and functional recovery. Here, we hypothesize that the signaling of cells by ensembles of molecules could be optimized by tuning their internal motions.

36 MATERIALS SCIENCE↗

DOE OSTI2018

Comparative genomics, infectivity and cytopathogenicity of Zika viruses produced by acutely and persistently infected human hematopoietic cell lines

Zika virus (ZIKV), an arthropod-borne virus, has emerged as a major human pathogen. Prolonged or persistent ZIKV infection of human cells and tissues may serve as a reservoir for the virus and present serious challenges to the safety of public health. Human hematopoietic cell lines with different developmental properties revealed differences in susceptibility and outcomes to ZIKV infection. In three separate studies involving the prototypic MR 766 ZIKV strain and the human monocytic leukemia U937 cell line, ZIKV initially developed only a low-grade infection at a slow rate. After continuous culture for several months, persistently ZIKV-infected cell lines were observed with most, if not all, cells testing positive for ZIKV antigen. The infected cultures produced ZIKV RNA (v-RNA) and infectious ZIKVs persistently (“persistent ZIKVs”) with distinct infectivity and pathogenicity when tested using various kinds of host cells. When the genomes of ZIKVs from the three persistently infected cell lines were compared with the genome of the prototypic MR 766 ZIKV strain, distinct sets of mutations specific to each cell line were found. Significantly, all three “persistent ZIKVs” were capable of infecting fresh U937 cells with high efficiency at rapid rates, resulting in the development of a new set of persistently ZIKV-infected U937 cell lines. The genomes of ZIKVs from the new set of persistently ZIKV-infected U937 cell lines were further analyzed for their different mutations. The 2nd generation of persistent ZIKVs continued to possess most of the distinct sets of mutations specific to the respective 1st generation of persistent ZIKVs. We anticipate that the study will contribute to the understanding of the fundamental biology of adaptive mutations and selection during viral persistence. The persistently ZIKVinfected human cell lines that we developed will also be useful to investigate critical molecular pathways of ZIKV persistence and to study drugs or countermeasures against ZIKV infections and transmission.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1991

Controlled evolution of an RNA enzyme

It is generally thought that prior to the origin of protein synthesis, life on earth was based on self-replicating RNA molecules. This idea has become especially popular recently due to the discovery of catalytic RNA (ribozymes). RNA has both genotypic and phenotypic properties, suggesting that it is capable of undergoing Darwinian evolution. RNA evolution is likely to have played a critical role in the early history of life on earth, and thus is important in considering the possibility of life elsewhere in the solar system. We have constructed an RNA-based evolving system in the laboratory, combining amplification and mutation of an RNA genotype with selection of a corresponding RNA phenotype. This system serves as a functional model of a primitive organism. It can also be used as a tool to explore the catalytic potential of RNA. By altering the selection constraints, we are attempting to modify the substrate specificity of an existing ribozyme in order to develop ribozymes with novel catalytic function. In this way, we hope to gain a better understanding of RNA's catalytic versatility and to assess its suitability for the role of primordial catalyst. All of the RNA enzymes that are known to exist in contemporary biology carry out cleavage/ligation reactions involving RNA substrates. The Tetrahymena ribozyme, for example, catalyzes phosphoester transfer between a guanosine containing and an oligopyrimidine containing substrate. We tested the ability of mutant forms of the Tetrahymena ribozyme to carry out a comparable reaction using DNA, rather than RNA substrate. An ensemble of structural variants of the ribozyme was prepared and tested for their ability to specifically cleave d(GGCCCTCT-A3TA3TA) at the phosphodiester bond following the sequence CCCTCT. We recovered a mutant form of the enzyme that cleaves DNA more efficiently than does the wild-type. Beginning with this selected mutant we have now scattered random mutations throughout the ribozyme and have begun an evolutionary search to further expand the catalytic repertoire of RNA.

Joyce, G. F.↗

DOE OSTI2018

Understanding the Species Selectivity of Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors

To test for on target toxicity of a new chemical entity, it is important to have comparable binding affinities of the compound in the target proteins from humans and the test species. To evaluate our myeloid cell leukemia-1 (Mcl-1) inhibitors, we tested them against rodent Mcl-1 and found a significant loss of binding affinity when compared to that seen with human Mcl-1. To understand the affinity loss, we used sequence alignments and structures of human Mcl-1/inhibitor complexes to identify the important differences in the amino acid sequences. One difference is human L246 (F226 in rat, F227 in mouse) in the ligand binding pocket. Mutating rat F226 to a Leu restores affinity, but the mouse F227L mutant still has a ligand affinity that is lower than that of human Mcl-1. Another mutation of mouse F267, located ~12 Å from the ligand pocket, to the human/rat cysteine, F267C, improved the affinity and combined with F227L resulted in a mutant mouse protein with a binding affinity similar to that of human Mcl-1. To help understand the structural components of the affinity loss, we obtained an X-ray structure of a mouse Mcl-1/inhibitor complex and identified how the residue changes reduced compound complementarity. Finally, we tested Mcl-1 of other preclinical animal models (canine, monkey, rabbit, and ferret) that are identical to humans in terms of these two residues and found that their Mcl-1 bound our compounds with affinities comparable to that of human Mcl-1. Furthermore, these results have implications for understanding ligand selectivity for similar proteins and for the interpretation of preclinical toxicology studies with Mcl-1 inhibitors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2026

High-Sensitivity DNA Aptasensors for Detecting Salivary Biomarker S100A7 in Heart Failure

Early detection of heart failure (HF) is vital for improving patient outcomes, lowering hospital readmission rates, and enabling prompt treatment. We present the first high-affinity DNA aptamer for the salivary HF biomarker S100A7 and its application in highly sensitive, noninvasive diagnostic tests. Iterative truncation of the initial 82-nt aptamer (17–82) produced a 43-nt core (17–43) with a binding affinity of 27 nM, which was further enhanced to 5.5 nM through dimerization. Biochemical and mutational studies confirmed that 17–43 adopts a G-quadruplex structure, which is essential for S100A7 recognition and resistance to enzymatic degradation in human saliva. Incorporating 17–43 into sandwich aptamer-ELISA and hybrid aptamer–antibody ELISA assays allowed detection of recombinant S100A7 in human saliva with limits of detection (LOD) of 7.4 ng mL–1 (0.6 nM) and 29 pg/mL (2.2 pM), respectively-outperforming commercial immunoassays in both sensitivity and dynamic range. The hybrid assay maintained its full performance after 2.5 months of room temperature storage. Additionally, a biolayer interferometry (BLI) sensor with 17–43 quantified S100A7 in patient saliva (n = 3), achieving a LOD of 3.2 ng mL–1 (0.3 nM) with a total assay time of less than 20 min. The aptamer’s stability, high specificity, and versatility across biosensing platforms establish it as a promising tool for noninvasive heart failure diagnostics, laying the groundwork for portable aptamer-based biosensors for multiplexed monitoring of HF biomarkers.

DNA aptamer↗

NASA NTRS2005

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.

Non-NASA Center↗

DOE OSTI2024

Investigating the mechanism of chloroplast singlet oxygen signaling in the Arabidopsis thaliana accelerated cell death 2 mutant

As sessile organisms, plants have evolved complex signaling mechanisms to sense stress and acclimate. This includes the use of reactive oxygen species (ROS) generated during dysfunctional photosynthesis to initiate signaling. One such ROS, singlet oxygen ( 1 O 2 ), can trigger retrograde signaling, chloroplast degradation, and programmed cell death. However, the signaling mechanisms are largely unknown. Several proteins (e.g. PUB4, OXI1, EX1) are proposed to play signaling roles across three Arabidopsis thaliana mutants that conditionally accumulate chloroplast 1 O 2 (fluorescent in blue light (flu), chlorina 1 (ch1), and plastid ferrochelatase 2 (fc2)). We previously demonstrated that these mutants reveal at least two chloroplast 1 O 2 signaling pathways (represented by flu and fc2/ch1). Here, we test if the 1 O 2 -accumulating lesion mimic mutant, accelerated cell death 2 (acd2), also utilizes these pathways. The pub4–6 allele delayed lesion formation in acd2 and restored photosynthetic efficiency and biomass. Conversely, an oxi1 mutation had no measurable effect on these phenotypes. acd2 mutants were not sensitive to excess light (EL) stress, yet pub4–6 and oxi1 both conferred EL tolerance within the acd2 background, suggesting that EL-induced 1 O 2 signaling pathways are independent from spontaneous lesion formation. Thus, 1 O 2 signaling in acd2 may represent a third (partially overlapping) pathway to control cellular degradation.

59 BASIC BIOLOGICAL SCIENCES↗