Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

Baghirova, Sabina; Hughes, Bryan; Hendzel, Michael J.; Schulz, Richard

doi:10.1016/j.mex.2015.11.001

Cited by 153 publications

(117 citation statements)

References 8 publications

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“…2). To confirm this, we biochemically separated cytosolic and membrane bound proteins by differential detergent fractionation (Baghirova et al, 2015). Indeed, Ykt6-3E was enriched in the membrane fraction, while Ykt6-WT was mainly in the cytoplasmic fraction ( Fig.…”

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confidence: 80%

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Linnemannstöns

Witte

et al. 2018

Preprint

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Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion.However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6 RNAi-mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. We show that phosphomimicking Ykt6 accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies in a dose-dependent manner. Taken together, we show that exosomal sorting of Wnts is fine-tuned by a regulatory switch in Ykt6 shifting it from a membrane-bound to cytosolic state at the level of endosomal maturation.

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mentioning

confidence: 80%

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Linnemannstöns

Witte

et al. 2018

Preprint

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“…The protein samples for specific cellular components (whole cell, cytosolic and nuclear fractions) were separated following standard sequential fractionation process as described by Baghirova et al [42]. Sample proteins (20 μg) isolated from the cardiac tissues of the experimental animals of different groups were subjected to SDS-PAGE (12%) for the separation of proteins and transferred into nitrocellulose membrane following standard transfer protocol [43].…”

Section: Methodsmentioning

confidence: 99%

Sansevieria roxburghiana Schult. & Schult. F. (Family: Asparagaceae) Attenuates Type 2 Diabetes and Its Associated Cardiomyopathy

et al. 2016

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BackgroundSansevieria roxburghiana Schult. & Schult. F. (Family: Asparagaceae) rhizome has been claimed to possess antidiabetic activity in the ethno-medicinal literature in India. Therefore, present experiments were carried out to explore the protective role of edible (aqueous) extract of S. roxburghiana rhizome (SR) against experimentally induced type 2 diabetes mellitus (T2DM) and its associated cardiomyopathy in Wistar rats.MethodsSR was chemically characterized by GC-MS analysis. Antidiabetic activity of SR (50 and 100 mg/kg, orally) was measured in high fat diets (ad libitum) + low-single dose of streptozotocin (35 mg/kg, intraperitoneal) induced type 2 diabetic (T2D) rat. Fasting blood glucose level was measured at specific intermissions. Serum biochemical and inflammatory markers were estimated after sacrificing the animals. Besides, myocardial redox status, expressions of signal proteins (NF-κB and PKCs), histological and ultrastructural studies of heart were performed in the controls and SR treated T2D rats.ResultsPhytochemical screening of the crude extract revealed the presence of phenolic compounds, sugar alcohols, sterols, amino acids, saturated fatty acids within SR. T2D rats exhibited significantly (p < 0.01) higher fasting blood glucose level with respect to control. Alteration in serum lipid profile (p < 0.01) and increased levels of lactate dehydrogenase (p < 0.01) and creatine kinase (p < 0.01) in the sera revealed the occurrence of hyperlipidemia and cell destruction in T2D rats. T2DM caused significant (p < 0.05–0.01) alteration in the biochemical markers in the sera. T2DM altered the redox status (p < 0.05–0.01), decreased (p < 0.01) the intracellular NAD and ATP concentrations in the myocardial tissues of experimental rats. While investigating the molecular mechanism, activation PKC isoforms was observed in the selected tissues. T2D rats also exhibited an up-regulation in nuclear NF-κB (p65) in the cardiac tissues. So, oral administration of SR (50 and 500 mg/kg) could reduce hyperglycemia, hyperlipidemia, membrane disintegration, oxidative stress, vascular inflammation and prevented the activation of oxidative stress induced signaling cascades leading to cell death. Histological and ultra-structural studies of cardiac tissues supported the protective characteristics of SR.ConclusionsFrom the present findings it can be concluded that, SR could offer protection against T2DM and its associated cardio-toxicity via multiple mechanisms viz. hypoglycemic, antioxidant and anti-inflammatory actions.

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“…The extracts were briefly sonicated and protein concentrations were determined using Bio-Rad protein assay. Cellular fractionation was done as described by S. Baghirova et al (Baghirova, Hughes et al, 2015) with some minor adjustments. In brief, cells were trypsinized and were subsequently treated with Nocodazole or DMSO (as control) for 30 minutes.…”

Section: Protein Analysismentioning

confidence: 99%

Reduced PABPN1 levels causes cytoskeleton disorganization and aberrant differentiation

Domagoj

et al. 2020

Preprint

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The polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, regulates muscle wasting and atrophy. Previously, we elucidated the PABPN1-dependent proteome and found that levels of structural proteins, sarcomeric and cytoskeletal, were highly altered. We identified MURC, a plasma membrane-associated protein, to be affected by the cytoskeletal stability and suggest that MURC is a novel marker for impaired regeneration in muscles. We also studied the spatial organization of muscle structural proteins in 2D and 3D cell models with reduced PABPN1 levels (named here as shPAB). We show that dysregulation of cytoskeletal proteins in the shPab proteome is associated with a cytoskeleton lacking a polarized organization in muscle cells. We show that consequently, the cell mechanical features as well as myogenic differentiation are significantly reduced. We then show that restoring cytoskeletal stability, by actin overexpression in shPAB was beneficial for cell fusion and for the expression of sarcomeric proteins in shPAB models. We suggest that poor cytoskeleton mechanical features are caused by altered expression levels and contribute to agingassociated muscle wasting and atrophy.

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Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

Abstract: Graphical abstract

Cited by 153 publications

References 8 publications

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

Sansevieria roxburghiana Schult. & Schult. F. (Family: Asparagaceae) Attenuates Type 2 Diabetes and Its Associated Cardiomyopathy

Reduced PABPN1 levels causes cytoskeleton disorganization and aberrant differentiation

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