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424 records · Page 24
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Genome-scale model development and genomic sequencing of the oleaginous clade Lipomyces

The Lipomyces clade contains oleaginous yeast species with advantageous metabolic features for biochemical and biofuel production. Limited knowledge about the metabolic networks of the species and limited tools for genetic engineering have led to a relatively small amount of research on the microbes. Here, a genome-scale metabolic model (GSM) of Lipomyces starkeyi NRRL Y-11557 was built using orthologous protein mappings to model yeast species. Phenotypic growth assays were used to validate the GSM (66% accuracy) and indicated that NRRL Y-11557 utilized diverse carbohydrates but had more limited catabolism of organic acids. The final GSM contained 2,193 reactions, 1,909 metabolites, and 996 genes and was thus named iLst996. The model contained 96 of the annotated carbohydrate-active enzymes. iLst996 predicted a flux distribution in line with oleaginous yeast measurements and was utilized to predict theoretical lipid yields. Twenty-five other yeasts in the Lipomyces clade were then genome sequenced and annotated. Sixteen of the Lipomyces species had orthologs for more than 97% of the iLst996 genes, demonstrating the usefulness of iLst996 as a broad GSM for Lipomyces metabolism. Pathways that diverged from iLst996 mainly revolved around alternate carbon metabolism, with ortholog groups excluding NRRL Y-11557 annotated to be involved in transport, glycerolipid, and starch metabolism, among others. Overall, this study provides a useful modeling tool and data for analyzing and understanding Lipomyces species metabolism and will assist further engineering efforts in Lipomyces .

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 OSTI2023

Site specific N- and O-glycosylation mapping of the spike proteins of SARS-CoV-2 variants of concern

Abstract The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N -terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N - and O - glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1992

The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

Non-NASA Center↗
NASA NTRS2003

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

NASA Discipline Evolutionary Biology↗
NASA NTRS2003

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.

Non-NASA Center↗
DOE OSTI2023

A High-Quality Genome-Scale Model for Rhodococcus opacus Metabolism

Rhodococcus opacus is a bacterium that has a high tolerance to aromatic compounds and can produce significant amounts of triacylglycerol (TAG). Here, we present iGR1773, the first genome-scale model (GSM) of R. opacus PD630 metabolism based on its genomic sequence and associated data. The model includes 1773 genes, 3025 reactions, and 1956 metabolites, was developed in a reproducible manner using CarveMe, and was evaluated through Metabolic Model tests (MEMOTE). We combine the model with two Constraint-Based Reconstruction and Analysis (COBRA) methods that use transcriptomics data to predict growth rates and fluxes: E-Flux2 and SPOT (Simplified Pearson Correlation with Transcriptomic data). Growth rates are best predicted by E-Flux2. Flux profiles are more accurately predicted by E-Flux2 than flux balance analysis (FBA) and parsimonious FBA (pFBA), when compared to 44 central carbon fluxes measured by 13C-Metabolic Flux Analysis (13C-MFA). Under glucose-fed conditions, E-Flux2 presents an R2 value of 0.54, while predictions based on pFBA had an inferior R2 of 0.28. We attribute this improved performance to the extra activity information provided by the transcriptomics data. For phenol-fed metabolism, in which the substrate first enters the TCA cycle, E-Flux2’s flux predictions display a high R2 of 0.96 while pFBA showed an R2 of 0.93. We also show that glucose metabolism and phenol metabolism function with similar relative ATP maintenance costs. These findings demonstrate that iGR1773 can help the metabolic engineering community predict aromatic substrate utilization patterns and perform computational strain design.

Roell, Garrett W.↗
DOE OSTI2024

MjCyc: Rediscovering the pathway-genome landscape of the first sequenced archaeon, Methanocaldococcus (Methanococcus) jannaschii

The genome of Methanocaldococcus (Methanococcus) jannaschii DSM 2661 was the first Archaeal genome to be sequenced in 1996. Subsequent sequence-based annotation cycles led to its first metabolic reconstruction in 2005. Leveraging new experimental results and function assignments, we have now re-annotated M. jannaschii, creating an updated resource with novel information and testable predictions in a pathway-genome database available at BioCyc.org. This reannotation effort has resulted in 652 function assignments with enzyme roles, accounting for a third of the total protein-coding entries for this genome. The updated resource includes 883 reactions, 540 enzymes, and 142 individual pathways. Despite notable progress in computational genomics, more than a third of the genome remains functionally uncharacterized. The publicly available MjCyc pathway-genome database holds great potential for the wider community to conduct research on the biology of methanogenic Archaea.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.

Cytochrome c Group/chemistry/genetics/metabolism↗
NASA NTRS1999

Correlation of Fos expression and circling asymmetry during gerbil vestibular compensation

Vestibular compensation is a central nervous system process resulting in recovery of functional movement and control following a unilateral vestibular lesion. Small pressure injections of phosphorothioate 20mer oligonucleotides were used to probe the role of the Fos transcription protein during vestibular compensation in the gerbil brainstem. During isoflurane gas anesthesia, antisense probes against the c-fos mRNA sequence were injected into the medial vestibular and prepositus nuclei unilaterally prior to a unilateral surgical labyrinthectomy. Anionic dyes, which did not interact with the oligonucleotides, were used to mark the injection site and help determine the extent of diffusion. The antiFos oligonucleotide injections reduced Fos expression at the injection site in neurons which normally express Fos after the lesion, and also affected circling behavior induced by hemilabyrinthectomy. With both ipsilateral and contralateral medial vestibular and prepositus nuclei injections, less ipsilateral and more contralateral circling was noted in animals injected with antiFos injections as compared to non-injected controls. The degree of change in these behaviors was dependent upon the side of the injection. Histologically, antiFos injections reduced the number of Fos immunolabeled neurons around the injection site, and increased Fos expression contralaterally. The correlation of the number of neurons with Fos expression to turning behavior was stronger for contralateral versus ipsilateral turns, and for neurons in the caudal and ipsilateral sub-regions of the medial vestibular and prepositus nuclei. The results are discussed in terms of neuronal firing activity versus translational activity based on the asymmetrical expression of the Fos inducible transcription factor in the medial vestibular and prepositus nuclei. Although ubiquitous in the brain, transcription factors like Fos can serve localized and specific roles in sensory-specific adaptive stimuli. Antisense injections can be an effective procedure for localized intervention into complex physiological functions, e.g. vestibular compensation. Copyright 1999 Elsevier Science B.V.

NASA Discipline Neuroscience↗