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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Differential timing of gene expression and recruitment in independent origins of CAM in the Agavoideae (Asparagaceae)

Crassulacean acid metabolism (CAM) photosynthesis has evolved repeatedly across the plant tree of life, however our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well watered and drought-stressed treatments. We further assessed gene expression and the molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. By contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2 , a copy of PPC that has never been shown to be recruited for CAM in angiosperms. Together the physiological and transcriptomic comparison of closely related C 3 and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS2011

Radiation Exposure Alters Expression of Metabolic Enzyme Genes in Mice

Most administered pharmaceuticals are metabolized by the liver. The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand the effects of spaceflight on the enzymes of the liver and exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments. Additionally, it has been previous noted that pre-exposure to small radiation doses seems to confer protection against later and larger radiation doses. This protective power of pre-exposure has been called a priming effect or radioadaptation. This study is an effort to examine the drug metabolizing effects of radioadaptation mechanisms that may be triggered by early exposure to low radiation doses.

Wotring, V. E.↗
NASA NTRS2012

Changes in Liver Metabolic Gene Expression after Radiation Exposure

The health of the liver, especially the rate of its metabolic enzymes, determines the concentration of circulating drugs as well as the duration of their efficacy. Most pharmaceuticals are metabolized by the liver, and clinically-used medication doses are given with normal liver function in mind. A drug overdose can result in the case of a liver that is damaged and removing pharmaceuticals from the circulation at a rate slower than normal. Alternatively, if liver function is elevated and removing drugs from the system more quickly than usual, it would be as if too little drug had been given for effective treatment. Because of the importance of the liver in drug metabolism, we want to understand any effects of spaceflight on the enzymes of the liver. Exposure to cosmic radiation is one aspect of spaceflight that can be modeled in ground experiments.

Peters, C. P.↗
DOE OSTI2020

Room-temperature X-ray crystallography reveals the oxidation and reactivity of cysteine residues in SARS-CoV-2 3CL Mpro: insights into enzyme mechanism and drug design

The emergence of the novel coronavirus SARS-CoV-2 has resulted in a worldwide pandemic not seen in generations. Creating treatments and vaccines to battle COVID-19, the disease caused by the virus, is of paramount importance in order to stop its spread and save lives. The viral main protease, 3CL M pro , is indispensable for the replication of SARS-CoV-2 and is therefore an important target for the design of specific protease inhibitors. Detailed knowledge of the structure and function of 3CL M pro is crucial to guide structure-aided and computational drug-design efforts. Here, the oxidation and reactivity of the cysteine residues of the protease are reported using room-temperature X-ray crystallography, revealing that the catalytic Cys145 can be trapped in the peroxysulfenic acid oxidation state at physiological pH, while the other surface cysteines remain reduced. Only Cys145 and Cys156 react with the alkylating agent N -ethylmaleimide. It is suggested that the zwitterionic Cys145–His45 catalytic dyad is the reactive species that initiates catalysis, rather than Cys145-to-His41 proton transfer via the general acid–base mechanism upon substrate binding. The structures also provide insight into the design of improved 3CL M pro inhibitors.

3CL Mpro↗
NASA NTRS1992

The contribution of the extracellular matrix to gravisensing in characean cells

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.

NASA Discipline Plant Biology↗
NASA NTRS1982

Bethanechol-Induced Water Intake in Rats: Possible Mechanisms of Induction

Acute administration of the parasympathomimetic agent, bethanechol, at 2, 4, 8 and 12 mg/kg body wt, IP, induced drinking and increased urine output of rats in a dose-dependent fashion. The first significant increases in both water intake and urine output above that of controls occurred when 4 mg/kg was administered. The drinking and increased urine output in response to administration of 8 mg bethanechol/kg was inhibited by atropine sulfate (3 and 6 mg/kg, IP). In addition, the 0-adrenergic antagonist, propranolol (6 mg/kg, IP, administered 30 min prior to treatment with bethanechol), inhibited bethanechol (8 mg/kg, IP)-induced drinking. Urine output, however, was unaffected by propranolol. Further, the angiotensin I converting enzyme inhibitor, captopril, inhibited significantly the drinking response, but not the increased urine output, accompanying administration of bethanechol (8 mg/kg). The effect of bethanechol and the beta-adrenergic agonist, isoproterenol (25 Ag/kg) separately and in combination, on water intake was also studied. Both compounds increased water intake but they exerted no interactive effect when administered simultaneously. Administration of bethanechol (8 mg/kg) to conscious rats was also accompanied by a significant reduction in both mean blood pressure and heart rate that reached minimal levels within 10 min after treatment. Both responses had returned to control level by one hour after treatment. These results suggest that bethanechol induces drinking in rats by way of the renin-angiotensin system.

Fregly, Melvin J.↗
DOE OSTI2020

Minimally disruptive optical control of protein tyrosine phosphatase 1B

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)—an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer—and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2011

System Re-set: High LET Radiation or Transient Musculoskeletal Disuse Cause Lasting Changes in Oxidative Defense Pathways Within Bone

Six months post-IR, there were no notable changes in skeletal expression of 84 principal genes in the p53 signaling pathway due to low dose IR (0.5Gy), HU, or both. In contrast, numerous genes relevant to oxidative stress were regulated by the treatments, typically in a direction indicative of increased oxidative stress and impaired defense. IR and HU independently reduced (between 0.46 to 0.88 fold) expression levels of Noxa1, Gpx3, Prdx2, Prdx3, and Zmynd17. Surprisingly, transient HU alone (sham-irradiated) decreased expression of several redox-related genes (Gpx1,Gstk1, Prdx1, Txnrd2), which were not affected significantly by IR alone. Irradiation increased (1.13 fold) expression of a gene responsible for production of superoxides by neutrophils (NCF2). Of interest, only combined treatment with HU and IR led to increased expression levels of Ercc2, (1.19 fold), a DNA excision repair enzyme. Differences in gene expression levels may reflect a change in gene expression on a per cell basis, a shift in the repertoire of specific cell types within the tissue, or both. Serum nitrite/nitrate levels were elevated to comparable levels (1.6-fold) due to IR, HU or both, indicative of elevated systemic nitrosyl stress. CONCLUSIONS The magnitude of changes in skeletal expression of oxidative stress-related genes six months after irradiation and/or transient unloading tended to be relatively modest (0.46-1.15 fold), whereas the p53 pathway was not affected. The finding that many different oxidative stress-related genes differed from controls at this late time point implicates a generalized impairment of oxidative defense within skeletal tissue, which coincides with both profound radiation damage to osteoprogenitors/stem cells in bone marrow and impaired remodeling of mineralized tissue.

Kumar, Akhilesh↗
NASA NTRS1995

Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages

Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

Non-NASA Center↗
NASA NTRS2000

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

NASA Discipline Cardiopulmonary↗
NASA NTRS1984

Isolation and characterization of beta-glucan synthase: A potential biochemical regulator of gravistimulated differential cell wall loosening

In plants, gravity stimulates differential growth in the upper and lower halves of horizontally oriented organs. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the activity of Golgi-localized Beta-1,4-glucan synthase, an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. The primary objective is to determine if auxin induces de novo formation of Golgi glucan synthase and increases the level of this glucan synthase mRNA. This shall be accomplished by (a) preparation of a monoclonal antibody to the synthase, (b) isolation, and characterization of the glucan synthase, and (c) examination for cross reactivity between the antibody and translation products of auxin induced mRNAs in pea tissue. The antibody will also be used to localize the glucan synthase in upper and lower halves of pea stem tissue before, during and after the response to gravity.

Kuzmanoff, K. M.↗
NASA NTRS1999

Angiotensin-converting enzyme and matrix metalloproteinase inhibition with developing heart failure: comparative effects on left ventricular function and geometry

The progression of congestive heart failure (CHF) is left ventricular (LV) myocardial remodeling. The matrix metalloproteinases (MMPs) contribute to tissue remodeling and therefore MMP inhibition may serve as a useful therapeutic target in CHF. Angiotensin converting enzyme (ACE) inhibition favorably affects LV myocardial remodeling in CHF. This study examined the effects of specific MMP inhibition, ACE inhibition, and combined treatment on LV systolic and diastolic function in a model of CHF. Pigs were randomly assigned to five groups: 1) rapid atrial pacing (240 beats/min) for 3 weeks (n = 8); 2) ACE inhibition (fosinopril, 2.5 mg/kg b.i.d. orally) and rapid pacing (n = 8); 3) MMP inhibition (PD166793 2 mg/kg/day p.o.) and rapid pacing (n = 8); 4) combined ACE and MMP inhibition (2.5 mg/kg b.i.d. and 2 mg/kg/day, respectively) and rapid pacing (n = 8); and 5) controls (n = 9). LV peak wall stress increased by 2-fold with rapid pacing and was reduced in all treatment groups. LV fractional shortening fell by nearly 2-fold with rapid pacing and increased in all treatment groups. The circumferential fiber shortening-systolic stress relation was reduced with rapid pacing and increased in the ACE inhibition and combination groups. LV myocardial stiffness constant was unchanged in the rapid pacing group, increased nearly 2-fold in the MMP inhibition group, and was normalized in the ACE inhibition and combination treatment groups. Increased MMP activation contributes to the LV dilation and increased wall stress with pacing CHF and a contributory downstream mechanism of ACE inhibition is an effect on MMP activity.

Non-NASA Center↗
DOE OSTI2021

Inhibitors of Mycobacterium tuberculosis EgtD target both substrate binding sites to limit hercynine production

Ergothioneine (EGT) is a low molecular weight histidine betaine essential in all domains of life but only synthesized by selected few organisms. Synthesis of EGT by Mycobacterium tuberculosis (M. tb) is critical for maintaining bioenergetic homeostasis and protecting the bacterium from alkylating agents, oxidative stress, and anti-tubercular drugs. EgtD, an S-adenosylmethionine-dependent methyltransferase (AdoMet), catalyzes the trimethylation of L-Histidine to initiate EGT biosynthesis and this reaction has been shown to be essential for EGT production in mycobacteria and for long-term infection of murine macrophages by M. tb. In this work, library screening and structure-guided strategies identified multiple classes of M. tb EgtD inhibitors that bind in various regions of the enzyme active site. X-ray crystal structures of EgtD-inhibitor complexes confirm that L-Histidine analogs bind solely to the L-Histidine binding site while drug-like inhibitors, such as TGX-221, and S-Glycyl-H-1152 span both the L-Histidine and AdoMet binding sites. These enzyme-inhibitor complexes provide detailed structural information of compound scaffolds useful for developing more potent inhibitors that could shorten Tuberculosis treatment regimens by weakening important bacterial defenses.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1985

Isolation of Hybridomas for Golgi-associated Proteins and a Plant Calmodulin

The demonstration of a role for calcium in the mechanism of the gravitropic response indicates a role for calmodulin. Localization studies indicate that plant cell walls have a high content of calmodulin which suggests a regulatory role for CaM in both gravitropic curvature and auxin-induced growth. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the ctivity of Golgi-localized B-1,4-glucan synthase (GS), an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. In order to determine whether auxin stimulates GS activity either by modulation of existing enzyme or induces de novo formation of Golgi glucan synthase, a study was undertaken to isolate and quantitate glucan synthase. This enzyme appears to be an integral protein of the Golgi membrane and has resisted isolation with retention of activity. The production of monoclonal antibody for glucan synthase was undertaken due to the inability to isolate GS by standard detergent/liposome techniques.

Kuzmanoff, K. M.↗
NASA NTRS2000

Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis

Induction of the biosynthesis of phenylpropanoids was monitored at the enzyme level through measurement of the temporal change in the activity of two marker enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, (PAL, E.C. 4.1.3.5) and 4-coumaryl-CoA ligase (4-CL, E.C. 6.2.1.12) and two marker enzymes for hydroxycinnamyl alcohol biosynthesis, cinnamoyl-CoA:NADP+ oxidoreductase (CCR, E.C. 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) in both suberizing potato (Solanum tuberosum) tubers and lignifying loblolly pine (Pinus taeda) cell cultures. While measurable activities of PAL, 4-CL and CAD increased upon initiation of suberization in potato tubers, that of CCR did not. By contrast, all four enzymes were induced upon initiation of lignification in pine cell cultures. The lack of CCR induction in potato by wound treatment is consistent with the channelling of hydroxycinnamoyl-CoA derivatives away from monolignol formation and toward other hydroxycinnamoyl derivatives such as those that accumulate during suberization.

Non-NASA Center↗
DOE OSTI2020

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Deubiquitinating enzyme USP46 suppresses the progression of hepatocellular carcinoma by stabilizing MST1

Highlights: • Low USP46 level is correlated with poor prognosis in HCC. • USP46 suppresses the proliferation and metastasis of HCC. • USP46 suppress the progression of HCC by inhibiting the expression of YAP1. • MST1 mediates the regulation of USP46 on YAP1. • USP46 stabilized MST1 protein through inhibiting its ubiquitination and degradation. The deubiquitinating enzyme USP46 (ubiquitin-specific protease 46) is implicated in various cancers. However, its role and regulatory mechanism in HCC (hepatocellular carcinoma) are still unknown. In this study, we showed that USP46 is downregulated in HCC tissues and that low USP46 levels are associated with poor prognosis in HCC patients. In functional experiments, overexpression of USP46 impaired proliferation and metastasis of HCC cells, whereas knockdown of USP46 enhanced cell proliferation and invasiveness in vitro and in vivo. Furthermore, we found that USP46 suppresses HCC cell proliferation and metastasis by inhibiting YAP1. Ectopic expression of YAP1 rescued the inhibition of cell proliferation and metastasis caused by USP46 overexpression. Mechanistically, USP46 promotes the degradation of YAP1 by increasing expression of MST1, and the increase in MST1 protein antagonizes YAP1 to suppress HCC progression. Finally, we demonstrated that USP46 stabilizes the MST1 protein by directly binding to it and decreasing its ubiquitination. Taken together, our results demonstrated that USP46 may be a novel tumor suppressor in HCC. Moreover, USP46 acts as a deubiquitinating enzyme of MST1 to potentiate MST1 kinase activity to suppress tumor growth and metastasis, indicating that USP46 activation may represent a potential treatment strategy for HCC.

60 APPLIED LIFE SCIENCES↗