scholarly journals The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of α-synuclein

2020 ◽  
Vol 295 (23) ◽  
pp. 8005-8016 ◽  
Author(s):  
Rachel Underwood ◽  
Bing Wang ◽  
Christine Carico ◽  
Robert H. Whitaker ◽  
William J. Placzek ◽  
...  
Keyword(s):  
Lewy Bodies ◽  
Neuronal Model ◽  
Primary Component ◽  
Size Exclusion ◽  
Autophagic Flux ◽  
Protein Levels ◽  
Exclusion Chromatography ◽  
Cell Release ◽  
The Impact

α-Synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). αsyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The cell-to-cell release and uptake of αsyn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in αsyn spread has yet to be characterized. In this study, we used a paracrine αsyn in vitro neuronal model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. Rab27b reduced αsyn release primarily through nonexosomal pathways, but the αsyn released after Rab27b KD was of higher-molecular-weight species, as determined by size-exclusion chromatography. Rab27b KD increased intracellular levels of insoluble αsyn and led to an accumulation of endogenous light chain 3 (LC3)-positive puncta. Rab27b KD also decreased LC3 turnover after treatment with an autophagosome-lysosome fusion inhibitor, chloroquine, indicating that Rab27b KD induces a defect in autophagic flux. Rab27b protein levels were increased in brain lysates obtained from postmortem tissues of individuals with PD and DLB compared with healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and, ultimately, in the pathogenesis of synucleinopathies.

scholarly journals Rab27b regulates the release, autophagic clearance, and toxicity of alpha-synuclein

2020 ◽  
Author(s):  
Rachel Underwood ◽  
Bing Wang ◽  
Christine Carico ◽  
Robert H. Whitaker ◽  
William J. Placzek ◽  
...  
Keyword(s):  
Lewy Bodies ◽  
Alpha Synuclein ◽  
Primary Component ◽  
Size Exclusion ◽  
Autophagic Flux ◽  
Postmortem Human Brain ◽  
Protein Levels ◽  
Exclusion Chromatography ◽  
The Impact

ABSTRACTAlpha synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy Bodies that pathologically define synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The release and uptake of αsyn from cell to cell are considered important processes for this prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance but has yet to be characterized in its role in αsyn spread. In this study, we used a paracrine αsyn in vitro model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. While Rab27b reduced αsyn release primarily through non-exosomal pathways, the αsyn released under KD conditions was of higher molecular weight species by size exclusion chromatography. Rab27b KD increased intracellular insoluble αsyn levels and led to an accumulation of endogenous LC3 positive puncta. Rab27b KD also decreased LC3 turnover with chloroquine treatment, indicating a defect in autophagic flux. Rab27b protein levels were increased in postmortem human brain lysates from PD and DLB subjects compared to healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and ultimately in the pathogenesis of synucleinopathies.

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 A glutaminyl cyclase-catalyzed α-synuclein modification identified in human synucleinopathies

2021 ◽  
Author(s):  
Maike Hartlage-Rübsamen ◽  
Alexandra Bluhm ◽  
Sandra Moceri ◽  
Lisa Machner ◽  
Janett Köppen ◽  
...  
Keyword(s):  
Cell Viability ◽  
Neurodegenerative Disorder ◽  
Lewy Bodies ◽  
Size Exclusion ◽  
Lewy Neurites ◽  
Enzymatic Assays ◽  
Aβ Peptides ◽  
Glutaminyl Cyclase ◽  
Affect Cell Viability ◽  
Exclusion Chromatography

AbstractParkinson’s disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79-α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may—in analogy to pGlu-Aβ peptides in Alzheimer’s disease—act as a seed for pathogenic protein aggregation.

Impact of Curcumin on Microsomal Enzyme Activities: Drug Interaction and Chemopreventive Studies

2021 ◽  
Vol 28 ◽  
Author(s):  
Habibeh Mashayekhi-Sardoo ◽  
Adeleh Mashayekhi‐Sardoo ◽  
Basil D. Roufogalis ◽  
Tannaz Jamialahmadi ◽  
Amirhossein Sahebkar
Keyword(s):  
Curcuma Longa ◽  
Significant Inhibition ◽  
Microsomal Enzyme ◽  
Scientific Databases ◽  
Protein Levels ◽  
Curcumin Analogs ◽  
The Us ◽  
Natural Polyphenol ◽  
The Impact

: Curcumin, a yellow pigment in Asian spice, is a natural polyphenol component of Curcuma longa rhizome. Curcuminoid components include curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Previous studies established curcumin as a safe agent based on preclinical and clinical evaluations and curcuminoids have been approved by the US Food and Drug Administration (FDA) as “Generally Recognized as Safe” (GRAS). The present review collects and summarizes clinical and preclinical studies of curcumin interactions, with an emphasis on the effect of curcumin and curcumin analogs on the mRNA and protein levels of microsomal CYP450 enzymes (phase I metabolism) and their interactions with toxicants, drugs and drug probes. The literature search was conducted using keywords in various scientific databases, including Web of Science, Scopus, PubMed, and Google Scholar. Studies concerning the impact of curcumin and curcumin analogs on microsomal enzyme activity are reviewed and include oral, topical, and systemic treatment in humans and experimental animals, as well as studies from in vitro research. When taken together the data identified some inconsistent results between various studies. The findings showed significant inhibition of CYP450 enzymes by curcumin and its analogs. However such effects often differed when curcumin and curcumin analogs were coadministered with toxicant and other drugs and drug probes. We conclude from this review that herb-drug interactions should be considered when curcumin and curcumin analogs are consumed.

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 Inhaled Anesthetics Promote Albumin Dimerization through Reciprocal Exchange of Subdomains

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Benjamin J. Pieters ◽  
Eugene E. Fibuch ◽  
Joshua D. Eklund ◽  
Norbert W. Seidler
Keyword(s):  
Fluorescence Emission ◽  
Size Exclusion ◽  
Inhaled Anesthetics ◽  
Protein Protein Interaction ◽  
Reciprocal Exchange ◽  
Proposed Model ◽  
Exclusion Chromatography ◽  
Spectral Shifting ◽  
And Control ◽  
The Impact

Inhaled anesthetics affect protein-protein interaction, but the mechanisms underlying these effects are still poorly understood. We examined the impact of sevoflurane and isoflurane on the dimerization of human serum albumin (HSA), a protein with anesthetic binding sites that are well characterized. Intrinsic fluorescence emission was analyzed for spectral shifting and self-quenching, and control first derivatives (spectral responses to changes in HSA concentration) were compared against those obtained from samples treated with sevoflurane or isoflurane. Sevoflurane increased dimer-dependent self-quenching and both decreased oligomer-dependent spectral shifting, suggesting that inhaled anesthetics promoted HSA dimerization. Size exclusion chromatography and polarization data were consistent with these observations. The data support the proposed model of a reciprocal exchange of subdomains to form an HSA dimer. The open-ended exchange of subdomains, which we propose occuring in HSA oligomers, was inhibited by sevoflurane and isoflurane.

A switch toward angiostatic gene expression impairs the angiogenic properties of endothelial progenitor cells in low birth weight preterm infants

Blood ◽  
2011 ◽  
Vol 118 (6) ◽  
pp. 1699-1709 ◽  
Author(s):  
Isabelle Ligi ◽  
Stéphanie Simoncini ◽  
Edwige Tellier ◽  
Paula Frizera Vassallo ◽  
Florence Sabatier ◽  
...  
Keyword(s):  
Birth Weight ◽  
Low Birth Weight ◽  
Profile Analysis ◽  
Preterm Neonates ◽  
Akt Phosphorylation ◽  
Protein Levels ◽  
Increased Risk ◽  
The Impact

Abstract Low birth weight (LBW) is associated with increased risk of cardiovascular diseases at adulthood. Nevertheless, the impact of LBW on the endothelium is not clearly established. We investigate whether LBW alters the angiogenic properties of cord blood endothelial colony forming cells (LBW-ECFCs) in 25 preterm neonates compared with 25 term neonates (CT-ECFCs). We observed that LBW decreased the number of colonies formed by ECFCs and delayed the time of appearance of their clonal progeny. LBW dramatically reduced LBW-ECFC capacity to form sprouts and tubes, to migrate and to proliferate in vitro. The angiogenic defect of LBW-ECFCs was confirmed in vivo by their inability to form robust capillary networks in Matrigel plugs injected in nu/nu mice. Gene profile analysis of LBW-ECFCs demonstrated an increased expression of antiangiogenic genes. Among them, thrombospondin 1 (THBS1) was highly expressed at RNA and protein levels in LBW-ECFCs. Silencing THBS1 restored the angiogenic properties of LBW-ECFCs by increasing AKT phosphorylation. The imbalance toward an angiostatic state provide a mechanistic link between LBW and the impaired angiogenic properties of ECFCs and allows the identification of THBS1 as a novel player in LBW-ECFC defect, opening new perspectives for novel deprogramming agents.

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