scholarly journals The N-terminal domain of rhamnosyltransferase EpsF influences exopolysaccharide chain length determination in Streptococcus thermophilus 05-34

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8524
Author(s):  
Guohong Wang ◽  
Jiaxi Li ◽  
Shuxin Xie ◽  
Zhengyuan Zhai ◽  
Yanling Hao
Keyword(s):  
Molecular Mass ◽  
Chain Length ◽  
Streptococcus Thermophilus ◽  
Monosaccharide Composition ◽  
Truncated Form ◽  
Terminal Domain ◽  
Homologous Overexpression ◽  
Expression Of Genes ◽  
Length Determination

Glycosyltransferases are key enzymes involved in the assembly of repeating units of exopolysaccharides (EPS). A glycosyltransferase generally consists of the N-terminal and the C-terminal domain, however, the functional role of these domains in EPS biosynthesis remains largely unknown. In this study, homologous overexpression was employed to investigate the effects of EpsFN, a truncated form of rhamnosyltransferase EpsF with only the N-terminal domain, on EPS biosynthesis in Streptococcus thermophilus 05-34. Reverse transcription qPCR and Western blotting analysis confirmed the successful expression of epsFN in 05-34 at the transcription and translation level, respectively. Further analysis showed that the monosaccharide composition and yield of EPS were not affected by the overexpression of epsFN, whereas the molecular mass decreased by 5-fold. Accordingly, the transcription levels of genes involved in EPS biosynthesis, including chain-length determination gene epsC, were down-regulated by 5- to 6-fold. These results indicated that the N-terminal domain of EpsF alone could influence the molecular mass of EPS, probably via lowering the concentration of sugar precursors, which may lead to decreased expression of genes responsible for chain-length determination.

scholarly journals Homologous Over-Expression of Chain Length Determination Protein EpsC Increases the Molecular Weight of Exopolysaccharide in Streptococcus thermophilus 05-34

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhengyuan Zhai ◽  
Shuxin Xie ◽  
Hongxing Zhang ◽  
Huaxi Yi ◽  
Yanling Hao
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
High Performance ◽  
Gel Permeation Chromatography ◽  
Streptococcus Thermophilus ◽  
Monosaccharide Composition ◽  
Molar Ratio ◽  
Gas Chromatography Mass Spectrometry ◽  
Over Expression ◽  
Length Determination

In Streptococcus thermophilus, EpsC is a polysaccharide co-polymerase which is involved in determining the chain length of EPS synthesized by the Wzx/Wzy-dependent pathway. Our previous study found that there was a positive correlation between transcription level of epsC and molecular weight of EPS in S. thermophilus 05-34. To further investigate the effects of EpsC on EPS biosynthesis, this gene was over-expressed in S. thermophilus 05-34 in this study. Reverse transcription qPCR and Western blotting confirmed the successful transcription and translation of epsC in 05-34, respectively. The yield of EPS was not affected by the over-expression of EpsC. Gas chromatography-mass spectrometry (GC-MS) showed that the monosaccharide composition was still composed of galactose and glucose in a molar ratio of 1.0:0.8, whereas high performance gel permeation chromatography (HPGPC) indicated that the molecular weight of EPS was increased from 4.62 × 105 Da to 9.17 × 105 Da by the over-expression of EpsC. In addition, S. thermophilus 05epsC which could produce higher molecular weight EPS improved the viscoelasticity and water-holding capacity of yogurt, but significantly reduced the level of syneresis in yogurt. In summary, these results indicated that homologous over-expression of EpsC in S. thermophilus could increase the molecular weight of EPS and improve the microrheological or physical properties of yogurt.

scholarly journals Arginine in the FARM and SARM: A Role in Chain-Length Determination for Arginine in the Aspartate-Rich Motifs of Isoprenyl Diphosphate Synthases from Mycobacterium tuberculosis

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2546 ◽  
Author(s):  
Raimund Nagel ◽  
Jill Thomas ◽  
Faith Adekunle ◽  
Francis Mann ◽  
Reuben Peters
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Chain Length ◽  
Site Directed Mutagenesis ◽  
Primary Role ◽  
Carbon Precursor ◽  
Geranyl Diphosphate ◽  
Length Determination ◽  
Isoprenyl Diphosphate

Isoprenyl chains are found in many important metabolites. These are derived from precursors of the appropriate length produced by isoprenyl diphosphate synthases (IDSs). The human pathogen Mycobacterium tuberculosis makes various isoprenoids/terpenoids, with important roles in their biosynthesis played by two closely related IDSs, encoded by grcC1 (Rv0562) and grcC2 (Rv0989c), with Rv0989c generating the 10-carbon precursor (E)-geranyl diphosphate (GPP), and Rv0562 the 20-carbon precursor (E,E,E)-geranylgeranyl diphosphate (GGPP). Intriguingly, while Rv0562 contains the prototypical trans-IDS first and second aspartate-rich (DDxxD) motifs (FARM and SARM, respectively), Rv0989c uniquely contains arginine in place of the second Asp in the FARM and first Asp in the SARM. Here site-directed mutagenesis of the corresponding residues in both Rv0562 and Rv0989c reveals that these play a role in determination of product chain length. Specifically, substitution of Asp for the Arg in the FARM and SARM of Rv0989c leads to increased production of the longer 15-carbon farnesyl diphosphate (FPP), while substitution of Arg for the corresponding Asp in Rv0562 leads to increased release of shorter products, both FPP and GPP. Accordingly, while the primary role of the FARM and SARM is known to be chelation of the divalent magnesium ion co-factors that assist substrate binding and catalysis, the Arg substitutions found in Rv0989c seem to provide a novel means by which product chain length is moderated, at least in these M. tuberculosis IDSs.

scholarly journals The role of histidine-114 of Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase in chain-length determination

FEBS Letters ◽  
2000 ◽  
Vol 481 (1) ◽  
pp. 68-72 ◽  
Author(s):  
Kazutake Hirooka ◽  
Tatsuya Kato ◽  
Jun-ichiro Matsu-ura ◽  
Hisashi Hemmi ◽  
Tokuzo Nishino
Keyword(s):  
Chain Length ◽  
Sulfolobus Acidocaldarius ◽  
Geranylgeranyl Diphosphate Synthase ◽  
Geranylgeranyl Diphosphate ◽  
Length Determination

Directive Effect of Chain Length in Modulating Peptide Nano-assemblies

2020 ◽  
Vol 27 (9) ◽  
pp. 923-929
Author(s):  
Gaurav Pandey ◽  
Prem Prakash Das ◽  
Vibin Ramakrishnan
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Light Scattering ◽  
Chain Length ◽  
Self Assembly ◽  
The Self ◽  
Ionic Charge ◽  
Self Assembling ◽  
Biophysical Techniques

Background: RADA-4 (Ac-RADARADARADARADA-NH2) is the most extensively studied and marketed self-assembling peptide, forming hydrogel, used to create defined threedimensional microenvironments for cell culture applications. Objectives: In this work, we use various biophysical techniques to investigate the length dependency of RADA aggregation and assembly. Methods: We synthesized a series of RADA-N peptides, N ranging from 1 to 4, resulting in four peptides having 4, 8, 12, and 16 amino acids in their sequence. Through a combination of various biophysical methods including thioflavin T fluorescence assay, static right angle light scattering assay, Dynamic Light Scattering (DLS), electron microscopy, CD, and IR spectroscopy, we have examined the role of chain-length on the self-assembly of RADA peptide. Results: Our observations show that the aggregation of ionic, charge-complementary RADA motifcontaining peptides is length-dependent, with N less than 3 are not forming spontaneous selfassemblies. Conclusion: The six biophysical experiments discussed in this paper validate the significance of chain-length on the epitaxial growth of RADA peptide self-assembly.

New trends in the pharmacological intervention of PPARs in obesity: Role of natural and synthetic compounds_

2020 ◽  
Vol 28 ◽  
Author(s):  
Seyed Mohammad Nabavi ◽  
Kasi Pandima Devi ◽  
Sethuraman Sathya ◽  
Ana Sanches-Silva ◽  
Listos Joanna ◽  
...  
Keyword(s):  
Tissue Expression ◽  
Health Concern ◽  
Pharmacological Intervention ◽  
Peroxisome Proliferator ◽  
Expression Of Genes ◽  
Ppar Agonists ◽  
Prevalence Of Obesity ◽  
Peroxisome Proliferator Activated Receptors ◽  
Natural And Synthetic

: Obesity is a major health concern for a growing fraction of the population, with the prevalence of obesity and its related metabolic disorders not being fully understood. Over the last decade, many attempts have been undertaken to understand the mechanisms at the basis of this condition, in which the accumulation of fat occurring in adipose tissue, leads to the pathogenesis of obesity related disorders. Among the most recent studies, those on Peroxisome Proliferator Activated Receptors (PPARs) revealed that these nuclear receptor proteins acting as transcription factors, among others, regulate the expression of genes involved in energy, lipid, and glucose metabolisms, and chronic inflammation. The three different isotypes of PPARs, with different tissue expression and ligand binding specificity, exert similar or overlapping functions directly or indirectly linked to obesity. In this study, we reviewed the available scientific reports concerning the PPARs structure and functions, especially in obesity, considering both natural and synthetic ligands and their role in the therapy of obesity and obesity-associated disorders. In the whole, the collected data show that there are both natural and synthetic compounds that show beneficial promising activity as PPAR agonists in chronic diseases related to obesity.

scholarly journals The role of the C-terminal domain in collagenase and stromelysin specificity.

1992 ◽  
Vol 267 (14) ◽  
pp. 9612-9618 ◽  
Author(s):  
G Murphy ◽  
J.A. Allan ◽  
F Willenbrock ◽  
M.I. Cockett ◽  
J.P. O'Connell ◽  
...  
Keyword(s):  
Terminal Domain

scholarly journals Catalytic effectiveness of human aldose reductase. Critical role of C-terminal domain.

1992 ◽  
Vol 267 (29) ◽  
pp. 20965-20970
Author(s):  
K.M. Bohren ◽  
C.E. Grimshaw ◽  
K.H. Gabbay
Keyword(s):  
Aldose Reductase ◽  
Critical Role ◽  
Terminal Domain

RNA secondary structure dependence in METTL3–METTL14 mRNA methylation is modulated by the N-terminal domain of METTL3

2020 ◽  
Vol 402 (1) ◽  
pp. 89-98
Author(s):  
Nathalie Meiser ◽  
Nicole Mench ◽  
Martin Hengesbach
Keyword(s):  
Secondary Structure ◽  
Rna Binding ◽  
Specific Protein ◽  
Structure Dependence ◽  
Terminal Domain ◽  
Methylation Assay ◽  
A Site ◽  
Methylation Activity

AbstractN6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3–METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.

scholarly journals The Role of Extracellular Vesicles as Shuttles of RNA and Their Clinical Significance as Biomarkers in Hepatocellular Carcinoma

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 902
Author(s):  
Eva Costanzi ◽  
Carolina Simioni ◽  
Gabriele Varano ◽  
Cinzia Brenna ◽  
Ilaria Conti ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Extracellular Vesicles ◽  
Tumor Metastasis ◽  
Biological Processes ◽  
Rna Molecules ◽  
Expression Of Genes ◽  
Non Coding Rna ◽  
Donor Cells ◽  
Relevant Role

Extracellular vesicles (EVs) have attracted interest as mediators of intercellular communication following the discovery that EVs contain RNA molecules, including non-coding RNA (ncRNA). Growing evidence for the enrichment of peculiar RNA species in specific EV subtypes has been demonstrated. ncRNAs, transferred from donor cells to recipient cells, confer to EVs the feature to regulate the expression of genes involved in differentiation, proliferation, apoptosis, and other biological processes. These multiple actions require accuracy in the isolation of RNA content from EVs and the methodologies used play a relevant role. In liver, EVs play a crucial role in regulating cell–cell communications and several pathophysiological events in the heterogeneous liver class of cells via horizontal transfer of their cargo. This review aims to discuss the rising role of EVs and their ncRNAs content in regulating specific aspects of hepatocellular carcinoma development, including tumorigenesis, angiogenesis, and tumor metastasis. We analyze the progress in EV-ncRNAs’ potential clinical applications as important diagnostic and prognostic biomarkers for liver conditions.

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