scholarly journals Bioinformatic Analyses to Uncover Genes Involved in Trehalose Metabolism in the Polyploid Sugarcane

2022 ◽  
Author(s):  
Lauana Oliveira ◽  
Bruno Navarro ◽  
João Pedro Pereira ◽  
Adriana Lopes ◽  
Marina Martins ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Essential Role ◽  
Plant Metabolism ◽  
Post Translational Modifications ◽  
Expression Levels ◽  
Glycosylation Sites ◽  
Trehalose Metabolism ◽  
Trehalose Biosynthesis ◽  
Binding Residues

Abstract Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

scholarly journals Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11

1994 ◽  
Vol 302 (2) ◽  
pp. 451-454 ◽  
Author(s):  
M H Lafrance ◽  
C Vézina ◽  
Q Wang ◽  
G Boileau ◽  
P Crine ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Neutral Endopeptidase ◽  
Soluble Form ◽  
Cell Extracts ◽  
Glycosylation Sites ◽  
Sugar Addition ◽  
Membrane Bound

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

scholarly journals Structural analysis of the endogenous glycoallergen Hev b 2 (endo-β-1,3-glucanase) fromHevea brasiliensisand its recognition by human basophils

2014 ◽  
Vol 70 (2) ◽  
pp. 329-341 ◽  
Author(s):  
Adela Rodríguez-Romero ◽  
Alejandra Hernández-Santoyo ◽  
Deyanira Fuentes-Silva ◽  
Laura A. Palomares ◽  
Samira Muñoz-Cruz ◽  
...  
Keyword(s):  
Binding Sites ◽  
Plant Defence ◽  
Time Lapse ◽  
Cross Reactivity ◽  
Basophil Activation ◽  
Post Translational Modifications ◽  
Glycosylation Sites ◽  
Binding Epitope ◽  
Human Basophils

Endogenous glycosylated Hev b 2 (endo-β-1,3-glucanase) fromHevea brasiliensisis an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most importantN-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.

scholarly journals Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity

2021 ◽  
Author(s):  
Krzysztof Mikolajczyk ◽  
Anna Bereznicka ◽  
Katarzyna Szymczak-Kulus ◽  
Katarzyna Haczkiewicz-Lesniak ◽  
Bozena Szulc ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Subcellular Localization ◽  
Posttranslational Modification ◽  
Regulatory Mechanism ◽  
Specific Activity ◽  
Sweet Spot ◽  
Dual Nature ◽  
Glycosylation Sites ◽  
Opposing Effects

AbstractN-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the role of N-glycans in the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Galβ (Gb3 and the P1 antigen) and Galα1→4GalNAcβ disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and a glycoform without it showed increased enzyme activity. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localize in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the ER. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling biological activity of proteins.

scholarly journals EZH2 abnormalities in lymphoid malignancies: underlying mechanisms and therapeutic implications

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Boheng Li ◽  
Wee-Joo Chng
Keyword(s):  
Gene Expression ◽  
Essential Role ◽  
Histone H3 ◽  
Epigenetic Silencing ◽  
Post Translational Modifications ◽  
Therapeutic Implications ◽  
Polycomb Repressive Complex 2 ◽  
Lymphoid Malignancies ◽  
Underlying Mechanisms

AbstractEZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which along with other PRC2 components mediates gene expression suppression via the methylation of Histone H3 at lysine 27. Recent studies have revealed a dichotomous role of EZH2 in physiology and in the pathogenesis of cancer. While it plays an essential role in the development of the lymphoid system, its deregulation, whether due to genetic or non-genetic causes, promotes B cell- and T cell-related lymphoma or leukemia. These findings triggered a boom in the development of therapeutic EZH2 inhibitors in recent years. Here, we discuss physiologic and pathogenic function of EZH2 in lymphoid context, various internal causes of EZH2 aberrance and how EZH2 modulates lymphomagenesis through epigenetic silencing, post-translational modifications (PTMs), orchestrating with surrounding tumor micro-environment and associating with RNA or viral partners. We also summarize different strategies to directly inhibit PRC2-EZH2 or to intervene EZH2 upstream signaling.

Essential role of Sharpin in TNFα dependent NFκB activation in primary hepatocytes

2012 ◽  
Vol 50 (01) ◽  
Author(s):  
N Lange ◽  
S Sieber ◽  
A Erhardt ◽  
G Sass ◽  
HJ Kreienkamp ◽  
...  
Keyword(s):  
Essential Role ◽  
Primary Hepatocytes

Different Abilities of Thrombin Receptor Activating Peptide and Thrombin to Induce Platelet Calcium Rise and Full Release Reaction

1995 ◽  
Vol 74 (05) ◽  
pp. 1323-1328 ◽  
Author(s):  
Dominique Lasne ◽  
José Donato ◽  
Hervé Falet ◽  
Francine Rendu
Keyword(s):  
Essential Role ◽  
Calcium Influx ◽  
Dense Granule ◽  
Receptor Interaction ◽  
Thrombin Receptor ◽  
Release Reaction ◽  
External Calcium ◽  
Maximum Rise ◽  
Granule Release

SummarySynthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-1/AM- labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 μM SFLLR, 5 μM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]j. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.

Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

1979 ◽  
Vol 42 (04) ◽  
pp. 1193-1206 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Essential Role ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
High Doses ◽  
Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

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