Detergent-resistant Fractions Research Articles

How does ionizing radiation affect amyloidogenesis in plants?

Purpose Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation of prion-like proteins in plant tissues. The object of this study, pea (Pisum sativum L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. Materials and methods Pea seeds were irradiated in the dose range 5–50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins. Results We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more β-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology. Conclusion Our data hinted that protein accumulation stimulated seedlings’ growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.

Oligomerization and aggregation of NAP-22 with several metal ions

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aβ peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

Integrin-Dependent Transient Density Increase in Detergent-Resistant Membrane Rafts in Platelets Activated by Thrombin.

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbβ3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin-integrin αIIbβ3-myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin.

#4868 MASS-SPECTROMETRY ANALYSIS OF CONGOPHILIC URINE AND ITS DETERGENT RESISTANT FRACTION IN PATIENTS WITH AMYLOID AND NON-AMYLOID NEPHROPATHIES

Abstract Background and Aims Being a well-known intrinsic property of amyloid, сongophilia was recently demonstrated in urine of patients with proteinuria of different etiology including renal amyloidosis (RA) and non-amyloid nephropathies (NANP) [1]. Urine proteins (UPs) responsible for congophilia in RA and NANP are supposed to have another property of amyloid such as resistance to ionic detergents and present in urine as detergent-resistant aggregates (DRA). In the pilot study we performed mass-spectrometry (MS) analysis of congophilic urine samples and its detergent-resistant fraction in patients with RA and NANP to investigate UPs and their specificity to the particular kidney disease. Method We collected first morning void urine samples from patients with RA (n = 4) and NANP (n = 4). Urine congophilia was assessed by Congo red Dot test as described previously [1]. To analyze bulk urine (BU) proteins, 30 μl of supernatant was precipitated with 80% acetone and boiled in 2% sodium dodecyl sulfate (SDS) for 30 minutes. Then 5 μg of UPs was separated in 10% polyacrylamide gel electrophoresis (PAGE) followed by Coomassie blue staining. The ultracentrifugation of 0.5-3 ml of urine at 300,000x g for 16h following by the treatment of precipitate with 3% sarcosyl in phosphate-buffered saline (PBS) for 10 min with subsequent washing of resistant aggregates in PBS were applied to obtain DRA. After boiling in SDS 8 μl of the sample was separated in 10% PAGE. UPs concentration in the DRA was estimated by densitometry regarding to 2 μg of bovine serum albumin. Then 6 μg of UPs was digested with trypsin, followed by purification on silicate (CDS Empore™ C18 Extraction Disks). Prepared samples of BU and DRA were analyzed by electrospray ionization tandem MS. We used MsFragger software to obtain lists of UPs for each sample. Most representative UPs in the sample were selected by the unique spectral count (USC). We considered the protein as having diagnosis-specific potential (DSP) if it was present in every sample of the particular disease group, i.e. AA, AL, NANP in BU or DRA, and was absent in any sample of another disease group. Results The patients had following characteristics: age 51±13 years, 3 male/ 5 female, eGFR = 45 (19; 95) ml/min/1.73 m2; 24 h proteinuria = 6.5 (4.5; 7.9) g. In PAGE analysis, BU proteins appeared to be similar in all 8 samples with 2 major bends in 70 kDa and 50 kDa regions, corresponding to albumin and immunoglobulin (Ig) heavy chains, respectively (Fig. 1A). The amount of protein in DRA was small and comprised 1.02 (0.71; 1.61) % of the total protein in the sample (4.7 (2.3; 5,1) g/l). Compared with BU PAGE analysis of DRA proteins revealed other bends with trace albumin bend, predominance of 45 kDA region bend and more apparent 30 kDa bend (Ig light chains) in the majority of patients (Fig. 1B). Results of MS analysis are shown on the Figure 2. There were more DSP revealed in DRA vs BU: 14 vs 5, 46 vs 2 and 4 vs 2 UPs in samples of AL, AA and NANP, respectively. When compare by a particular disease group, there were no DSP found either in BU or DRA in RA samples. One protein (aminopeptidase N) and 3 proteins (isoform 3 of unconventional myosin-LC, protein S100-A8, elongation factor 1-α 1) were detected as DSP in BU and DRA in AL, respectively. Alpha-1-acid glycoprotein 1 was only DSP for AA in DRA as well as serum paraoxonase 1 for NANP in BU. Venn-diagrams of shared and divergent UPs are present on the Figures 2D-G. Conclusion Although DRA represented a small portion of UPs, its composition significantly differed from BU and could contain more specific disease markers that makes urine detergent-resistant fraction promising for the future research. Understanding the role of DRA proteins in the pathogenesis of amyloid and non-amyloid renal disease and their diagnostic utility requires further studies.

Abstract B072: Induction of PARP7 creates a vulnerability for growth inhibition by RBN2397 in prostate cancer cells

Abstract The ADP-ribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids. PARP7 has been shown to affect gene expression in prostate cancer cells and certain other cell types by mechanisms that include transcription factor ADP-ribosylation. Here, we use a recently developed catalytic inhibitor to PARP7, RBN2397 (NCT04053673), to study the effects of PARP7 inhibition in androgen receptor-positive (AR+) and androgen receptor-negative (AR-) prostate cancer cells. Ribon Therapeutics developed RBN2397 as a first-in-class mono-ADP-ribosyltransferase inhibitor, and showed that it blocks PARP7 negative regulation of TBK1 [1]. We find that RBN2397 has nanomolar potency for inhibiting androgen-induced ADP-ribosylation of the androgen receptor. RBN2397 inhibits the growth of prostate cancer cells in culture when cells are treated with ligands that activate the androgen receptor (PC3-AR, VCaP, CWR22Rv1), or the aryl hydrocarbon receptor (PC3, DU145, NCI-H660), and induce PARP7 expression. We show that the growth inhibitory effects of RBN2397 are distinct from its enhancement of interferon signaling recently shown to promote tumor immunogenicity in lung cancer models [1]. Chemical inhibitors to PARP1 exert effects on cells by blocking enzyme function, but also via cytotoxic effects attributed to stabilizing PARP1-chromatin interactions in a process termed trapping [2]. Drug-induced trapping of PARP1 can be detected biochemically by immunoblotting the detergent-resistant chromatin fraction. We found that RBN2397 treatment of AR+ and AR- prostate cancer cells induces biochemical trapping of PARP7 within the nucleus, which was also detected by confocal microscopy. Potential therapeutic benefits of RBN2397 are likely to depend on the level of PARP7 expression, given its induction is necessary for growth inhibitory effects of RBN2397 in cell culture. As a first step towards evaluating whether PARP7 levels in human prostate cancer may be actionable with RBN2397, we used computational methods to analyze PARP7 gene expression data from primary prostate tumors and metastatic AR+ and AR- prostate tumors. To assess PARP7 mRNA levels, we used data from the online resource recount3, which uniformly reprocesses publicly available RNA-seq datasets using a Monorail analysis pipeline. Using the level of PARP7 expression in VCaP cells that confers sensitivity to RBN2397 as a threshold, 50% of primary tumors, 41% of metastatic AR- and 11% of AR+ tumors are predicted to have PARP7 expression levels that are sufficient for a response to RBN2397. Because RBN2397 can inhibit the growth of castration-resistant and neuroendocrine prostate cancer cells, PARP7 may be an actionable target in advanced prostate cancer. 1. Gozgit, J.M., et al., PARP7 negatively regulates the Type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell, 2021. 39(9): p. 1214-1226 e10 2. Murai, J., et al., Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors. Cancer Res, 2012. 72(21): p. 5588-99. Citation Format: Chunsong Yang, Krzysztof Wierbilowicz, Natalia M. Dworak, Song Yi Bae, Sachi B. Tengse, Nicki Abianeh, Justin M. Drake, Tarek Abbas, Aakrosh Ratan, David Wotton, Bryce M. Paschal. Induction of PARP7 creates a vulnerability for growth inhibition by RBN2397 in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B072.

Differential distribution of cholesterol pools across arteries under high-cholesterol diet

Excessive cholesterol constitutes a major risk factor for vascular disease. Within cells, cholesterol is distributed in detergent-sensitive and detergent-resistant fractions, with the largest amount of cholesterol residing in cellular membranes. We set out to determine whether various arteries differ in their ability to accumulate esterified and non-esterified cholesterol in detergent-sensitive versus detergent-resistant fractions throughout the course of a high-cholesterol diet. Male Sprague-Dawley rats were placed on 2 % cholesterol diet while a control group was receiving iso-caloric standard chow. Liver, aorta, and pulmonary, mesenteric, and cerebral arteries were collected at 2–6, 8–12, 14–18, and 20–24 weeks from the start of high-cholesterol diet. After fraction separation, esterified and free non-esterified cholesterol levels were measured. In all arteries, largest cholesterol amounts were present in detergent-sensitive fractions in the non-esterified form. Overall, cholesterol in aorta and cerebral arteries was elevated during 14–18 weeks of high-cholesterol diet. Cerebral arteries also exhibited increase in esterified cholesterol within detergent-sensitive domains, as well as increase in cholesterol level in the detergent-resistant fraction at earlier time-points of diet. Pulmonary artery and mesenteric artery were largely resistant to cholesterol accumulation. Quantitative polymerase chain reaction (qPCR) analysis revealed up-regulation of low-density lipoprotein receptor (Ldlr) and low-density lipoprotein receptor-related protein 1 (Lrp1) gene expression in cerebral arteries when compared to mesenteric and pulmonary arteries, respectively. In summary, we unveiled the differential ability of arteries to accumulate cholesterol over the course of a high-cholesterol diet. The differential accumulation of cholesterol seems to correlate with the up-regulated gene expression of proteins responsible for cholesterol uptake.

Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport.

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

Stearic acid blunts growth-factor signaling via oleoylation of GNAI proteins

Covalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

Dual functionality of the amyloid protein TasA in Bacillus physiology and fitness on the phylloplane

Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiology and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytological symptoms indicative of excessive cellular stress leading to increased cell death. TasA associates to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addition to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.

Increase in fatty acids and flotillins upon resveratrol treatment of human breast cancer cells

Flotillin-1 and flotillin-2 are highly conserved proteins that localize into cholesterol-rich microdomains in cellular membranes. Flotillins are closely related to the occurrence and development of various types of human cancers. Flotillin-1 is highly expressed in breast cancer, and the high expression level of flotillin-1 is significantly correlated with poorer patient survival. Here we studied the relationship between the formation of lipid rafts and the expression of flotillins and lipids in human breast cancer cells. We used the polyphenol compound resveratrol to alter the structure and function of the plasma membrane. Our data revealed an increase in fatty acids in MCF-7 and MDA-MB-231 cells upon resveratrol treatment. Interestingly, we also found an increase in the expression of both flotillin-1 and flotillin-2 in breast tumor cells after treatment. Resveratrol also induced changes in the pattern of flotillin distribution among detergent-resistant lipid rafts fractions in both cell lines and induced the nuclear translocation of flotillin-2. Since resveratrol has been pointed out as a putative cancer therapy agent, our results could have an impact on the understanding of the effects of resveratrol in tumor cells.

GM1 promotes TrkA-mediated neuroblastoma cell differentiation by occupying a plasma membrane domain different from TrkA.

Recently, we highlighted that the ganglioside GM1 promotes neuroblastoma cells differentiation by activating the TrkA receptor through the formation of a TrkA-GM1 oligosaccharide complex at the cell surface. To study the TrkA-GM1 interaction, we synthesized two radioactive GM1 derivatives presenting a photoactivable nitrophenylazide group at the end of lipid moiety, 1 or at position 6 of external galactose, 2; and a radioactive oligosaccharide portion of GM1 carrying the nitrophenylazide group at position 1 of glucose, 3. The three compounds were singly administered to cultured neuroblastoma Neuro2a cells under established conditions that allow cell surface interactions. After UV activation of photoactivable compounds, the proteins were analyzed by PAGE separation. The formation of cross-linked TrkA-GM1 derivatives complexes was identified by both radioimaging and immunoblotting. Results indicated that the administration of compounds 2 and 3, carrying the photoactivable group on the oligosaccharide, led to the formation of a radioactive TrkA complex, while the administration of compound 1 did not. This underlines that the TrkA-GM1 interaction directly involves the GM1 oligosaccharide, but not the ceramide. To better understand how GM1 relates to the TrkA, we isolated plasma membrane lipid rafts. As expected, GM1 was found in the rigid detergent-resistant fractions, while TrkA was found as a detergent soluble fraction component. These results suggest that TrkA and GM1 belong to separate membrane domains: probably TrkA interacts by 'flopping' down its extracellular portion onto the membrane, approaching its interplay site to the oligosaccharide portion of GM1.

Interaction of dynamin I with NAP-22, a neuronal protein enriched in the presynaptic region

Neurons have well-developed membrane microdomains called “rafts” that are recovered as a detergent-resistant low-density membrane microdomain fraction (DRM). NAP-22 is one of the major protein components of neuronal DRM and localizes in the presynaptic region. In order to know the role of NAP-22 in the synaptic transmission, NAP-22 binding proteins in the cytosol were searched with an affinity screening with NAP-22 as a bait and several protein bands were detected. Using mass-analysis and western blotting, one of the main band of ∼90 kDa was identified as dynamin I. The GTPase activity of dynamin I was partly inhibited by NAP-22 expressed in bacteria and this inhibition was recovered by the addition of calmodulin, a NAP-22 binding protein. The GTPase activity of dynamin was known to be activated with acidic membrane lipids such as phosphatidylserine and the addition of NAP-22, a phosphatidylserine binding protein, inhibited the activation of the GTPase by this lipid. Since NAP-22 localizes on the presynaptic plasma membrane and on synaptic vesicles, these results suggest the participation of NAP-22 in the membrane cycling through binding to dynamin and acidic membrane lipids at the presynaptic region.

Inhibitory effect of several sphingolipid metabolites on calcineurin

Neurons have well-developed membrane microdomains called “rafts” that are recovered as a detergent-resistant membrane microdomain fraction (DRM). NAP-22 is one of the major protein components of neuronal DRM. In a previous study, we showed that DRM-derived NAP-22 binds ganglioside and the inhibitory effect of ganglioside to calcineurin (CaN), a neuron-enriched calmodulin-regulated phosphoprotein phosphatase. Considering the important roles of CaN in neurons, identification of other cellular regulators of CaN could be a good clue to understand the molecular background of neuronal function. In this study, we screened the effect of several membrane lipid-derived molecules on the CaN activity and found sphingosine and some sphingosine-derived metabolites such as sphingosylphosphorylcholine, galactosylsphingosine (psychosine), and glucosylsphingosine, have inhibitory effect on CaN through the interaction with calmodulin.

Size and Shape of Amyloid Fibrils Induced by Ganglioside Nanoclusters: Role of Sialyl Oligosaccharide in Fibril Formation.

Ganglioside-enriched microdomains in the presynaptic neuronal membrane play a key role in the initiation of amyloid ß-protein (Aß) assembly related to Alzheimer's disease. We previously isolated lipids from a detergent-resistant membrane microdomain fraction of synaptosomes prepared from aged mouse brain and found that spherical Aß assemblies were formed on Aß-sensitive ganglioside nanoclusters (ASIGN) of reconstituted lipid bilayers in the synaptosomal fraction. In the present study, we investigated the role of oligosaccharides in Aß fibril formation induced by ganglioside-containing mixed lipid membranes that mimic the features of ASIGN. Ganglioside nanoclusters were constructed as ternary mixed lipid bilayers composed of ganglioside (GM1, GM2, GM3, GD1a, or GT1b), sphingomyelin, and cholesterol, and their surface topography was visualized by atomic force microscopy. Aß fibril formation on the nanocluster was strongly induced in the presence of 10 mol % ganglioside, and Aß-sensitive features were observed at cholesterol contents of 35-55 mol %. GM1-, GD1a-, and GT1b-containing membranes induced longer fibrils than those containing GD1b and GM2, indicating that the terminal galactose of GM1 along with N-acetylneuraminic acid accelerates protofibril elongation. These results demonstrate that Aß fibril formation is induced by ASIGN that are highly enriched ganglioside nanoclusters with a limited number of components and that the generation and elongation of Aß protofibrils are regulated by the oligosaccharide structure of gangliosides.

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton - a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

Lipid profiles of detergent resistant fractions of the plasma membrane in oat and rye in association with cold acclimation and freezing tolerance

Cold acclimation (CA) results in alteration of the plasma membrane (PM) lipid composition in plants, which plays a crucial role in the acquisition of freezing tolerance via membrane stabilization. Recent studies have indicated that PM structure is consistent with the fluid mosaic model but is laterally non-homogenous and contains microdomains enriched in sterols, sphingolipids and specific proteins. In plant cells, the function of these microdomains in relation to CA and freezing tolerance is not yet fully understood. The present study aimed to investigate the lipid compositions of detergent resistant fractions of the PM (DRM) which are considered to represent microdomains. They were prepared from leaves of low-freezing tolerant oat and high-freezing tolerant rye. The DRMs contained higher proportions of sterols, sphingolipids and saturated phospholipids than the PM. In particular, one of the sterol lipid classes, acylated sterylglycoside, was the predominant sterol in oat DRM while rye DRM contained free sterol as the major sterol. Oat and rye showed different patterns (or changes) of sterols and 2-hydroxy fatty acids of sphingolipids of DRM lipids during CA. Taken together, these results suggest that CA-induced changes of lipid classes and molecular species in DRMs are associated with changes in the thermodynamic properties and physiological functions of microdomains during CA and hence, influence plant freezing tolerance.

Preparation of the Nuclear Matrix for Parallel Microscopy and Biochemical Analyses.

Immobilized proteins within the nucleus are usually identified by treating cells with detergent. The detergent-resistant fraction is often assumed to be chromatin and is described as such in many studies. However, this fraction consists of both chromatin-bound and nuclear-matrix-bound proteins. To investigate nuclear-matrix-bound proteins alone, further separation of these fractions is required; the DNA must be removed so that the remaining proteins can be compared with those from untreated cells. This protocol uses a nonionic detergent (Triton X-100) to remove membranes and soluble proteins from cells under physiologically relevant salt concentrations, followed by extraction with 0.5 m NaCl, digestion with DNase I, and removal of fragmented DNA. It uses a specialized buffer (cytoskeletal buffer) to stabilize the cytoskeleton and nuclear matrix in relatively gentle conditions. Nuclear matrix proteins can then be assessed by either immunofluorescence (IF) and immunoblotting (IB). IB has the advantage of resolving different forms of a protein of interest, and the soluble fractions can be analyzed. The major advantage of IF analysis is that individual cells (rather than homogenized populations) can be monitored, and the spatial arrangement of proteins bound to residual nuclear structures can be revealed.

Transbilayer asymmetry and sphingomyelin composition modulate the preferential membrane partitioning of the nicotinic acetylcholine receptor in Lo domains

We have previously shown that the intact nicotinic acetylcholine receptor (AChR) lacks preference for Lo domains when reconstituted in a sphingomyelin (SM), cholesterol (Chol) and POPC (1:1:1) model system (Bermúdez V, Antollini SS, Fernández-Nievas GA, Aveldaño MI, Barrantes FJ. J. Lipid Res. 2010; 51: 2629–2641). Here, we have furthered our studies by characterizing the influence of different lipid host compositions on the distribution of purified AChR reconstituted in two model systems (POPC:Chol, 1:1 and POPC:Chol:SM, 1:1:1), involving a) different SM species (porcine brain SM (bSM), 16:0-SM, 18:0-SM or 24:1-SM); or b) induced transbilayer asymmetry, resulting from enrichment in bSM in the external hemilayer. AChR distribution was evaluated by fluorescence resonance energy transfer efficiency between the AChR intrinsic fluorescence and Laurdan or dehydroergosterol fluorescence, and by analyzing the distribution of AChR in detergent-resistant and detergent-soluble fractions (1% Triton X-100, 4 °C). bSM-induced transbilayer asymmetry or the presence of 16:0-SM and/or 18:0-SM (unlike bSM or 24:1-SM) resulted in the preferential partitioning of AChR in Lo domains, suggesting that the localization of AChR in ordered domains strongly depends on the characteristics of the host lipid membrane, and in particular on the sphingolipid composition and transbilayer asymmetry.

Lipids that determine detergent resistance of MDCK cell membrane fractions

A comparative lipidomic study has been performed of whole Madin-Darby canine kidney epithelial cells and of the detergent-resistant membrane fraction (DRM) obtained after treating the cells with the non-ionic detergent Triton X-100. The DRM were isolated following a standard procedure that is extensively used in cell biology studies. Significant differences were found in the lipid composition of the whole cells and of DRM. The latter were enriched in all the analyzed sphingolipid classes: sphingomyelins, ceramides and hexosylceramides. Diacylglycerols were also preferentially found in DRM. The detergent-resistant fraction was also enriched in saturated over unsaturated fatty acyl chains, and in sn-1 acyl chains containing 16 carbon atoms, over the longer and shorter ones. The glycerophospholipid species phosphatidylethanolamines and phosphatidylinositols, that were mainly unsaturated, did not show a preference for DRM. Phosphatidylcholines were an intermediate case: the saturated, but not the unsaturated species were found preferentially in DRM. The question remains on whether these DRM, recovered from detergent-membrane mixtures by floatation over a sucrose gradient, really correspond to membrane domains existing in the cell membrane prior to detergent treatment.