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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

SERTA Domain Containing Protein 1 (SERTAD1) Interacts with Classical Swine Fever Virus Structural Glycoprotein E2, Which Is Involved in Virus Virulence in Swine

E2 is the major structural glycoprotein of the classical swine fever virus (CSFV). E2 has been shown to be involved in important virus functions such as replication and virulence in swine. Using the yeast two-hybrid system, we previously identified several host proteins specifically interacting with CSFV E2. Here, we analyze the protein interaction of E2 with SERTA domain containing protein 1 (SERTAD1), a factor involved in the stimulation of the transcriptional activities of different host genes. We have confirmed that the interaction between these two proteins occurs in CSFV-infected swine cells by using a proximity ligation assay and confocal microscopy. Amino acid residues in the CSFV E2 protein that are responsible for mediating the interaction with SERTAD1 were mapped by a yeast two-hybrid approach using a randomly mutated E2 library. Using that information, a recombinant CSFV mutant (E2ΔSERTAD1v) that harbors substitutions in those residues mediating the protein-interaction with SERTAD1 was developed and used to study the role of the E2-SERTAD1 interaction in viral replication and virulence in swine. CSFV E2ΔSERTAD1v, when compared to the parental BICv, showed a clearly decreased ability to replicate in the SK6 swine cell line and a more severe replication defect in primary swine macrophage cultures. Importantly, 80% of animals infected with E2ΔSERTAD1v survived infection, remaining clinically normal during the 21-day observational period. This result would indicate that the ability of CSFV E2 to bind host SERTAD1 protein during infection plays a critical role in virus virulence.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structures of the TMC-1 complex illuminate mechanosensory transduction

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid–protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Viscoelastic analysis of mussel threads reveals energy dissipative mechanisms

Mussels use byssal threads to secure themselves to rocks and as shock absorbers during cyclic loading from wave motion. Byssal threads combine high strength and toughness with extensibility of nearly 200%. Researchers attribute tensile properties of byssal threads to their elaborate multi-domain collagenous protein cores. Because the elastic properties have been previously scrutinized, we instead examined byssal thread viscoelastic behaviour, which is crucial for withstanding cyclic loading. By targeting protein domains in the collagenous core via chemical treatments, stress relaxation experiments provided insights on domain contributions and were coupled with in situ small-angle X-ray scattering to investigate relaxation-specific molecular reorganizations. Results show that when silk-like domains in the core were disrupted, the stress relaxation of the threads decreased by nearly 50% and lateral molecular spacing also decreased, suggesting that these domains are essential for energy dissipation and assume a compressed molecular rearrangement when disrupted. A generalized Maxwell model was developed to describe the stress relaxation response. The model predicts that maximal damping (energy dissipation) occurs at around 0.1 Hz which closely resembles the wave frequency along the California coast and implies that these materials may be well adapted to the cyclic loading of the ambient conditions.

36 MATERIALS SCIENCE↗
NASA NTRSDate not supplied

Understanding amyloids to prevent biofilm formation in space

There is a pressing need to search for novel approaches to combat biofilm formation, both in space and in medical applications. Many proteins have the ability to form ordered aggregates called amyloids. Amyloids are known to be an important part of biofilms. The use of anti-amyloid drugs is a novel venue for the development of antimicrobial agents. The ultrastructure of the amyloid aggregate shows a high packing of proteins, the second-order structure of which is dominated by β-sheets. The ability to form an amyloid aggregate is especially typical for proteins containing domains (protein fragments) with sufficient lability to arrange themselves in a tight β-sheet structure. Bioinformatics tools allow the prediction of such behavior of proteins in genomic data. We use GeneLab data of microbial populations identified aboard the International Space Station and other spacecraft to look for bacterial species that utilize amyloid aggregation in biofilm formation. We use a combined bioinformatic approach with a relatively high throughput molecular biology assay and biophysical assays to evaluate the anti-amyloid anti-biofilm approach. The significance of the research extends from understanding basic microbial community responses to spaceflight, to biofouling of the built environments in space as well as the long-term health of astronauts. Bioinformatics shows that onboard the ISS, bacterial species produce far more amyloid and prion proteins than are currently verified, hence their role in bacterial ecosystems is largely unknown. As we propose there is a link between amyloid formation in space and biofilm production, this research should lead to new paths for biofilm remediation in space.

Tomasz Zajkowski↗
DOE OSTI2025

Archaeal protein containing domain of unknown function 2193 undergoes oligomeric reconfiguration upon iron–sulfur cluster binding

Methanogenic archaea are particularly rich in iron–sulfur proteins, yet their roles remain largely enigmatic. Here, we characterized aMethanococcus voltae(Mvo) protein from the domain of unknown function (DUF) 2193 family, a group of proteins present primarily in archaea and characterized by a conserved cysteine‐rich C‐terminal motif.MvoDUF2193 was heterologously expressed and characterized by a range of spectroscopic and analytical methods. The results demonstrate thatMvoDUF2193 binds a single [4Fe–4S] cluster per subunit and that cluster occupancy regulates the transition from an apo tetramer to a [4Fe–4S] monomeric form. We hypothesize thatMvoDUF2193 serves a regulatory role in the cell, mediated by [Fe–S] cluster binding and changes in oligomeric state.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Targeting a proteolytic neoepitope on CUB domain containing protein 1 ( CDCP1 ) for RAS -driven cancers

Extracellular proteolysis is frequently dysregulated in disease and can generate proteoforms with unique neoepitopes not found in healthy tissue. Here, we demonstrate that Abs that selectively recognize a proteolytic neoepitope on CUB domain containing protein 1 (CDCP1) could enable more effective and safer treatments for solid tumors. CDCP1 is highly overexpressed in RAS-driven cancers, and its ectodomain is cleaved by extracellular proteases. Biochemical, biophysical, and structural characterization revealed that the 2 cleaved fragments of CDCP1 remain tightly associated with minimal proteolysis-induced conformational change. Using differential phage display, we generated recombinant Abs that are exquisitely selective to cleaved CDCP1 with no detectable binding to the uncleaved form. These Abs potently targeted cleaved CDCP1-expressing cancer cells as an Ab-drug conjugate, an Ab-radionuclide conjugate, and a bispecific T cell engager. In a syngeneic pancreatic tumor model, these cleaved-specific Abs showed tumor-specific localization and antitumor activity with superior safety profiles compared with a pan-CDCP1 approach. Targeting proteolytic neoepitopes could provide an orthogonal "AND" gate for improving the therapeutic index.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1992

Evolution of EF-hand calcium-modulated proteins. II. Domains of several subfamilies have diverse evolutionary histories

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

Non-NASA Center↗
DOE OSTI2021

Biotin attachment domain-containing proteins mediate hydroxy fatty acid-dependent inhibition of acetyl CoA carboxylase

Hundreds of naturally occurring specialized fatty acids (FAs) have potential as desirable chemical feedstocks if they could be produced at large scale by crop plants; however, transgenic expression of their biosynthetic genes has generally been accompanied by dramatic reductions in oil yield. For example, expression of castor (Ricinus communis) FA hydroxylase (FAH) in the Arabidopsis thaliana FA elongation mutant fae1 resulted in a 50% reduction of FA synthesis rate that was attributed to inhibition of acetyl-CoA carboxylase (ACCase) by an undefined mechanism. Here, we tested the hypothesis that the ricinoleic acid-dependent decrease in ACCase activity is mediated by biotin attachment domain-containing (BADC) proteins. BADCs are inactive homologs of biotin carboxy carrier protein that lack a biotin cofactor and can inhibit ACCase. Arabidopsis contains three BADC genes. To reduce expression levels of BADC1 and BADC3 in fae1/FAH plants, a homozygous badc1,3/fae1/FAH line was created. The rate of FA synthesis in badc1,3/fae1/FAH seeds doubled relative to fae1/FAH, restoring it to fae1 levels, increasing both native FA and HFA accumulation. Total FA per seed, seed oil content, and seed yield per plant all increased in badc1,3/fae1/FAH, to 5.8 µg, 37%, and 162 mg, respectively, relative to 4.9 µg, 33%, and 126 mg, respectively, for fae1/FAH. Transcript levels of FA synthesis-related genes, including those encoding ACCase subunits, did not significantly differ between badc1,3/fae1/FAH and fae1/FAH. These results demonstrate that BADC1 and BADC3 mediate ricinoleic acid-dependent inhibition of FA synthesis. We propose that BADC-mediated FAS inhibition as a general mechanism that limits FA accumulation in specialized FA-accumulating seeds.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1998

Calcium-binding proteins and development

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

Review↗
DOE OSTI2020

Gene content evolution in the arthropods

Background: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. Results: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. Conclusions: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Arthropods↗
DOE OSTI2022

Graph identification of proteins in tomograms ( GRIP‐Tomo )

Abstract In this study, we present a method of pattern mining based on network theory that enables the identification of protein structures or complexes from synthetic volume densities, without the knowledge of predefined templates or human biases for refinement. We hypothesized that the topological connectivity of protein structures is invariant, and they are distinctive for the purpose of protein identification from distorted data presented in volume densities. Three‐dimensional densities of a protein or a complex from simulated tomographic volumes were transformed into mathematical graphs as observables. We systematically introduced data distortion or defects such as missing fullness of data, the tumbling effect, and the missing wedge effect into the simulated volumes, and varied the distance cutoffs in pixels to capture the varying connectivity between the density cluster centroids in the presence of defects. A similarity score between the graphs from the simulated volumes and the graphs transformed from the physical protein structures in point data was calculated by comparing their network theory order parameters including node degrees, betweenness centrality, and graph densities. By capturing the essential topological features defining the heterogeneous morphologies of a network, we were able to accurately identify proteins and homo‐multimeric complexes from 10 topologically distinctive samples without realistic noise added. Our approach empowers future developments of tomogram processing by providing pattern mining with interpretability, to enable the classification of single‐domain protein native topologies as well as distinct single‐domain proteins from multimeric complexes within noisy volumes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Domain architecture and catalysis of the Staphylococcus aureus fatty acid kinase

Fatty acid kinase (Fak) is a two-component enzyme that generates acyl-phosphate for phospholipid synthesis. Fak consists of a kinase domain protein (FakA) that phosphorylates a fatty acid enveloped by a fatty acid binding protein (FakB). The structural basis for FakB function has been established, but little is known about FakA. Here, we used limited proteolysis to define three separate FakA domains: the amino terminal FakA_N, the central FakA_L, and the carboxy terminal FakA_C. The isolated domains lack kinase activity, but activity is restored when FakA_N and FakA_L are present individually or connected as FakA_NL. The X-ray structure of the monomeric FakA_N captures the product complex with ADP and two Mg 2+ ions bound at the nucleotide site. The FakA_L domain encodes the dimerization interface along with conserved catalytic residues Cys240, His282, and His284. AlphaFold analysis of FakA_L predicts the catalytic residues are spatially clustered and pointing away from the dimerization surface. Furthermore, the X-ray structure of FakA_C shows that it consists of two subdomains that are structurally related to FakB. Analytical ultracentrifugation demonstrates that FakA_C binds FakB, and site-directed mutagenesis confirms that a positively charged wedge on FakB meshes with a negatively charged groove on FakA_C. Finally, small angle X-ray scattering analysis is consistent with freely rotating FakA_N and FakA_C domains tethered by flexible linkers to FakA_L. These data reveal specific roles for the three independently folded FakA protein domains in substrate binding and catalysis.

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

Structural basis for Zika envelope domain III recognition by a germline version of a recurrent neutralizing antibody

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. In this work, we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Intelligent resolution: Integrating Cryo-EM with AI-driven multi-resolution simulations to observe the severe acute respiratory syndrome coronavirus-2 replication-transcription machinery in action

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication transcription complex (RTC) is a multi-domain protein responsible for replicating and transcribing the viral mRNA inside a human cell. Attacking RTC function with pharmaceutical compounds is a pathway to treating COVID-19. Conventional tools, e.g., cryo-electron microscopy and all-atom molecular dynamics (AAMD), do not provide sufficiently high resolution or timescale to capture important dynamics of this molecular machine. Consequently, we develop an innovative workflow that bridges the gap between these resolutions, using mesoscale fluctuating finite element analysis (FFEA) continuum simulations and a hierarchy of AI-methods that continually learn and infer features for maintaining consistency between AAMD and FFEA simulations. We leverage a multi-site distributed workflow manager to orchestrate AI, FFEA, and AAMD jobs, providing optimal resource utilization across HPC centers. Our study provides unprecedented access to study the SARS-CoV-2 RTC machinery, while providing general capability for AI-enabled multi-resolution simulations at scale.

Trifan, Anda↗
DOE OSTI2021

Tissue distribution and subcellular localization of the family of Kidney Ankyrin Repeat Domain (KANK) proteins

Kidney Ankyrin Repeat-containing Proteins (KANKs) comprise a family of four evolutionary conserved proteins (KANK1 to 4) that localize to the belt of mature focal adhesions (FAs) where they regulate integrin-mediated adhesion, actomyosin contractility, and link FAs to the cortical microtubule stabilization complex (CMSC). The human KANK proteins were first identified in kidney and have been associated with kidney cancer and nephrotic syndrome. Here, we report the distributions and subcellular localizations of the four Kank mRNAs and proteins in mouse tissues. We found that the KANK family members display distinct and rarely overlapping expression patterns. Whereas KANK1 is expressed at the basal side of epithelial cells of all tissues tested, KANK2 expression is mainly observed at the plasma membrane and/or cytoplasm of mesenchymal cells and KANK3 exclusively in vascular and lymphatic endothelial cells. KANK4 shows the least widespread expression pattern and when present, overlaps with KANK2 in contractile cells, such as smooth muscle cells and pericytes. Our findings show that KANKs are widely expressed in a cell type-specific manner, which suggests that they have cell- and tissue-specific functions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). This study describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

59 BASIC BIOLOGICAL SCIENCES↗