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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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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont

ABSTRACT Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus , which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes . IMPORTANCE The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

TU-FG-209-12: Treatment Site and View Recognition in X-Ray Images with Hierarchical Multiclass Recognition Models

Purpose: To investigate an approach of automatically recognizing anatomical sites and imaging views (the orientation of the image acquisition) in 2D X-ray images. Methods: A hierarchical (binary tree) multiclass recognition model was developed to recognize the treatment sites and views in x-ray images. From top to bottom of the tree, the treatment sites are grouped hierarchically from more general to more specific. Each node in the hierarchical model was designed to assign images to one of two categories of anatomical sites. The binary image classification function of each node in the hierarchical model is implemented by using a PCA transformation and a support vector machine (SVM) model. The optimal PCA transformation matrices and SVM models are obtained by learning from a set of sample images. Alternatives of the hierarchical model were developed to support three scenarios of site recognition that may happen in radiotherapy clinics, including two or one X-ray images with or without view information. The performance of the approach was tested with images of 120 patients from six treatment sites – brain, head-neck, breast, lung, abdomen and pelvis – with 20 patients per site and two views (AP and RT) per patient. Results: Given two images in known orthogonal views (AP and RT), the hierarchical model achieved a 99% average F1 score to recognize the six sites. Site specific view recognition models have 100 percent accuracy. The computation time to process a new patient case (preprocessing, site and view recognition) is 0.02 seconds. Conclusion: The proposed hierarchical model of site and view recognition is effective and computationally efficient. It could be useful to automatically and independently confirm the treatment sites and views in daily setup x-ray 2D images. It could also be applied to guide subsequent image processing tasks, e.g. site and view dependent contrast enhancement and image registration. The senior author received research grants from ViewRay Inc. and Varian Medical System.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

PHD Finger Recognition of Unmodified Histone H3R2 Links UHRF1 to Regulation of Euchromatic Gene Expression

Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHD{sub UHRF1}), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHD{sub UHRF1} bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHD{sub UHRF1} binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.

08 HYDROGEN↗
DOE OSTI2019

The three-dimensional structure and recognition mechanism of Manduca sexta peptidoglycan recognition protein-1

Peptidoglycan recognition proteins (PGRPs) recognize bacteria through their unique cell wall constituent, peptidoglycans (PGs). PGRPs are conserved from insects to mammals and all function in antibacterial defense. In the tobacco hornworm Manduca sexta, PGRP1 and microbe binding protein (MBP) interact with PGs and hemolymph protease-14 precursor (proHP14) to yield active HP14. HP14 triggers a serine protease network that produces active phenoloxidase (PO), Spätzle, and other cytokines to stimulate immune responses. PGRP1 binds preferentially to diaminopimelic acid (DAP)-PGs of Gram-negative bacteria and Gram-positive Bacillus and Clostridium species than Lys-PGs of other Gram-positive bacteria. In this study, we synthesized DAP- and Lys-muramyl pentapeptide (MPP) and monitored their associations with M. sexta PGRP1 by surface plasmon resonance. The K d values (0.57 μM for DAP-MPP and 45.6 μM for Lys-MPP) agree with the differential recognition of DAP- and Lys-PGs. To reveal its structural basis, we produced the PGRP1 in insect cells and determined its structure at a resolution of 2.1 Å. The protein adopts a fold similar to those from other PGRPs with a classical L-shaped PG-binding groove. A unique loop lining the shallow groove suggests a different ligand-binding mechanism. In summary, this study provided new insights into the PG recognition by PGRPs, a critical first step that initiates the serine protease cascade.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Structural Basis for Recognition of H3T3ph and Smac/DIABLO N-terminal Peptides by Human Survivin

Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth

Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate–protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell‐based assays, glycan microarrays, and X‐ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI ‐60 panel; in particular, it inhibited growth of melanoma cancer cells ( LOX IMVI ) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N ‐acetylgalactosamine (Gal NA c). BfL was found to have especially strong binding (apparent K d = 0.5–1.0 n m ) to the tumor‐associated Tn antigen. High‐resolution crystal structures were determined for the ligand‐free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of Gal NA c recognition. Of special note, the carboxylate group of Glu126, lining the glycan‐binding pocket, forms H‐bonds with both the N ‐acetyl of Gal NA c and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any Gal NA c‐specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. Database Structural data are available in the PDB under the accession numbers 5T50 , 5T52 , 5T55 , 5T54 , 5T5L , 5T5J , 5T5P , and 5T5O .

Lubkowski, Jacek↗
DOE OSTI2011

Structural Basis for Promoter ;#8722;10 Element Recognition by the Bacterial RNA Polymerase [sigma] Subunit

The key step in bacterial promoter opening is recognition of the -10 promoter element (T-{sub 12}A-{sub 11}T-{sub 10}A-{sub 9}A-{sub 8}T{sub -7} consensus sequence) by the RNA polymerase {alpha} subunit. We determined crystal structures of {alpha} domain 2 bound to single-stranded DNA bearing -10 element sequences. Extensive interactions occur between the protein and the DNA backbone of every -10 element nucleotide. Base-specific interactions occur primarily with A{sub -11} and T{sub -7}, which are flipped out of the single-stranded DNA base stack and buried deep in protein pockets. The structures, along with biochemical data, support a model where the recognition of the -10 element sequence drives initial promoter opening as the bases of the nontemplate strand are extruded from the DNA double-helix and captured by {alpha}. These results provide a detailed structural basis for the critical roles of A{sub -11} and T{sub -7} in promoter melting and reveal important insights into the initiation of transcription bubble formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Structural basis of RNA recognition and activation by innate immune receptor RIG-I

Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

ATRX ADD Domain Links an Atypical Histone Methylation Recognition Mechanism to Human Mental-Retardation Syndrome

ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD{sub ATRX}), whose function has remained elusive. Here we identify ADD{sub ATRX} as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD{sub ATRX} bound to H3{sub 1-15}K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Specific Recognition of Arginine Methylated Histone Tails by JMJD5 and JMJD7

We have reported that JMJD5 and JMJD7 (JMJD5/7) are responsible for the clipping of arginine methylated histone tails to generate "tailless nucleosomes", which could release the pausing RNA polymerase II (Pol II) into productive transcription elongation. JMJD5/7 function as endopeptidases that cleave histone tails specifically adjacent to methylated arginine residues and continue to degrade N-terminal residues of histones via their aminopeptidase activity. Here, we report structural and biochemical studies on JMJD5/7 to understand the basis of substrate recognition and catalysis mechanism by this JmjC subfamily. Recognition between these enzymes and histone substrates is specific, which is reflected by the binding data between enzymes and substrates. High structural similarity between JMJD5 and JMJD7 is reflected by the shared common substrates and high binding affinity. However, JMJD5 does not bind to arginine methylated histone tails with additional lysine acetylation while JMJD7 does not bind to arginine methylated histone tails with additional lysine methylation. Furthermore, the complex structures of JMJD5 and arginine derivatives revealed a Tudor domain-like binding pocket to accommodate the methylated sidechain of arginine, but not lysine. There also exists a glutamine close to the catalytic center, which may suggest a unique imidic acid mediated catalytic mechanism for proteolysis by JMJD5/7.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Recognition of Multivalent Histone States Associated with Heterochromatin by UHRF1 Protein

Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16{sup INK4A}, when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

A computer aided treatment event recognition system in radiation therapy

Purpose: To develop an automated system to safeguard radiation therapy treatments by analyzing electronic treatment records and reporting treatment events. Methods: CATERS (Computer Aided Treatment Event Recognition System) was developed to detect treatment events by retrieving and analyzing electronic treatment records. CATERS is designed to make the treatment monitoring process more efficient by automating the search of the electronic record for possible deviations from physician's intention, such as logical inconsistencies as well as aberrant treatment parameters (e.g., beam energy, dose, table position, prescription change, treatment overrides, etc). Over a 5 month period (July 2012–November 2012), physicists were assisted by the CATERS software in conducting normal weekly chart checks with the aims of (a) determining the relative frequency of particular events in the authors’ clinic and (b) incorporating these checks into the CATERS. During this study period, 491 patients were treated at the University of Iowa Hospitals and Clinics for a total of 7692 fractions. Results: All treatment records from the 5 month analysis period were evaluated using all the checks incorporated into CATERS after the training period. About 553 events were detected as being exceptions, although none of them had significant dosimetric impact on patient treatments. These events included every known event type that was discovered during the trial period. A frequency analysis of the events showed that the top three types of detected events were couch position override (3.2%), extra cone beam imaging (1.85%), and significant couch position deviation (1.31%). The significant couch deviation is defined as the number of treatments where couch vertical exceeded two times standard deviation of all couch verticals, or couch lateral/longitudinal exceeded three times standard deviation of all couch laterals and longitudinals. On average, the application takes about 1 s per patient when executed on either a desktop computer or a mobile device. Conclusions: CATERS offers an effective tool to detect and report treatment events. Automation and rapid processing enables electronic record interrogation daily, alerting the medical physicist of deviations potentially days prior to performing weekly check. The output of CATERS could also be utilized as an important input to failure mode and effects analysis.

62 RADIOLOGY AND NUCLEAR MEDICINE↗