scholarly journals Exploring Extracellular Vesicles Biogenesis in Hypothalamic Cells through a Heavy Isotope Pulse/Trace Proteomic Approach

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1320 ◽  
Author(s):  
Chee Fan Tan ◽  
Hui San Teo ◽  
Jung Eun Park ◽  
Bamaprasad Dutta ◽  
Shun Wilford Tse ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Quantitative Proteomics ◽  
Energy Homeostasis ◽  
Protein Composition ◽  
Heavy Isotope ◽  
Stable Isotope Labelling ◽  
Proteomic Approach ◽  
Preferential Localization ◽  
Hypothalamic Cells ◽  
Chemical Inhibition

Studies have shown that the process of extracellular vesicles (EVs) secretion and lysosome status are linked. When the lysosome is under stress, the cells would secrete more EVs to maintain cellular homeostasis. However, the process that governs lysosomal activity and EVs secretion remains poorly defined and we postulated that certain proteins essential for EVs biogenesis are constantly synthesized and preferentially sorted to the EVs rather than the lysosome. A pulsed stable isotope labelling of amino acids in cell culture (pSILAC) based quantitative proteomics methodology was employed to study the preferential localization of the newly synthesized proteins into the EVs over lysosome in mHypoA 2/28 hypothalamic cell line. Through proteomic analysis, we found numerous newly synthesized lysosomal enzymes—such as the cathepsin proteins—that preferentially localize into the EVs over the lysosome. Chemical inhibition against cathepsin D promoted EVs secretion and a change in the EVs protein composition and therefore indicates its involvement in EVs biogenesis. In conclusion, we applied a heavy isotope pulse/trace proteomic approach to study EVs biogenesis in hypothalamic cells. The results demonstrated the regulation of EVs secretion by the cathepsin proteins that may serve as a potential therapeutic target for a range of neurological disorder associated with energy homeostasis.

scholarly journals Analysis of purified Wild type and mutant adenovirus particles by SILAC based quantitative proteomics

2014 ◽  
Vol 95 (11) ◽  
pp. 2504-2511 ◽  
Author(s):  
Ali Alqahtani ◽  
Kate Heesom ◽  
Jonathan L. Bramson ◽  
David Curiel ◽  
Hideyo Ugai ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Quantitative Proteomics ◽  
Core Protein ◽  
Protein Composition ◽  
Genetically Engineered ◽  
Protein Abundance ◽  
Wild Type ◽  
Stable Isotope Labelling ◽  
Gene Therapy Vector ◽  
Viral Core Protein

We used SILAC (stable isotope labelling of amino acids in cell culture) and high-throughput quantitative MS mass spectrometry to analyse the protein composition of highly purified WT wild type adenoviruses, mutant adenoviruses lacking an internal protein component (protein V) and recombinant adenoviruses of the type commonly used in gene therapy, including one virus that had been used in a clinical trial. We found that the viral protein abundance and composition were consistent across all types of virus examined except for the virus lacking protein V, which also had reduced amounts of another viral core protein, protein VII. In all the samples analysed we found no evidence of consistent packaging or contamination with cellular proteins. We believe this technique is a powerful method to analyse the protein composition of this important gene therapy vector and genetically engineered or synthetic virus-like particles. The raw data have been deposited at proteomexchange, identifer PXD001120.

scholarly journals Temporal Quantitative Proteomics Analysis of Neuroblastoma Cells Treated with Bovine Milk-Derived Extracellular Vesicles Highlights the Anti-Proliferative Properties of Milk-Derived Extracellular Vesicles

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 750
Author(s):  
Pamali Fonseka ◽  
Taeyoung Kang ◽  
Sing Chee ◽  
Sai V. Chitti ◽  
Rahul Sanwlani ◽  
...  
Keyword(s):  
High Risk ◽  
Extracellular Vesicles ◽  
Quantitative Proteomics ◽  
Cell Metabolism ◽  
Bovine Milk ◽  
Neuroblastoma Cells ◽  
Treatment Strategies ◽  
Treatment Resistance ◽  
Label Free ◽  
The Impact

Neuroblastoma (NBL) is a pediatric cancer that accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc occurs in 20% of NBL patients and is considered high risk as it correlates with aggressiveness, treatment resistance and poor prognosis. Even though the treatment strategies have improved in the recent years, the survival rate of high-risk NBL patients remain poor. Hence, it is crucial to explore new therapeutic avenues to sensitise NBL. Recently, bovine milk-derived extracellular vesicles (MEVs) have been proposed to contain anti-cancer properties. However, the impact of MEVs on NBL cells is not understood. In this study, we characterised MEVs using Western blotting, NTA and TEM. Importantly, treatment of NBL cells with MEVs decreased the proliferation and increased the sensitivity of NBL cells to doxorubicin. Temporal label-free quantitative proteomics of NBL cells highlighted the depletion of proteins involved in cell metabolism, cell growth and Wnt signalling upon treatment with MEVs. Furthermore, proteins implicated in cellular senescence and apoptosis were enriched in NBL cells treated with MEVs. For the first time, this study highlights the temporal proteomic profile that occurs in cancer cells upon MEVs treatment.

scholarly journals Exercise alters the mitochondrial proteostasis and induces the mitonuclear imbalance and UPRmt in the hypothalamus of mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Renata R. Braga ◽  
Barbara M. Crisol ◽  
Rafael S. Brícola ◽  
Marcella R. Sant’ana ◽  
Susana C. B. R. Nakandakari ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Body Weight ◽  
Physical Training ◽  
Energy Homeostasis ◽  
Mitochondrial Activity ◽  
In Vivo Experiments ◽  
Reduced Body ◽  
Hypothalamic Cells ◽  
Bxd Mice

AbstractThe maintenance of mitochondrial activity in hypothalamic neurons is determinant to the control of energy homeostasis in mammals. Disturbs in the mitochondrial proteostasis can trigger the mitonuclear imbalance and mitochondrial unfolded protein response (UPRmt) to guarantee the mitochondrial integrity and function. However, the role of mitonuclear imbalance and UPRmt in hypothalamic cells are unclear. Combining the transcriptomic analyses from BXD mice database and in vivo experiments, we demonstrated that physical training alters the mitochondrial proteostasis in the hypothalamus of C57BL/6J mice. This physical training elicited the mitonuclear protein imbalance, increasing the mtCO-1/Atp5a ratio, which was accompanied by high levels of UPRmt markers in the hypothalamus. Also, physical training increased the maximum mitochondrial respiratory capacity in the brain. Interestingly, the transcriptomic analysis across several strains of the isogenic BXD mice revealed that hypothalamic mitochondrial DNA-encoded genes were negatively correlated with body weight and several genes related to the orexigenic response. As expected, physical training reduced body weight and food intake. Interestingly, we found an abundance of mt-CO1, a mitochondrial DNA-encoded protein, in NPY-producing neurons in the lateral hypothalamus nucleus of exercised mice. Collectively, our data demonstrated that physical training altered the mitochondrial proteostasis and induced the mitonuclear protein imbalance and UPRmt in hypothalamic cells.

scholarly journals Dysregulated miRNome of plasmacytoid dendritic cells from patients with Sjögren’s syndrome is associated with processes at the centre of their function

Rheumatology ◽  
2019 ◽  
Vol 58 (12) ◽  
pp. 2305-2314 ◽  
Author(s):  
Maarten R Hillen ◽  
Eleni Chouri ◽  
Maojie Wang ◽  
Sofie L M Blokland ◽  
Sarita A Y Hartgring ◽  
...  
Keyword(s):  
Cell Death ◽  
Dendritic Cells ◽  
Mammalian Target Of Rapamycin ◽  
Plasmacytoid Dendritic Cells ◽  
Type I ◽  
Large Set ◽  
Type I Ifn ◽  
Stable Isotope Labelling ◽  
Proteomic Approach ◽  
Novel Targets

Abstract Objective A considerable body of evidence supports a role for type-I IFN in the pathogenesis of primary SS (pSS). As plasmacytoid dendritic cells (pDCs) are a major source of type-I IFN, we investigated their molecular regulation by measuring expression of a large set of miRNAs. Methods pDCs were isolated from peripheral blood of pSS patients (n = 30) and healthy controls (n = 16) divided into two independent cohorts (discovery and replication). Screening of 758 miRNAs was assessed by an OpenArray quantitative PCR-based technique; replication of a set of identified miRNAs was performed by custom array. Functional annotation of miRNA targets was performed using pathway enrichment. Novel targets of miR-29a and miR-29c were identified using a proteomic approach (stable isotope labelling with amino acids in cell culture). Results In the discovery cohort, 20 miRNAs were differentially expressed in pSS pDCs compared with healthy control pDCs. Of these, differential expression of 10 miRNAs was confirmed in the replication cohort. The dysregulated miRNAs were involved in phosphoinositide 3-kinase-Ak strain transforming and mammalian target of rapamycin signalling, as well as regulation of cell death. In addition, a set of novel protein targets of miR-29a and miR-29c were identified, including five targets that were regulated by both miRs. Conclusion The dysregulated miRNome in pDCs of patients with pSS is associated with aberrant regulation of processes at the centre of pDC function, including type-I IFN production and cell death. As miR-29a and miR-29c are pro-apoptotic factors and several of the novel targets identified here are regulators of apoptosis, their downregulation in patients with pSS is associated with enhanced pDC survival.

scholarly journals The Fatty Acid and Protein Profiles of Circulating CD81-Positive Small Extracellular Vesicles Are Associated with Disease Stage in Melanoma Patients

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4157
Author(s):  
Giovanni Paolino ◽  
Veronica Huber ◽  
Serena Camerini ◽  
Marialuisa Casella ◽  
Alberto Macone ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Extracellular Vesicles ◽  
Ad Hoc ◽  
Early Stage ◽  
Disease Onset ◽  
Protein Composition ◽  
Disease Stage ◽  
Biological Macromolecules ◽  
Healthy Donors ◽  
Melanoma Patients

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II–IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0–I, II and III–IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0–I) from late (III–IV) stages’ CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III–IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV’ FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.

scholarly journals A systematic characterization of intrinsically formed microglia-like cells during retinal organoid differentiation.

2022 ◽  
Author(s):  
Katarina Bartalska ◽  
Verena Hübschmann ◽  
Medina Korkut-Demirbaş ◽  
Ryan John Abat Cubero ◽  
Alessandro Venturino ◽  
...  
Keyword(s):  
Protein Composition ◽  
Human Microglia ◽  
Development Organization ◽  
Cellular Environment ◽  
Preferential Localization ◽  
Circuit Development ◽  
And Migration ◽  
Induced Pluripotent ◽  
Insight Into

Brain organoids differentiated from human induced pluripotent stem cells provide a unique opportunity to investigate the development, organization and connectivity of neurons in a complex cellular environment. However, organoids usually lack microglia, brain-resident immune cells which are both present in the early human embryonic brain and participate in neuronal circuit development. Here, we find that microglia innately develop in unguided retinal organoid differentiation between week 3 and 4 in 2.5D culture and appear later in floating, non-pigmented, 3D-cystic compartments. We enriched for cystic structures using a low-dosed BMP4 application and performed mass spectrometry, thus defining the protein composition of microglia-containing compartments. We found that cystic compartments expressed both mesenchymal and epithelial markers with microglia enriched in the mesenchymal region. Interestingly, microglia-like cells started to express the border-associated macrophage marker CD163. The preferential localization of human microglia to a mesenchymal compartment provides insight into the behavior and migration of microglia. The model will ultimately allow detailed study of these enigmatic cells and how they enter and distribute within the human brain.

scholarly journals Na2CO3-Responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

2019 ◽  
Author(s):  
Jinwei Suo ◽  
Heng Zhang ◽  
Qi Zhao ◽  
Nan Zhang ◽  
Yongxue Zhang ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Energy Homeostasis ◽  
Thermal Dissipation ◽  
Wild Type ◽  
Ros Scavenging ◽  
Fructose Bisphosphate ◽  
Ps Ii ◽  
Ros Homeostasis ◽  
Fructose Bisphosphate Aldolase ◽  
Regulation Of Photosynthesis

Alkali-salinity exerts severe osmotic, ionic and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na2CO3-responsive proteins. A total of 104 and 102 Na2CO3-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na2CO3-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na2CO3 tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na2CO3 stress, and they have improved our understanding of the Na2CO3-responsive mechanisms in halophytes.

scholarly journals An atlas of protein-protein interactions across mammalian tissues

2018 ◽  
Author(s):  
Michael A. Skinnider ◽  
Nichollas E. Scott ◽  
Anna Prudova ◽  
Nikolay Stoynov ◽  
R. Greg Stacey ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Cellular Mechanisms ◽  
Stable Isotope Labelling ◽  
Proteomic Approach ◽  
Mouse Tissues ◽  
Mammalian Tissues ◽  
Genetically Manipulated

SummaryCellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the complete network of biologically relevant protein-protein interactions, the interactome, has therefore been a central objective of high-throughput biology. Yet, because widely used methods for high-throughput interaction discovery rely on heterologous expression or genetically manipulated cell lines, the dynamics of protein interactions across physiological contexts are poorly understood. Here, we use a quantitative proteomic approach combining protein correlation profiling with stable isotope labelling of mammals (PCP SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide the first proteome-scale survey of interactome dynamics across mammalian tissues, revealing over 27,000 unique interactions with an accuracy comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewiring of protein interactions across tissues is widespread, and is poorly predicted by gene expression or coexpression. Rewired proteins are tightly regulated by multiple cellular mechanisms and implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 723-732 ◽  
Author(s):  
Marianna Prokopi ◽  
Giordano Pula ◽  
Ursula Mayr ◽  
Cécile Devue ◽  
Joy Gallagher ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Mononuclear Cells ◽  
Lectin Binding ◽  
Large Population ◽  
Protein Composition ◽  
Population Based ◽  
Endothelial Progenitor ◽  
Cardiovascular Research ◽  
Proteomic Approach ◽  
Cellular Phenotypes

Abstract The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography–tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires “endothelial” characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

The high-resolution mass spectrometry study of the protein composition of amyloid-like urine aggregates associated with preeclampsia

2019 ◽  
Vol 26 (2) ◽  
pp. 158-161 ◽  
Author(s):  
Victoria A Sergeeva ◽  
Natalia V Zakharova ◽  
Anna E Bugrova ◽  
Natalia L Starodubtseva ◽  
Maria I Indeykina ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Pregnant Women ◽  
Congo Red ◽  
Molecular Mechanisms ◽  
High Resolution Mass Spectrometry ◽  
Protein Composition ◽  
High Resolution Mass ◽  
Proteomic Approach ◽  
Resolution Mass

The study of protein misfolding and post-translational processing abnormalities is a promising diagnostic approach for socially significant pathologies associated with the accumulation of abnormal forms of proteins. Recently, it was shown that amyloid-like aggregates can be observed in the urine of pregnant women with preeclampsia, which is the most severe hypertensive complication that can lead to fateful outcomes. The protein composition of urine aggregates may clarify the molecular mechanisms underlying the pathology and has not yet been studied in detail. Using a proteomic approach based on high-resolution mass spectrometry, we studied the protein composition of amyloid-like structures that aggregate in the presence of Congo red azo-dye in the urine of pregnant women with preeclampsia. Fragments of β-sheets of α-1-antitrypsin, complement 3, haptoglobin, ceruloplasmin, and trypstatin were identified as most likely targets for Congo red binding.

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