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At least 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Protein N -Glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease

Diabetes is expected to directly affect renal glycosylation; yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). Here, we used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in formalin-fixed paraffin-embedded sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single-nucleus RNA sequencing (snRNAseq) data were used to inform on transcript levels of glycosylation machinery in different cell types and states. We detected 120 N-glycans, and among them, we identified 12 of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that were common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and N-acetyllactosamine synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Protein engineering for critical metal recovery beyond REEs

Achieving decarbonization and electrification goals will require expanded production of critical minerals (CM), including Li, Co, Cu, rare earths, Ni, and graphite, whose supply chains are geopolitically vulnerable. Problematically, current extraction and separation processes pose severe environmental burdens that impede the development of a diversified domestic supply chain and undercut the environmental benefits of energy technologies [1, 2]. The development of efficient, economical, and environmentally sustainable processing technologies is thus important for meeting the CM demand of the emerging energy technology market. To this end, we have recently developed an all-aqueous protein-based process for rare earth element (REE) extraction and separation. To extend our protein-based approach to critical metals beyond REEs, the goal of this project was to develop a protein discovery and engineering pipeline to generate a panel of proteins that selectively bind target critical metals.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

New alignment method for remote protein sequences by the direct use of pairwise sequence correlations and substitutions

Understanding protein sequences and how they relate to the functions of proteins is extremely important. One of the most basic operations in bioinformatics is sequence alignment and usually the first things learned from these are which positions are the most conserved and often these are critical parts of the structure, such as enzyme active site residues. In addition, the contact pairs in a protein usually correspond closely to the correlations between residue positions in the multiple sequence alignment, and these usually change in a systematic and coordinated way, if one position changes then the other member of the pair also changes to compensate. In the present work, these correlated pairs are taken as anchor points for a new type of sequence alignment. The main advantage of the method here is its combining the remote homolog detection from our method PROST with pairwise sequence substitutions in the rigorous method from Kleinjung et al. We show a few examples of some resulting sequence alignments, and how they can lead to improvements in alignments for function, even for a disordered protein.

Jia, Kejue↗
DOE OSTI2023

Allosterically coupled conformational dynamics in solution prepare the sterol transfer protein StarD4 to release its cargo upon interaction with target membranes

Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe 2+ utilizing O 2 or H 2 O 2 as oxidant, and the resultant Fe 3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Characterization of Plasma Protein Alterations in Pregnant and Postpartum Individuals Living With HIV to Support Physiologically-Based Pharmacokinetic Model Development

Alterations in plasma protein concentrations in pregnant and postpartum individuals can influence antiretroviral (ARV) pharmacokinetics. Physiologically-based pharmacokinetic (PBPK) models can serve to inform drug dosing decisions in understudied populations. However, development of such models requires quantitative physiological information (e.g., changes in plasma protein concentration) from the population of interest. Objective: To quantitatively describe the time-course of albumin and α1-acid glycoprotein (AAG) concentrations in pregnant and postpartum women living with HIV. Serum and plasma protein concentrations procured from the International Maternal Pediatric Adolescent AIDS Clinical Trial Protocol 1026s (P1026s) were analyzed using a generalized additive modeling approach. Separate non-parametric smoothing splines were fit to albumin and AAG concentrations as functions of gestational age or postpartum duration. The analysis included 871 and 757 serum albumin concentrations collected from 380 pregnant (~20 to 42 wks gestation) and 354 postpartum (0 to 46 wks postpartum) women, respectively. Thirty-six and 32 plasma AAG concentrations from 31 pregnant (~24 to 38 wks gestation) and 30 postpartum women (~2–13 wks postpartum), respectively, were available for analysis. Estimated mean albumin concentrations remained stable from 20 wks gestation to term (33.4 to 34.3 g/L); whereas, concentrations rapidly increased postpartum until stabilizing at ~42.3 g/L 15 wk after delivery. Estimated AAG concentrations slightly decreased from 24 wks gestation to term (53.6 and 44.9 mg/dL) while postpartum levels were elevated at two wks after delivery (126.1 mg/dL) and subsequently declined thereafter. Computational functions were developed to quantitatively communicate study results in a form that can be readily utilized for PBPK model development. By characterizing the trajectory of plasma protein concentrations in pregnant and postpartum women living with HIV, our analysis can increase confidence in PBPK model predictions for HIV antiretrovirals and better inform drug dosing decisions in this understudied population.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Optimizing the Design of Diatom Biosilica-Targeted Fusion Proteins in Biosensor Construction for Bacillus anthracis Detection

In vivo functionalization of diatom biosilica frustules by genetic manipulation requires careful consideration of the overall structure and function of complex fusion proteins. Although we previously had transformed Thalassiosira pseudonana with constructs containing a single domain antibody (sdAb) raised against the Bacillus anthracis Sterne strain, which detected an epitope of the surface layer protein EA1 accessible in lysed spores, we initially were unsuccessful with constructs encoding a similar sdAb that detected an epitope of EA1 accessible in intact spores and vegetative cells. This discrepancy limited the usefulness of the system as an environmental biosensor for B. anthracis. We surmised that to create functional biosilica-localized biosensors with certain constructs, the biosilica targeting and protein trafficking functions of the biosilica-targeting peptide Sil3T8 had to be uncoupled. We found that retaining the ER trafficking sequence at the N-terminus and relocating the Sil3T8 targeting peptide to the C-terminus of the fusion protein resulted in successful detection of EA1 with both sdAbs. Homology modeling of antigen binding by the two sdAbs supported the hypothesis that the rescue of antigen binding in the previously dysfunctional sdAb was due to removal of steric hindrances between the antigen binding loops and the diatom biosilica for that particular sdAb.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Protein Structure Inspired Discovery of a Novel Inducer of Anoikis in Human Melanoma

Drug discovery historically starts with an established function, either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function, we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds, we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures, we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast, prostate, colon, or lung cancer, and for toxicity on normal human bone marrow stem cells, both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound, Dxr2-017, exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays, Dxr2-017 had an IC50 of 12 nM against melanoma cells, while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis, a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable.

Oncology↗
DOE OSTI2022

The Magnetosome Protein, Mms6 from Magnetospirillum magneticum Strain AMB-1, Is a Lipid-Activated Ferric Reductase

Magnetosomes of magnetotactic bacteria consist of magnetic nanocrystals with defined morphologies enclosed in vesicles originated from cytoplasmic membrane invaginations. Although many proteins are involved in creating magnetosomes, a single magnetosome protein, Mms6 from Magnetospirillum magneticum strain AMB-1, can direct the crystallization of magnetite nanoparticles in vitro. The in vivo role of Mms6 in magnetosome formation is debated, and the observation that Mms6 binds Fe3+ more tightly than Fe2+ raises the question of how, in a magnetosome environment dominated by Fe3+, Mms6 promotes the crystallization of magnetite, which contains both Fe3+ and Fe2+. Here we show that Mms6 is a ferric reductase that reduces Fe3+ to Fe2+ using NADH and FAD as electron donor and cofactor, respectively. Reductase activity is elevated when Mms6 is integrated into either liposomes or bicelles. Analysis of Mms6 mutants suggests that the C-terminal domain binds iron and the N-terminal domain contains the catalytic site. Although Mms6 forms multimers that involve C-terminal and N-terminal domain interactions, a fusion protein with ubiquitin remains a monomer and displays reductase activity, which suggests that the catalytic site is fully in the monomer. However, the quaternary structure of Mms6 appears to alter the iron binding characteristics of the C-terminal domain. These results are consistent with a hypothesis that Mms6, a membrane protein, promotes the formation of magnetite in vivo by a mechanism that involves reducing iron.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Note on the Effects of Linear Topology Preservation in Monte Carlo Simulations of Knotted Proteins

Monte Carlo simulations are a powerful technique and are widely used in different fields. When applied to complex molecular systems with long chains, such as those in synthetic polymers and proteins, they have the advantage of providing a fast and computationally efficient way to sample equilibrium ensembles and calculate thermodynamic and structural properties under desired conditions. Conformational Monte Carlo techniques employ a move set to perform the transitions in the simulation Markov chain. While accepted conformations must preserve the sequential bonding of the protein chain model and excluded volume among its units, the moves themselves may take the chain across itself. We call this a break in linear topology preservation. In this manuscript, we show, using simple protein models, that there is no difference in equilibrium properties calculated with a move set that preserves linear topology and one that does not. However, for complex structures, such as those of deeply knotted proteins, the preservation of linear topology provides correct equilibrium results but only after long relaxation. In any case, to analyze folding pathways, knotting mechanisms and folding kinetics, the preservation of linear topology may be an unavoidable requirement.

Especial, João N. C. (ORCID:0000000254765370)↗
DOE OSTI2021

Utilizing Amino Acid Composition and Entropy of Potential Open Reading Frames to Identify Protein-Coding Genes

One of the main steps in gene-finding in prokaryotes is determining which open reading frames encode for a protein, and which occur by chance alone. There are many different methods to differentiate the two; the most prevalent approach is using shared homology with a database of known genes. This method presents many pitfalls, most notably the catch that you only find genes that you have seen before. The four most popular prokaryotic gene-prediction programs (GeneMark, Glimmer, Prodigal, Phanotate) all use a protein-coding training model to predict protein-coding genes, with the latter three allowing for the training model to be created ab initio from the input genome. Different methods are available for creating the training model, and to increase the accuracy of such tools, we present here GOODORFS, a method for identifying protein-coding genes within a set of all possible open reading frames (ORFS). Our workflow begins with taking the amino acid frequencies of each ORF, calculating an entropy density profile (EDP), using KMeans to cluster the EDPs, and then selecting the cluster with the lowest variation as the coding ORFs. To test the efficacy of our method, we ran GOODORFS on 14,179 annotated phage genomes, and compared our results to the initial training-set creation step of four other similar methods (Glimmer, MED2, PHANOTATE, Prodigal). We found that GOODORFS was the most accurate (0.94) and had the best F1-score (0.85), while Glimmer had the highest precision (0.92) and PHANOTATE had the highest recall (0.96).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Identification and Characterization of Lipid Droplet-Associated Protein (LDAP) Isoforms from Tung Tree (Vernicia fordii)

Lipid droplets (LDs) are cytoplasmic organelles responsible primarily for the storage of neutral lipids, such as triacyclglycerols (TAGs). Derived from the endoplasmic reticulum bilayer, LDs are composed of a hydrophobic lipid core encased by a phospholipid monolayer and surface-associated proteins. To date, only a relatively few LD ‘coat’ proteins in plants have been identified and characterized, most of which come from studies of the model plant Arabidopsis thaliana. To expand our knowledge of the plant LD proteome, the LD-associated protein (LDAP) family from the tung tree (Vernicia fordii), whose seeds are rich in a commercially valuable TAG containing the conjugated fatty acid α-eleostearic acid (C18:3Δ9cis,11trans,13trans [α-ESA]), was identified and characterized. Based on the tung tree transcriptome, three LDAP isoforms (VfLDAP1-3) were elucidated and the encoded proteins distinctly clustered into three clades along with their respective isoforms from other angiosperm species. Ectopic expression of the VfLDAPs in Nicotiana benthamiana leaves revealed that they localized specifically to LDs and influenced LD numbers and sizes, as well as increasing TAG content and altering TAG fatty acid composition. Interestingly, in a partially reconstructed TAG-ESA biosynthetic pathway, the co-expression of VfLDAP3 and, to a lesser degree, VfLDAP2, significantly increased the content of α-ESA stored within the LDs. These results suggest that the VfLDAPs can influence the steady-state content and composition of TAG in plant cells and that certain LDAP isoforms may have evolved to more efficiently package TAGs into LDs containing unusual fatty acids, such as α-ESA.

Plant Sciences↗
DOE OSTI2021

Protection against Different Genotypes of Newcastle Disease Viruses (NDV) Afforded by an Adenovirus-Vectored Fusion Protein and Live NDV Vaccines in Chickens

The efficacy of an adenovirus-vectored Newcastle disease virus (NDV) vaccine expressing the fusion (F) NDV protein (adeno-F) was evaluated against challenges with virulent heterologous and homologous NDV strains to the F protein. In a preliminary study, two different doses (low and high) of adeno-F were tested against a virulent NDV strain containing the homologous NDV F protein, CA02. In a second study, at three weeks post-vaccination, the efficacy of the high dose of adeno-F was compared to a live attenuated NDV vaccine strain (LaSota) against three antigenically distinct virulent NDV challenge strains, one homologous (CA02) and two heterologous (TZ12, EG14) to F in the vectored vaccine. In both experiments, clinical signs, mortality, virus shedding, and humoral response were evaluated. In the first experiment, the survival rates from birds vaccinated with adeno-F at a high and low dose were 100% and 25%, respectively. In the second experiment, birds vaccinated with the high dose of adeno-F had a survival rate of 80%, 75%, and 65% after challenge with the CA02, TZ12, and EG14 viruses, respectively. All of the LaSota-vaccinated birds survived post-challenge no matter the NDV challenge strain. High antibody titers were detected after vaccination with LaSota by HI and ELISA tests. The majority of adeno-F-vaccinated birds had detectable antibodies using the ELISA test, but not using the HI test, before the challenge. The data show that both the similarity of the F protein of the adeno-F vaccine to the challenge virus and the adeno-F vaccination dose affect the efficacy of an adenovirus-vectored NDV vaccine against a virulent NDV challenge.

Ferreira, Helena L. (ORCID:0000000232613712)↗
DOE OSTI2021

Resin Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types including cultured cells (e.g. mammalian, prokaryotic) and whole tissues (e.g. heart, lung, muscle) which are initially lysed/homogenized with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol affinity resin which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves our understanding of the role of redox dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

RAC, TMT, thiol redox proteomics, cysteine, PTM st↗
DOE OSTI2021

Design of Broadly Cross-Reactive M Protein–Based Group A Streptococcal Vaccines

Group A streptococcal infections are a significant cause of global morbidity and mortality. A leading vaccine candidate is the surface M protein, a major virulence determinant and protective Ag. An obstacle to the development of M protein–based vaccines is the >200 different M types defined by the N-terminal sequences that contain protective epitopes. Despite sequence variability, M proteins share coiled-coil structural motifs that bind host proteins required for virulence. In this study, we exploit this potential Achilles heel of conserved structure to predict cross-reactive M peptides that could serve as broadly protective vaccine Ags. Combining sequences with structural predictions, six heterologous M peptides in a sequence-related cluster were predicted to elicit cross-reactive Abs with the remaining five nonvaccine M types in the cluster. The six-valent vaccine elicited Abs in rabbits that reacted with all 11 M peptides in the cluster and functional opsonic Abs against vaccine and nonvaccine M types in the cluster. We next immunized mice with four sequence-unrelated M peptides predicted to contain different coiled-coil propensities and tested the antisera for cross-reactivity against 41 heterologous M peptides. Based on these results, we developed an improved algorithm to select cross-reactive peptide pairs using additional parameters of coiled-coil length and propensity. The revised algorithm accurately predicted cross-reactive Ab binding, improving the Matthews correlation coefficient from 0.42 to 0.74. These results form the basis for selecting the minimum number of N-terminal M peptides to include in potentially broadly efficacious multivalent vaccines that could impact the overall global burden of group A streptococcal diseases.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1972

Disproportional changes in hematocrit, plasma volume, and proteins during exercise and bed rest.

The interrelationships between the changes in plasma volume, hematocrit, and plasma proteins during muscular exercise and bed rest were investigated. Proportionally, the changes in hematocrit are always smaller than the changes in plasma volume. For this reason changes in the concentration of blood constituents can only be quantitated on the basis of plasma volume changes. During short periods of intensive exercise, there was a small loss of plasma proteins. With prolonged submaximal exercise there was a net gain in plasma protein, which contributes to stabilization of the vascular volume. Prolonged bed rest induced hypoproteinemia; this loss of plasma protein probably plays an important role in recumbency hypovolemia.

Van Beaumont, W.↗
NASA NTRS1979

Isoelectric focusing of proteins and peptides

Egg-white solution was chosen as the reference solution in order to assess the effects of operational parameters (voltage, flow rate, ampholine pH range and concentration, and protein concentration) of the RIEF apparatus on protein resolution. Topics of discussion include: (1) comparison of RIEF apparatus to conventional IEF techniques (column and PAG) with respect to resolution and throughput; (2) peptide and protein separation (AHF, Thymosin - Fraction 5, vasoactive peptide, L-asparaginase and ACP); and (3) detection of peptides - dansyl derivatives of amino acids and peptides, post-focusing fluorescent labeling of amino acids, peptides and proteins, and ampholine extraction from focused gels.

Egen, N.↗
NASA NTRS1980

Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

Pevzner, L. Z.↗