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At least 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Identification and preliminary characterization of conserved uncharacterized proteins from Chlamydomonas reinhardtii , Arabidopsis thaliana , and Setaria viridis

Abstract The rapid accumulation of sequenced plant genomes in the past decade has outpaced the still difficult problem of genome‐wide protein‐coding gene annotation. A substantial fraction of protein‐coding genes in all plant genomes are poorly annotated or unannotated and remain functionally uncharacterized. We identified unannotated proteins in three model organisms representing distinct branches of the green lineage (Viridiplantae): Arabidopsis thaliana (eudicot), Setaria viridis (monocot), and Chlamydomonas reinhardtii (Chlorophyte alga). Using similarity searching, we identified a subset of unannotated proteins that were conserved between these species and defined them as Deep Green proteins. Bioinformatic, genomic, and structural predictions were performed to begin classifying Deep Green genes and proteins. Compared to whole proteomes for each species, the Deep Green set was enriched for proteins with predicted chloroplast targeting signals predictive of photosynthetic or plastid functions, a result that was consistent with enrichment for daylight phase diurnal expression patterning. Structural predictions using AlphaFold and comparisons to known structures showed that a significant proportion of Deep Green proteins may possess novel folds. Though only available for three organisms, the Deep Green genes and proteins provide a starting resource of high‐value targets for further investigation of potentially new protein structures and functions conserved across the green lineage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

YPK9 and WHI2 Negatively Interact during Oxidative Stress

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson’s disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741 COM ). In contrast, deleting YPK9 from BY4741 COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741 COM and BY4741 COM ypk9Δ contain a novel premature stop codon near the 3' end of WHI2 (WHI2 G1324T ), whereas the collection’s YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2p E442* ) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2 G1324T strain’s genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure-aware annotation of leucine-rich repeat domains

Protein domain annotation is typically done by predictive models such as HMMs trained on sequence motifs. However, sequence-based annotation methods are prone to error, particularly in calling domain boundaries and motifs within them. These methods are limited by a lack of structural information accessible to the model. With the advent of deep learning-based protein structure prediction, existing sequenced-based domain annotation methods can be improved by taking into account the geometry of protein structures. We develop dimensionality reduction methods to annotate repeat units of the Leucine Rich Repeat solenoid domain. The methods are able to correct mistakes made by existing machine learning-based annotation tools and enable the automated detection of hairpin loops and structural anomalies in the solenoid. The methods are applied to 127 predicted structures of LRR-containing intracellular innate immune proteins in the model plant Arabidopsis thaliana and validated against a benchmark dataset of 172 manually-annotated LRR domains.

Xu, Boyan↗
DOE OSTI2020

Structure-based group A streptococcal vaccine design: Helical wheel homology predicts antibody cross-reactivity among streptococcal M protein–derived peptides

Group A streptococcus (Strep A) surface M protein, an α-helical coiled-coil dimer, is a vaccine target and a major determinant of streptococcal virulence. The sequence-variable N-terminal region of the M protein defines the M type and also contains epitopes that promote opsonophagocytic killing of streptococci. Recent reports have reported considerable cross-reactivity among different M types, suggesting the prospect of identifying cross-protective epitopes that would constitute a broadly protective multivalent vaccine against Strep A isolates. In this work, we have used a combination of immunological assays, structural biology, and cheminformatics to construct a recombinant M protein–based vaccine that included six Strep A M peptides that were predicted to elicit antisera that would cross-react with an additional 15 nonvaccine M types of Strep A. Rabbit antisera against this recombinant vaccine cross-reacted with 10 of the 15 nonvaccine M peptides. Two of the five nonvaccine M peptides that did not cross-react shared high sequence identity (≥50%) with the vaccine peptides, implying that high sequence identity alone was insufficient for cross-reactivity among the M peptides. Additional structural analyses revealed that the sequence identity at corresponding polar helical-wheel heptad sites between vaccine and nonvaccine peptides accurately distinguishes cross-reactive from non–cross-reactive peptides. On the basis of these observations, we developed a scoring algorithm based on the sequence identity at polar heptad sites. When applied to all epidemiologically important M types, this algorithm should enable the selection of a minimal number of M peptide–based vaccine candidates that elicit broadly protective immunity against Strep A.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria ( Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (C i ), but some evidence indicates that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of C i incorporation into different taxa, mat samples were incubated with [ 13 C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13 C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with 13 C. Most 13 C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins associated with photosynthesis, C i fixation, and N 2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for C i fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to C i fixation. Other taxa showed much lower [ 13 C]bicarbonate incorporation. IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven C i fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in C i fixation. Because this pathway imparts a lower degree of selection of isotopically heavy C i than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of 13 C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13 C/ 12 C ratios of mat biomass will help geochemists interpret the 13 C/ 12 C ratios of organic carbon in the fossil record.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

High-Performance Deep Learning Toolbox for Genome-Scale Prediction of Protein Structure and Function

Computational biology is one of many scientific disciplines ripe for innovation and acceleration with the advent of high-performance computing (HPC). In recent years, the field of machine learning has also seen significant benefits from adopting HPC practices. In this work, we present a novel HPC pipeline that incorporates various machine-learning approaches for structure-based functional annotation of proteins on the scale of whole genomes. Our pipeline makes extensive use of deep learning and provides computational insights into best practices for training advanced deep-learning models for high-throughput data such as proteomics data. We showcase methodologies our pipeline currently supports and detail future tasks for our pipeline to envelop, including large-scale sequence comparison using SAdLSA and prediction of protein tertiary structures using AlphaFold2.

Gao, Mu↗
DOE OSTI2024

Birth of protein folds and functions in the virome

The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence. Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database. Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts. Structural comparisons suggested putative functions for up to 25% of unannotated viral proteins, including those with roles in the evasion of innate immunity. In particular, RNA ligase T-like phosphodiesterases were found to resemble phage-encoded proteins that hydrolyse the host immune-activating cyclic dinucleotides 3',3'- and 2',3'-cyclic GMP-AMP (cGAMP). Experimental analysis showed that RNA ligase T homologues encoded by avian poxviruses similarly hydrolyse cGAMP, showing that RNA ligase T-mediated targeting of cGAMP is an evolutionarily conserved mechanism of immune evasion that is present in both bacteriophage and eukaryotic viruses. Together, the viral protein structural database and analyses presented here afford new opportunities to identify mechanisms of virus–host interactions that are common across the virome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

CasCollect: targeted assembly of CRISPR-associated operons from high-throughput sequencing data

Abstract CRISPR arrays and CRISPR-associated (Cas) proteins comprise a widespread adaptive immune system in bacteria and archaea. These systems function as a defense against exogenous parasitic mobile genetic elements that include bacteriophages, plasmids and foreign nucleic acids. With the continuous spread of antibiotic resistance, knowledge of pathogen susceptibility to bacteriophage therapy is becoming more critical. Additionally, gene-editing applications would benefit from the discovery of new cas genes with favorable properties. While next-generation sequencing has produced staggering quantities of data, transitioning from raw sequencing reads to the identification of CRISPR/Cas systems has remained challenging. This is especially true for metagenomic data, which has the highest potential for identifying novel cas genes. We report a comprehensive computational pipeline, CasCollect, for the targeted assembly and annotation of cas genes and CRISPR arrays—even isolated arrays—from raw sequencing reads. Benchmarking our targeted assembly pipeline demonstrates significantly improved timing by almost two orders of magnitude compared with conventional assembly and annotation, while retaining the ability to detect CRISPR arrays and cas genes. CasCollect is a highly versatile pipeline and can be used for targeted assembly of any specialty gene set, reconfigurable for user provided Hidden Markov Models and/or reference nucleotide sequences.

Podlevsky, Joshua D.↗
DOE OSTI2020

Positive modulator of bone morphogenic protein-2

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Modular protein-oligonucleotide signal exchange

Abstract While many methods are available to measure the concentrations of proteins in solution, the development of a method to quantitatively report both increases and decreases in different protein concentrations in real-time using changes in the concentrations of other molecules, such as DNA outputs, has remained a challenge. Here, we present a biomolecular reaction process that reports the concentration of an input protein in situ as the concentration of an output DNA oligonucleotide strand. This method uses DNA oligonucleotide aptamers that bind either to a specific protein selectively or to a complementary DNA oligonucleotide reversibly using toehold-mediated DNA strand-displacement. It is possible to choose the sequence of output strand almost independent of the sensing protein. Using this strategy, we implemented four different exchange processes to report the concentrations of clinically relevant human α-thrombin and vascular endothelial growth factor using changes in concentrations of DNA oligonucleotide outputs. These exchange processes can operate in tandem such that the same or different output signals can indicate changes in concentration of distinct or identical input proteins. The simplicity of our approach suggests a pathway to build devices that can direct diverse output responses in response to changes in concentrations of specific proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Five key aspects of metaproteomics as a tool to understand functional interactions in host-associated microbiomes

Host-associated microbial communities (microbiomes) play critical roles in human, animal, and plant health and development. However, interactions between the host, members of the microbiome, and invading pathogens are in most cases still poorly understood. Such interactions are multidimensional and can alter the taxonomic composition and/or the functional metabolic activities of the microbiome in response to disease or treatment conditions. For example, after 2 days of antibiotic treatment, the mouse gut microbiome is altered and more susceptible to invasion by the pathogen Clostridioides difficile. Studies of these multidimensional interactions have been fueled by the ability to use high-throughput sequencing of phylogenetic marker genes to profile microbial community composition and shotgun metagenomics to profile functional potential. However, many protein-coding genes predicted from metagenomes are not necessarily expressed under a given condition, and thus, it is difficult to assess the activities and functional interactions in microbial communities based on DNA sequencing data alone. The physiological and pathological processes expressed in these communities under specific conditions are better reflected by the abundances of transcripts or proteins. In this Pearl, we provide a brief introduction to metaproteomics, which is a tool for the large-scale analysis of proteins in microbiomes that allows researchers to address a diversity of questions related to functions and interactions in microbiomes. The term “metaproteomics” was first used in 2004 for “the large-scale characterization of the entire protein complement of environmental microbiota at a given point in time”, and since then, a large array of metaproteomics approaches have been developed. Our objective in this Pearl is to highlight what we feel are 5 essential elements to be considered for a metaproteomics research campaign and to introduce nonexpert readers to the topic without going into too much technical detail.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Uncovering DNA-PKcs ancient phylogeny, unique sequence motifs and insights for human disease

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to the assumption that DNA-PKcs is a vertebrate-specific PIKK. Here, we find that DNA-PKcs is widely distributed in invertebrates, fungi, plants, and protists, and that threonines 2609, 2638, and 2647 of the ABCDE cluster of phosphorylation sites are highly conserved amongst most Eukaryotes. Furthermore, we identify highly conserved amino acid sequence motifs and domains that are characteristic of DNA-PKcs relative to other PIKKs. These include residues in the Forehead domain and a novel motif we have termed YRPD, located in an α helix C-terminal to the ABCDE phosphorylation site loop. Combining sequence with biochemistry plus structural data on human DNA-PKcs unveils conserved sequence and conformational features with functional insights and implications. The defined generally progressive DNA-PKcs sequence diversification uncovers conserved functionality supported by Evolutionary Trace analysis, suggesting that for many organisms both functional sites and evolutionary pressures remain identical due to fundamental cell biology. The mining of cancer genomic data and germline mutations causing human inherited disease reveal that robust DNA-PKcs activity in tumors is detrimental to patient survival, whereas germline mutations compromising function are linked to severe immunodeficiency and neuronal degeneration. We anticipate that these collective results will enable ongoing DNA-PKcs functional analyses with biological and medical implications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Chromosome assembled and annotated genome sequence of Aspergillus flavus NRRL 3357

Abstract Aspergillus flavus is an opportunistic pathogen of crops, including peanuts and maize, and is the second leading cause of aspergillosis in immunocompromised patients. A. flavus is also a major producer of the mycotoxin, aflatoxin, a potent carcinogen, which results in significant crop losses annually. The A. flavus isolate NRRL 3357 was originally isolated from peanut and has been used as a model organism for understanding the regulation and production of secondary metabolites, such as aflatoxin. A draft genome of NRRL 3357 was previously constructed, enabling the development of molecular tools and for understanding population biology of this particular species. Here, we describe an updated, near complete, telomere-to-telomere assembly and re-annotation of the eight chromosomes of A. flavus NRRL 3357 genome, accomplished via long-read PacBio and Oxford Nanopore technologies combined with Illumina short-read sequencing. A total of 13,715 protein-coding genes were predicted. Using RNA-seq data, a significant improvement was achieved in predicted 5’ and 3’ untranslated regions, which were incorporated into the new gene models.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

A novel bacterial protein family that catalyses nitrous oxide reduction

Nitrous oxide (N 2 O), a driver of global warming and climate change, has reached unprecedented concentrations in Earth’s atmosphere. Current N 2 O sources outpace N 2 O sinks, emphasizing the need for comprehensive understanding of processes that consume N 2 O. Microbes that express the enzyme N 2 O reductase (N 2 OR) convert N 2 O to climate change-neutral dinitrogen (N 2 ). Known N 2 ORs belong to the canonical clade I and clade II NosZ reductases and are considered key enzymes for N 2 O reduction. Here we report a previously unrecognized protein family with a role in N 2 O reduction, clade III lactonase-type N 2 OR (L-N 2 OR), which diverges in sequence from canonical NosZ but conserves three-dimensional protein structural features. Integrated physiological, metagenomic, proteomic and structural modelling studies demonstrate that L-N 2 ORs catalyse N 2 O reduction. L-N 2 OR genes occur in several phyla, predominantly in uncultured taxa with broad geographic distribution. Our findings expand the known diversity of N 2 ORs and implicate previously unrecognized taxa (for example, Nitrospinota) in N 2 O consumption. In conclusion, the expansion of N 2 OR diversity and the identification of a novel type of catalyst for N 2 O reduction advances the understanding of N 2 O sinks, has implications for greenhouse gas emission and climate change modelling, and expands opportunities for innovative biotechnologies aimed at curbing N 2 O emissions.

He, Guang 何广 [Univ. of Tennessee, Knoxville, TN (U↗
DOE OSTI2025

Matrix Metalloproteinases as Candidate Antigenic Determinants for Anti‐Tumor Autoantibodies in Human Ovarian Cancer: A Post Hoc Analysis

Circulating antibodies in patients with cancer can facilitate the identification of accessible epitopes on autoantigens expressed by tumors. To identify previously unrecognized protein targets in ovarian cancer, we computationally assessed a heptapeptide consensus motif (VPELGHE, flanked by two cysteine residues yielding a cyclic nonapeptide under oxidizing conditions) previously discovered via phage display-based epitope mapping of autoantibodies in patients. Eight proteins associated with ovarian cancer encompass amino acid sequences similar to the consensus motif and were, therefore, considered as candidate native autoantigens. Among these candidate targets, however, matrix metalloproteinase 14 (MMP14) demonstrates gene expression that is both high and negatively correlated with survival in ovarian cancer patient cohorts. MMP14 protein levels are also stable in tumor versus non-tumor tissues. Moreover, the corresponding heptapeptide mimic in MMP14 occurs within an α-helical secondary structural element observed in its catalytic domain. These findings demonstrate that a subset of patient-derived autoantibodies may interact with a previously unknown antigenic epitope found in MMP14 and other MMPs, thereby providing opportunities for the development of new targeted agents.

Biochemistry & Molecular Biology↗
DOE OSTI2025

Sequence Programmable Order–Disorder Transitions in Supramolecular Assembly of Peptide Nanofibers

Protein–protein interactions determine the assembly of complexes that are responsible for numerous key biological processes. The assembly of many natural protein complexes is mediated by post-translational structural changes and environmental stimuli. In this study, we show that incorporation of adjacent lysine residues results in the pH-tunable stability of peptide secondary structure and assembly, allowing for the incorporation of complementary order-inducing motifs. The strategic placement of cysteine pairs in the same peptide sequence results in redox-dependent disulfide staple formation, inducing a transition from random coil to β-sheet conformation and subsequent supramolecular nanofiber assembly from otherwise disordered peptide monomers. Spectroscopic, imaging, molecular dynamics, and kinetic studies highlight the critical role of sequence motif location, oligomerization, and the competitive interplay between intra- and interpeptide disulfide bonding in determining assembly outcomes. We extend this approach to demonstrate phosphorylation-dependent assembly from the design of the same parent peptide sequence, suggesting a general approach to the design of diverse stimulus-responsive peptide sequences for supramolecular assembly. Furthermore, these findings also provide a framework for investigating sequence-dependent pathways in amyloid fiber formation with potential implications for neurodegenerative disease research.

Disulfides↗