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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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84 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu 1+ to the less toxic Cu 2+ . Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb 3 -type cytochrome oxidase ( cbb 3 -Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb 3 -Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A non-traditional crystal-based compound screening method targeting the ATP binding site of Plasmodium falciparum GRP78 for identification of novel nucleoside analogues

Drug resistance to front-line malarial treatments represents an ongoing threat to control malaria, a vector borne infectious disease. The malarial parasite, Plasmodium falciparum has developed genetic variants, conferring resistance to the current standard therapeutic artemisinin and its derivatives commonly referred to as artemisinin-combination therapies (ACTs). Emergence of multi-drug resistance parasite genotypes is a warning of potential treatment failure, reaffirming the urgent and critical need to find and validate alternate drug targets to prevent the spread of disease. An attractive and novel drug target includes glucose-regulated protein 78 kDa (GRP78, or BiP), an essential molecular chaperone protein involved in the unfolded protein response that is upregulated in ACT treated P. falciparum parasites. We have shown that both sequence and structure are closely related to human GRP78 (hGRP78), a chaperone belonging to the HSP70 class of ATPase proteins, which is often upregulated in cellular stress responses and cancer. By screening a library of nucleoside analogues, we identified eight ‘hit’ compounds binding at the active site of the ATP binding domain of P. falciparum GRP78 using a high-throughput ligand soaking screen using x-ray crystallography. These compounds were further evaluated using protein thermal shift assays to assess target binding activity. The nucleoside analogues identified from our screen provide a starting point for the development of more potent and selective antimalarial inhibitors. In addition, we have established a well-defined, high-throughput crystal-based screening approach that can be applied to many crystallizable P. falciparum proteins for generating anti-Plasmodium specific compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor

Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA− was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.

Borovsky, Dov↗
DOE OSTI2022

Advances in Genes-Encoding Transporters for Cadmium Uptake, Translocation, and Accumulation in Plants

Cadmium (Cd) is a heavy metal that is highly toxic for plants, animals, and human beings. A better understanding of the mechanisms involved in Cd accumulation in plants is beneficial for developing strategies for either the remediation of Cd-polluted soils using hyperaccumulator plants or preventing excess Cd accumulation in the edible parts of crops and vegetables. As a ubiquitous heavy metal, the transport of Cd in plant cells is suggested to be mediated by transporters for essential elements such as Ca, Zn, K, and Mn. Identification of the genes encoding Cd transporters is important for understanding the mechanisms underlying Cd uptake, translocation, and accumulation in either crop or hyperaccumulator plants. Recent studies have shown that the transporters that mediate the uptake, transport, and accumulation of Cd in plants mainly include members of the natural resistance-associated macrophage protein (Nramp), heavy metal-transporting ATPase (HMA), zinc and iron regulated transporter protein (ZIP), ATP-binding cassette (ABC), and yellow stripe-like (YSL) families. Here, we review the latest advances in the research of these Cd transporters and lay the foundation for a systematic understanding underlying the molecular mechanisms of Cd uptake, transport, and accumulation in plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a ‘pincer-like’ conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5–nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6

Significance The loading of the core Mcm2-7 helicase onto origin DNA is essential for the formation of replication forks and genomic stability. Here, we report two cryo-electron microscopy (cryo-EM) structures that capture helicase loader–helicase complexes just prior to DNA insertion. These pre-loading structures, combined with a computational simulation of the dynamic transition from the pre-loading state to the loaded state, provide crucial insights into the mechanism required for topologically linking the helicase to DNA. The helicase loading system is highly conserved from yeast to human, which means that the molecular principles described here for the yeast system are likely applicable to the human system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Leveraging computational genomics to understand the molecular basis of metal homeostasis

Genome-based data is helping to reveal the diverse strategies plants and algae use to maintain metal homeostasis. In addition to acquisition, distribution and storage of metals, acclimating to feast or famine can involve a wealth of genes that we are just now starting to understand. The fast-paced acquisition of genome-based data, however, is far outpacing our ability to experimentally characterize protein function. Computational genomic approaches are needed to fill the gap between what is known and unknown. To avoid misconstruing bioinformatically derived data, which is the root cause of the inaccurate functional annotations that plague databases, functional inferences from diverse sources and contextualization of that evidence with a robust understanding of protein family evolution is needed. Phylogenomic- and comparative-genomic-based studies can aid in the interpretation of experimental data or provide a spark for the discovery of a new function. These analyses not only lead to novel insight into a target protein's function but can generate thought-provoking insights across protein families.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Morphological and Genomic Features of the New Klosneuvirinae Isolate Fadolivirus IHUMI-VV54

Since the discovery of Mimivirus, viruses with large genomes encoding components of the translation machinery and other cellular processes have been described as belonging to the nucleocytoplasmic large DNA viruses. Recently, genome-resolved metagenomics led to the discovery of more than 40 viruses that have been grouped together in a proposed viral subfamily named Klosneuvirinae. Members of this group had genomes of up to 2.4Mb in size and featured an expanded array of translation system genes. Yet, despite the large diversity of the Klosneuvirinae in metagenomic data, there are currently only two isolates available. Here, we report the isolation of a novel giant virus known as Fadolivirus from an Algerian sewage site and provide morphological data throughout its replication cycle in amoeba and a detailed genomic characterization. The Fadolivirus genome, which is more than 1.5Mb in size, encodes 1,452 predicted proteins and phylogenetic analyses place this viral isolate as a near relative of the metagenome assembled Klosneuvirus and Indivirus. The genome encodes for 66 tRNAs, 23 aminoacyl-tRNA synthetases and a wide range of transcription factors, surpassing Klosneuvirus and other giant viruses. The Fadolivirus genome also encodes putative vacuolar-type proton pumps with the domains D and A, potentially constituting a virus-derived system for energy generation. The successful isolation of Fadolivirus will enable future hypothesis-driven experimental studies providing deeper insights into the biology of the Klosneuvirinae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular mechanism of Rubisco activase: Dynamic assembly and Rubisco remodeling

Ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) enzyme is the limiting step of photosynthetic carbon fixation, and its activation is regulated by its co-evolved chaperone, Rubisco activase (Rca). Rca removes the intrinsic sugar phosphate inhibitors occupying the Rubisco active site, allowing RuBP to split into two 3-phosphoglycerate (3PGA) molecules. This review summarizes the evolution, structure, and function of Rca and describes the recent findings regarding the mechanistic model of Rubisco activation by Rca. New knowledge in these areas can significantly enhance crop engineering techniques used to improve crop productivity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes’ protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗