scholarly journals The interaction of the mitochondrial protein importer TOMM34 with HSP70 is regulated by TOMM34 phosphorylation and binding to 14-3-3 adaptors

2020 ◽  
Vol 295 (27) ◽  
pp. 8928-8944 ◽  
Author(s):  
Filip Trcka ◽  
Michal Durech ◽  
Pavla Vankova ◽  
Veronika Vandova ◽  
Oliver Simoncik ◽  
...  
Keyword(s):  
Structural Changes ◽  
Protein Transport ◽  
Mitochondrial Protein ◽  
Inhibitory Effect ◽  
Size Exclusion ◽  
Tetratricopeptide Repeat ◽  
Interaction Interface ◽  
Exclusion Chromatography ◽  
Hydrogen Deuterium

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90–mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro. In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

scholarly journals Eduscho Coffee Extract Effectively Inhibits the Formation of Amyloid-like Fibrils by Trypsin in Aqueous Ethanol

2018 ◽  
Vol 13 (12) ◽  
pp. 1934578X1801301 ◽  
Author(s):  
Phanindra Babu Kasi ◽  
Attila Borics ◽  
Kinga Molnár ◽  
Lajos László ◽  
Márta Kotormán
Keyword(s):  
Binding Assay ◽  
Phenolic Content ◽  
Aqueous Ethanol ◽  
Inhibitory Effect ◽  
Size Exclusion ◽  
Total Phenolic ◽  
Transmission Electron ◽  
Oligomeric Form ◽  
Exclusion Chromatography

In this work we used an in vitro trypsin aggregation model to show that certain commercial coffee extracts can inhibit protein aggregation. Aggregation experiments were performed using several spectroscopic methods and a dye binding assay, such as turbidity, Congo red (CR) and electronic circular dichroism (ECD), that was further supported by transmission electron microscopy (TEM). A correlation was found between the anti-aggregation properties and the total phenolic content of the coffee extracts. The results revealed that the greatest effect was exerted by the Eduscho coffee extract. It was found that the inhibitory effect of this extract was concentration dependent. Using size exclusion chromatography, we demonstrated that the inhibitory effect of the Eduscho coffee extract on the formation of amyloid-like fibrils was due to its capacity to stabilize the oligomeric form of the protein.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

scholarly journals A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 157
Author(s):  
Kinga Böszörményi ◽  
Janet Hirsch ◽  
Gwendoline Kiemenyi Kayere ◽  
Zahra Fagrouch ◽  
Nicole Heijmans ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Neutralizing Antibodies ◽  
Size Exclusion ◽  
Vitro Assay ◽  
Usutu Virus ◽  
Transmission Electron ◽  
Exclusion Chromatography ◽  
Efficacious Vaccine ◽  
Human Infections

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

scholarly journals Garlic Extract: Inhibition of Biochemical and Biophysical Changes in Glycated HSA

2021 ◽  
Vol 11 (22) ◽  
pp. 11028
Author(s):  
Mohd W. A. Khan ◽  
Ahmed A. Otaibi ◽  
Arwa F. M. Alhumaid ◽  
Abdulmohsen K. D. Alsukaibi ◽  
Asma K. Alshamari ◽  
...  
Keyword(s):  
Structural Changes ◽  
Therapeutic Potential ◽  
Inhibitory Effect ◽  
Significant Inhibition ◽  
Garlic Extract ◽  
Diabetic Patients ◽  
Age Related ◽  
Health Complications ◽  
Biophysical Changes

Glycation of various biomolecules contributes to structural changes and formation of several high molecular weight fluorescent and non-fluorescent, advanced glycation end products (AGEs). AGEs and glycation are involved in various health complications. Synthetic medicines, including metformin, have several adverse effects. Natural products and their derivatives are used in the treatment of various diseases due to their significant therapeutic qualities. Allium sativum (garlic) is used in traditional medicines because of its antioxidant, anti-inflammatory, and anti-diabetic properties. This study aimed to determine the anti-glycating and AGEs inhibitory activities of garlic. Biochemical and biophysical analyses were performed for in vitro incubated human serum albumin (HSA) with 0.05 M of glucose for 1, 5, and 10 weeks. Anti-glycating and AGEs inhibitory effect of garlic was investigated in glycated samples. Increased biochemical and biophysical changes were observed in glycated HSA incubated for 10 weeks (G-HSA-10W) as compared to native HSA (N-HSA) as well as glycated HSA incubated for 1 (G-HSA-1W) and 5 weeks (G-HSA-5W). Garlic extract with a concentration of ≥6.25 µg/mL exhibited significant inhibition in biophysical and biochemical changes of G-HSA-10W. Our findings demonstrated that garlic extract has the ability to inhibit biochemical and biophysical changes in HSA that occurred due to glycation. Thus, garlic extract can be used against glycation and AGE-related health complications linked with chronic diseases in diabetic patients due to its broad therapeutic potential.

scholarly journals Isolation, characterization and in vitro anti-salmonellal activity of compounds from stem bark extract of Canarium schweinfurthii

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Jean Baptiste SOKOUDJOU ◽  
Olubunmi ATOLANI ◽  
Guy Sedar Singor NJATENG ◽  
Afsar KHAN ◽  
Cyrille Ngoufack TAGOUSOP ◽  
...  
Keyword(s):  
Gallic Acid ◽  
Food Poisoning ◽  
Ethanolic Extract ◽  
Stem Bark ◽  
Size Exclusion ◽  
Bark Extract ◽  
Main Stream ◽  
Exclusion Chromatography ◽  
Stream Control

Abstract Background Bacteria belonging to the Salmonella genus are major concern for health, as they are widely reported in many cases of food poisoning. The use of antibiotics remains a main stream control strategy for avian salmonellosis as well as typhoid and paratyphoid fevers in humans. Due to the growing awareness about drug resistance and toxicities, the use of antibiotics is being discouraged in many countries whilst advocating potent benign alternatives such as phyto-based medicine. The objective of this work was to isolate, characterise the bioactive compounds of Canarium schweinfurthii; and evaluate their anti-salmonellal activity. Methods The hydro-ethanolic extract of Canarium schweinfurthii was fractionated and tested for their anti-salmonellal activity. The most active fractions (i.e. chloroform and ethyl acetate partition fractions) were then explored for their phytochemical constituents. Fractionation on normal phase silica gel column chromatography and size exclusion chromatography on Sephadex LH-20 led to the isolation of four compounds (maniladiol, scopoletin, ethyl gallate and gallic acid) reported for the first time in Canarium schweinfurthii. Results Result indicated that scopoletin and gallic acid had greater activity than the crude extracts and partition fractions. Among the isolated compounds, scopoletin showed the highest inhibitory activity with a MIC of 16 μg/ml against Salmonella Typhimurium and Salmonella Enteritidis. Conclusions The overall results of this study indicates that the hydro-ethanolic extract as well as some of isolated compounds have interesting anti-salmonellal activities that could be further explored for the development of potent therapy for salmonellosis. Furthermore, the study adds credence to the folkloric applications of the plant.

scholarly journals Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

2020 ◽  
Vol 21 (7) ◽  
pp. 2400 ◽  
Author(s):  
René Stürmer ◽  
Jana Reising ◽  
Werner Hoffmann
Keyword(s):  
Molecular Mass ◽  
Complex I ◽  
Size Exclusion ◽  
Trefoil Factor ◽  
Binding Studies ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Exclusion Chromatography ◽  
Trefoil Factor Family

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

scholarly journals Prebiotic Activity of Poly- and Oligosaccharides Obtained from Plantago major L. Leaves

2020 ◽  
Vol 10 (8) ◽  
pp. 2648 ◽  
Author(s):  
Paolina Lukova ◽  
Mariana Nikolova ◽  
Emmanuel Petit ◽  
Redouan Elboutachfaiti ◽  
Tonka Vasileva ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Galacturonic Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Plantago Major ◽  
Prebiotic Activity ◽  
Molecular Fraction ◽  
Exclusion Chromatography

The aim of the present study was to evaluate the prebiotic potential of Plantago major L. leaves water-extractable polysaccharide (PWPs) and its lower molecular fractions. The structure of PWPs was investigated by high pressure anion exchange chromatography (HPAEC), size exclusion chromatography coupled with multi-angle laser light scattering detector (SEC-MALLS) and Fourier-transform infrared (FTIR) spectroscopy. The chemical composition and monosaccharide analyses showed that galacturonic acid was the main monosaccharide of PWPs followed by glucose, arabinose, galactose, rhamnose and xylose. FTIR study indicated a strong characteristic absorption peak at 1550 cm−1 corresponding to the vibration of COO− group of galacturonic acid. The PWPs was subjected to hydrolysis using commercial enzymes to obtain P. major low molecular fraction (PLM) which was successively separated by size exclusion chromatography on Biogel P2. PWPs and PLM were examined for in vitro prebiotic activity using various assays. Results gave evidence for changes in optical density of the bacteria cells and pH of the growth medium. A heterofermentative process with a lactate/acetate ratio ranged from 1:1 to 1:5 was observed. The ability of PLM to stimulate the production of certain probiotic bacteria glycohydrolases and to be fermented by Lactobacillus sp. strains was successfully proved.

scholarly journals Preliminary Characterization of a Homogeneous Polysaccharide with Anticomplement Activity from Sijunzi Decoction

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Ruijun Wang ◽  
Yunfei Ji ◽  
Ying Peng ◽  
Xiaobo Li
Keyword(s):  
Immune System ◽  
Size Exclusion ◽  
Molar Ratio ◽  
Classical Pathway ◽  
Glycosidic Bonds ◽  
Atomic Force ◽  
Exclusion Chromatography ◽  
Ft Ir ◽  
Immune System Regulation

Sijunzi decoction (SJZD) is a classical herbal prescription in traditional Chinese medicine (TCM) used for enhancing the function of immune system. In previous studies, a polysaccharide fraction S-3 was screened from SJZD by assessment of immune system regulation, intestinal microbiota, and SCFA in order to explore the immune active ingredients in SJZD. In the present study, S-3 was further purified, and a homogeneous polysaccharide S-3-1 with a molecular mass of 13.5 × 104 Da was obtained after further fractionation by Sephadex G-150 size-exclusion chromatography. The immunological activities of S-3-1 were assayed in vitro for the first time. The determination of the anticomplement activity showed that S-3-1 displayed inhibitory effects on classical pathway of the complement system, with CH50 values of 530 μg/mL. The FT-IR analysis showed that S-3-1 had absorptive peaks characteristic of polysaccharides. The methylation and GC-MS analysis showed that it is comprised of Rha, Ara, Xyl, Man, Gal, and Glc in a relative molar ratio of 0.35 : 0.37 : 1.4 : 0.31 : 3 : 0.8 and that it mainly contained 1,4-linked-Glc and 1,6-linked-Gal glycosidic bonds. The morphology of S-3-1 was observed by atomic force microscope (AFM). These results provided evidences for tracking the material basis of SJZD immune activity.

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