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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

The congenital APOA1 K107del mutation disrupts the lipid-free conformation of monomeric APOA1 and impairs oligomerization

Apolipoprotein A-I (APOA1) oligomerization is thought to be essential for high-density lipoprotein (HDL) formation and metabolism. Naturally occurring mutations can disrupt normal APOA1 folding and self-association, leading to dysfunctional HDL formation and cardiovascular disease. The congenital APOA1 variant p.K131del (APOA1 K107del ) has been associated with cardiovascular pathologies such as low HDL-cholesterol levels and aortic amyloidosis, and multiple studies indicate structural changes in APOA1 conformation underlie associated dysfunction. In the current study, we confirmed that APOA1 K107del exhibits no notable defect in lipid-binding. However, using polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC), we found that loss of lysine 107 resulted in a remarkable shift in the distribution of APOA1 oligomers with a much higher proportion of monomers present in APOA1 K107del compared to wild-type APOA1. Further investigation using quantitative cross-linking revealed a major disruption of interactions in helical regions reported to participate in domain swaps necessary for proper self-association. This structural disruption appears to impair N- and C-termini interactions and dynamics that lead to non-specific aggregation. In conclusion, these findings support the hypothesis that lysine 107 is critical for proper folding and self-association of lipid-free APOA1 which could impact HDL biogenesis.

APOA1 (p.K131del)↗
DOE OSTI2024

RNA language models predict mutations that improve RNA function

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. RNA structure prediction is not yet possible due to a lack of high-quality reference data associated with organismal phenotypes that could inform RNA function. We present GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences to experimental and predicted optimal growth temperatures of GTDB reference organisms. Using GARNET, we develop sequence- and structure-aware RNA generative models, with overlapping triplet tokenization providing optimal encoding for a GPT-like model. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identify mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Evidence for accelerated aging in mammary epithelia of women carrying germline BRCA1 or BRCA2 mutations

During aging in the human mammary gland, luminal epithelial cells lose lineage fidelity by expressing markers normally expressed in myoepithelial cells. We hypothesize that loss of lineage fidelity is a general manifestation of epithelia that are susceptible to cancer initiation. In the present study, we show that histologically normal breast tissue from younger women who are susceptible to breast cancer, as a result of harboring a germline mutation in BRCA1, BRCA2 or PALB2 genes, exhibits hallmarks of accelerated aging. These include proportionately increased luminal epithelial cells that acquired myoepithelial markers, decreased proportions of myoepithelial cells and a basal differentiation bias or failure of differentiation of cKit + progenitors. High-risk luminal and myoepithelial cells are transcriptionally enriched for genes of the opposite lineage, inflammatory- and cancer-related pathways. We have identified breast-aging hallmarks that reflect a convergent biology of cancer susceptibility, regardless of the specific underlying genetic or age-dependent risk or the associated breast cancer subtype.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Interrogating biomineralization one amino acid at a time: amplification of mutational effects in protein-aided titania morphogenesis through reaction-diffusion control

To emulate the control that biomineralizing organisms exert over reactant transport, we construct a countercurrent reaction-diffusion chamber in which an agarose hydrogel regulates the fluxes of inorganic precursor and precipitating solid-binding protein. Furthermore, we show that the morphology of the bioprecipitated titania can be changed from monolithic to interconnected particle networks and dispersed nanoparticles either by decreasing reaction time or by increasing agarose weight percentage at constant precursor and protein concentrations. More strikingly, protein variants with one or two substitutions in their metal oxide-binding domain yield unique peripheral morphologies (needles, threads, plates, and peapods) with distinct crystallography and photocatalytic activity. Our results suggest that diffusional control can magnify otherwise subtle mutational effects in biomineralizing proteins and provide a path for the green synthesis of morphologically and functionally diverse inorganic materials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Variable domain mutational analysis to probe the molecular mechanisms of high viscosity of an IgG 1 antibody

Subcutaneous injection is the preferred route of administration for many antibody therapeutics for reasons that include its speed and convenience. However, the small volume limit (typically ≤2 mL) for subcutaneous delivery often necessitates antibody formulations at high concentrations (commonly ≥100 mg/mL), which may lead to physicochemical problems. For example, antibodies with large hydrophobic or charged patches can be prone to self-interaction giving rise to high viscosity. Here, we combined X-ray crystallography with computational modeling to predict regions of an anti-glucagon receptor (GCGR) IgG 1 antibody prone to self-interaction. An extensive mutational analysis was undertaken of the complementarity-determining region residues residing in hydrophobic surface patches predicted by spatial aggregation propensity, in conjunction with residue-level solvent accessibility, averaged over conformational ensembles from molecular dynamics simulations. Dynamic light scattering (DLS) was used as a medium throughput screen for self-interaction of ~200 anti-GCGR IgG 1 variants. A negative correlation was found between the viscosity determined at high concentration (180 mg/mL) and the DLS interaction parameter measured at low concentration (2–10 mg/mL). Additionally, anti-GCGR variants were readily identified with reduced viscosity and antigen-binding affinity within a few fold of the parent antibody, with no identified impact on overall developability. The methods described here may be useful in the optimization of other antibodies to facilitate their therapeutic administration at high concentration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

PYK-SubstitutionOME: an integrated database containing allosteric coupling, ligand affinity and mutational, structural, pathological, bioinformatic and computational information about pyruvate kinase isozymes

Interpreting changes in patient genomes, understanding how viruses evolve and engineering novel protein function all depend on accurately predicting the functional outcomes that arise from amino acid substitutions. To that end, the development of first-generation prediction algorithms was guided by historic experimental datasets. However, these datasets were heavily biased toward substitutions at positions that have not changed much throughout evolution (i.e. conserved). Although newer datasets include substitutions at positions that span a range of evolutionary conservation scores, these data are largely derived from assays that agglomerate multiple aspects of function. To facilitate predictions from the foundational chemical properties of proteins, large substitution databases with biochemical characterizations of function are needed. We report here a database derived from mutational, biochemical, bioinformatic, structural, pathological and computational studies of a highly studied protein family—pyruvate kinase (PYK). A centerpiece of this database is the biochemical characterization—including quantitative evaluation of allosteric regulation—of the changes that accompany substitutions at positions that sample the full conservation range observed in the PYK family. We have used these data to facilitate critical advances in the foundational studies of allosteric regulation and protein evolution and as rigorous benchmarks for testing protein predictions. We trust that the collected dataset will be useful for the broader scientific community in the further development of prediction algorithms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A semidominant point mutation of Mediator tail subunit MED5b in Arabidopsis leads to altered enrichment of H3K27me3 and reduced expression of targets of MYC2

Abstract The Mediator complex coordinates regulatory input for transcription driven by RNA polymerase II in eukaryotes. reduced epidermal fluorescence4-3 (ref4-3) is a semidominant mutation that results in a single amino acid substitution in the Mediator tail subunit Med5b. Previous characterization of ref4-3 revealed altered expression of a variety of loci in Arabidopsis, including those contributing to phenylpropanoid biosynthesis. Examination of existing RNA-seq data indicated that loci enriched for the transcriptionally repressive chromatin modification H3K27me3 are overrepresented among genes that are misregulated in ref4-3. We used ChIP-seq and RNA-seq to examine the possibility that perturbation of H3K27me3 homeostasis in ref4-3 plants contributed to altered transcript levels. We observed that ref4-3 results in a modest global reduction of H3K27me3 at enriched loci and that this reduction is not dependent on gene expression; however, altered H3K27me3 was not strongly predictive of altered expression in ref4-3 plants. Instead, our analyses revealed a substantial enrichment of targets of the MYC2 transcriptional regulator among genes that exhibit decreased expression in ref4-3. Consistent with previous characterization of ref4-3, we observed that ref4-3-dependent decreased expression of MYC2 targets can be suppressed by loss of another Mediator tail subunit, MED25. This observation is consistent with previous biochemical characterization of MYC2. Our data highlight the diverse and distinct impacts that a single amino acid change in the tail subunit of Mediator can have on transcriptional circuits and raise the prospect that Mediator directly contributes to H3K27me3 homeostasis in plants.

Long, Jiaxin (ORCID:0000000335150706)↗
DOE OSTI2025

Mutations in the proximal binding site and F-loop of AdeJ confer resistance to efflux pump inhibitors

Multidrug efflux is one of the major mechanisms of antibiotic resistance in gram-negative bacteria. Inhibitors of efflux pumps potentiate the activities of antibiotics, and their discovery could lead to new therapeutic options. The AdeIJK pump in Acinetobacter baumannii is a promising target for efflux pump inhibitors (EPIs) due to its high clinical importance and conservation, and several classes of EPIs targeting this and other A. baumannii efflux transporters have been recently reported. However, the mechanisms of action of these EPIs and their resistance liability remain underexplored. Here, we analyzed the impact of site-specific substitutions in the substrate/EPI translocation path of the inner membrane transporter AdeJ on efflux of substrate antibiotics and fluorescent probes and activities of substituted 4,6-diaminoquinoline EPIs. We found that substitutions in amino acid residues located in the entrance cleft (R701) and the flexible loop (E675) of AdeJ lead to resistance specifically against biphenyl-substituted EPIs, whereas the substitution of F178 in the distal binding pocket increased AdeJ sensitivity to certain naphthyl- and biphenyl-substituted EPIs. No major differences in docking scores and poses of substrates and EPIs were observed between the wild type and corresponding AdeJ variants for any of the mutations considered. This study concludes that substrates and EPIs bound along the translocation path of AdeJ participate in its conformational transitions and can either increase or decrease the rate of transport and therefore the efficiency of EPIs.

Acinetobacter↗
DOE OSTI2025

Revisiting synthetic lethality of Gcn5-related N-acetyltransferase (GNAT) family mutations in Haloferax volcanii

ABSTRACT Lysine acetylation is a post-translational modification that occurs in all domains of life, highlighting its evolutionary significance. Previous genome comparison identified three Gcn5-related N-acetyltransferase (GNAT) family members as lysine acetyltransferase homologs (Pat1, Pat2, and Elp3) and two deacetylase homologs (Sir2 and HdaI) in the halophilic archaeonHaloferax volcanii, withelp3andpat2proposed as a synthetic lethal gene pair. Here, we advance these findings by performing single and double mutagenesis ofelp3with thepat1andpat2lysine acetyltransferase gene homologs. Genome sequencing and PCR screens of these strains reveal successful generation of Δelp3,Δpat1Δelp3, and Δpat2Δelp3mutant strains. Although these mutant strains exhibited a reduced growth rate compared to the parent, they remained viable. Overall, this study provides genetic evidence thatelp3andpat2, while impacting cell growth, are not a synthetic lethal gene pair as previously reported. IMPORTANCE Here, we reveal by whole-genome sequencing that the GNAT family gene homologselp3andpat2can be deleted in the sameHaloferax volcaniistrain. Beyond the targeted deletions, minimal differences between the parent and Δelp3Δpat2mutant were observed, suggesting that suppressor mutations are not responsible for our ability to generate this double mutant strain. Elp3 and Pat2, thus, may not share as close a functional relationship as implied by earlier study. Our finding is significant as Elp3 is thought to function in acetylation in tRNA modification, while Pat2 likely functions in the lysine acetylation of proteins.

Microbiology↗
DOE OSTI2024

Novel mutation leading to splice donor loss in a conserved site of DMD gene causes Duchenne muscular dystrophy with cryptorchidism

Background As one of the most common congenital abnormalities in male births, cryptorchidism has been found to have a polygenic aetiology according to previous studies of common variants. However, little is known about genetic predisposition of rare variants for cryptorchidism, since rare variants have larger effective size on diseases than common variants. Methods In this study, a cohort of 115 Chinese probands with cryptorchidism was analysed using whole-genome sequencing, alongside 19 parental controls and 2136 unaffected men. Additionally, CRISPR-Cas9 editing of a conserved variant was performed in a mouse model, with MRI screening used to observe the phenotype. Results In 30 of 115 patients (26.1%), we identified four novel genes ( ARSH , DMD , MAGEA4 and SHROOM2 ) affecting at least five unrelated patients and four known genes ( USP9Y , UBA1 , BCORL1 and KDM6A ) with the candidate rare pathogenic variants affecting at least two cases. Burden tests of rare variants revealed the genome-wide significances for newly identified genes (p<2.5×10 −6 ) under the Bonferroni correction. Surprisingly, novel and known genes were mainly found on X chromosome (seven on X and one on Y) and all rare X-chromosomal segregating variants exhibited a maternal inheritance rather than de novo origin. CRISPR-Cas9 mouse modelling of a splice donor loss variant in DMD (NC_000023.11:g.32454661C>G), which resides in a conserved site across vertebrates, replicated bilateral cryptorchidism phenotypes, confirmed by MRI at 4 and 10 weeks. The movement tests further revealed symptoms of Duchenne muscular dystrophy (DMD) in transgenic mice. Conclusion Our results revealed the role of the DMD gene mutation in causing cryptorchidism. The results also suggest that maternal-X inheritance of pathogenic defects could have a predominant role in the development of cryptorchidism.

Chen, Jianhai (ORCID:0000000300932003)↗
DOE OSTI2024

MUPPET: An automated OpenMP mutation testing framework for performance optimization

MUPPET is a tool for OpenMP programs that identifies program modifications, called mutations, aimed at improving program performance. Existing performance optimization techniques, including profiling-based and auto-tuning techniques, fail to indicate program modifications at the source level thus preventing their portability across compilers. MUPPET aims to help HPC developers reason about performance defects and missed opportunities to improve performance at the source code level.

Parasyris, Konstantinos↗
DOE OSTI2020

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Predicting Drug Resistance Using Deep Mutational Scanning

Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Mapping the functional landscape of the receptor binding domain of T7 bacteriophage by deep mutational scanning

The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP (1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many of which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against a urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence–function relationships in many phage–bacterial systems.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1972

Mutations induced in Tradescantia by small doses of X-rays and neutrons - Analysis of dose-response curves.

Dose-response curves for pink somatic mutations in Tradescantia stamen hairs were analyzed after neutron and X-ray irradiation with doses ranging from a fraction of a rad to the region of saturation. The dose-effect relation for neutrons indicates a linear dependence from 0.01 to 8 rads; between 0.25 and 5 rads, a linear dependence is indicated for X-rays also. As a consequence the relative biological effectiveness reaches a constant value (about 50) at low doses. The observations are in good agreement with the predictions of the theory of dual radiation action and support its interpretation of the effects of radiation on higher organisms. The doubling dose of X-rays was found to be nearly 1 rad.

Sparrow, A. H.↗
NASA NTRS1978

The point mutation process in proteins

An optimized scoring matrix for residue-by-residue comparisons of distantly related protein sequences has been developed. The scoring matrix is based on observed exchanges and mutabilities of amino acids in 1572 closely related sequences derived from a cross-section of protein groups. Very few superimposed or parallel mutations are included in the data. The scoring matrix is most useful for demonstrating the relatedness of proteins between 65 and 85% different.

Schwartz, R. M.↗