Masaki Wakabayashi

Formation of Toxic Aβ(1–40) Fibrils on GM1 Ganglioside-Containing Membranes Mimicking Lipid Rafts: Polymorphisms in Aβ(1–40) Fibrils

The abnormal aggregation and deposition of amyloid beta protein (Abeta) on neuronal cells are critical to the onset of Alzheimers disease. The entity (oligomers or fibrils) of toxic Abeta species responsible for the pathogenesis of the disease has been controversial. We have reported that the Abeta aggregates on ganglioside-rich domains of neuronal PC12 cells as well as in raft-like model membranes. Here, we identified toxic Abeta(1-40) aggregates formed with GM1-ganglioside-containing membranes. Abeta(1-40) was incubated with raft-like liposomes composed of GM1/cholesterol/sphingomyelin at 1:2:2 and 37 degrees C. After a lag period, toxic amyloid fibrils with a width of 12 nm were formed and subsequently laterally assembled with slight changes in their secondary structure as confirmed by viability assay, thioflavin-T fluorescence, circular dichroism, and transmission electron microscopy. In striking contrast, Abeta fibrils formed without membranes were thinner (6.7 nm) and much less toxic because of weaker binding to cell membranes and a smaller surface hydrophobicity. This study suggests that toxic Abeta(1-40) species formed on membranes are not soluble oligomers but amyloid fibrils and that Abeta(1-40) fibrils exhibit polymorphisms.

Ganglioside-induced amyloid formation by human islet amyloid polypeptide in lipid rafts

Human islet amyloid polypeptide (hIAPP) is the primary component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus. However, it is unknown how amyloid fibrils are formed in vivo. In this study, we demonstrate that gangliosides play an essential role in the formation of amyloid deposits by hIAPP on plasma membranes. Amyloid fibrils accumulated in ganglioside‐ and cholesterol‐rich microscopic domains (‘lipid rafts’). The depletion of gangliosides or cholesterol significantly reduced the amount of amyloid deposited. These results clearly showed that the formation of amyloid fibrils was mediated by gangliosides in lipid rafts.

Short-chain fatty acid receptor GPR41-mediated activation of sympathetic neurons involves synapsin 2b phosphorylation

Synapsins are neuronal phosphoproteins that coat synaptic vesicles and are believed to function in the regulation of neurotransmitter release. The signaling mechanism for short‐chain free fatty acid (SCFA)‐stimulated NE release was examined using primary‐cultured mouse sympathetic cervical ganglion neurons. Pharmacological and knockdown experiments showed that activation of sympathetic neurons by SCFA propionate involves SCFA receptor GPR41 linking to Gβγ‐PLCβ3‐ERK1/2‐synapsin 2 signaling. Further, synapsin 2b directly interacts with activated ERK1/2 and can be phosphorylated on serine when SCFA activates sympathetic neurons.

Hydrophilic Interaction Chromatography Using a Meter-Scale Monolithic Silica Capillary Column for Proteomics LC-MS

A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

Large-scale identification of phosphorylation sites for profiling protein kinase selectivity.

Protein kinase selectivity is largely governed by direct binding to the target site(s) on the substrate. Thus, substrate determinants identified from sequences around phosphorylation sites are desirable resources for matching kinases to their substrates. In this study, we tried to identify kinase-selective substrate determinants, including motif sequences, based on large-scale discovery of kinase/substrate pairs. For this purpose, we employed a combination strategy of in vitro kinase reaction followed by LC-MS/MS analysis and applied it to three well-studied kinases: c-AMP regulated protein kinase A (PKA), extracellular signal-regulated kinase 1 (ERK1), and RAC-alpha serine/threonine-protein kinase (AKT1). Cellular proteins were fractionated, dephosphorylated with thermosensitive alkaline phosphatase, phosphorylated with the target kinase, and digested with Lys-C/trypsin, and then phosphopeptides were enriched using TiO2-based hydroxy acid-modified metal oxide chromatography (HAMMOC) and subjected to LC-MS/MS. As a result, 3585, 4347, and 1778 in vitro phosphorylation sites were identified for PKA, ERK1, and AKT1, respectively. As expected, these extensive identifications of phosphorylation sites enabled extraction of both known and novel motif sequences, and this in turn permitted fine discrimination of the specificities of PKA and AKT1, which both belong to the AGC kinase family. Other unique features of the kinases were also characterized, including phospho-acceptor preference (Ser or Thr) and bias ratio of singly/multiply phosphorylated peptides. More motifs were found with this methodology as compared with target kinase phosphorylation of peptides obtained by predigestion of proteins with Lys-C/trypsin. Thus, this approach to characterization of kinase substrate determinants is effective for identification of kinases associated with particular phosphorylation sites.

Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.

Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC-MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases.

Temporal Profiling of Lapatinib-suppressed Phosphorylation Signals in EGFR/HER2 Pathways

Lapatinib is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5–7%) was regulated at least twofold by 1–10 μm lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including nine novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049–1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e. hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.

Analytical strategies for shotgun phosphoproteomics: status and prospects.

New analytical strategies for phosphoproteomics, both experimental and computational, have been rapidly introduced in recent years, leading to novel biological findings on the role of protein phosphorylation, which have in turn stimulated further development of the analytical techniques. In this review, we describe the development of analytical strategies for LC-MS/MS-based phosphoproteomics, focusing particularly on recent progress in phosphopeptide enrichment, LC-MS/MS measurement and the subsequent computational analysis. High-coverage analysis of the phosphoproteome has largely been achieved by combining pre-fractionation methods with multiple phosphopeptide enrichment approaches, at some cost in LC-MS/MS measurement time and increased sample loss. Key points for the future will be to further increase the selectivity and the recovery of enrichment methods to achieve higher sensitivity and efficiency in LC-MS/MS analysis in order to detect protein phosphorylation comprehensively, including low-abundance proteins. This is expected to lead to a more detailed understanding of the mechanisms and interactions of phosphorylation-mediated regulatory pathways in biological systems.

Effects of Lipid Membrane Curvature on Lipid Packing State Evaluated by Isothermal Titration Calorimetry

In this report, we present a novel approach for the elucidation of the physicochemical properties of lipid membranes by isothermal titration calorimetry (ITC) to quantify the heat absorbed during the solubilization of vesicles into TritonX-100 micelles. By using large and small unilamellar vesicles for comparison, this method provides calorimetric data on the gel-to-liquid-crystalline phase transition and its curvature effects and, in particular, the enthalpy change upon membrane deformation from a planar to a curved shape, which cannot be obtained by the conventional approach using differential scanning calorimetry. The results showed quantitatively that the increase in membrane curvature increases the enthalpy of 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes both below and above the phase-transition temperature, and that the effect is more significant for the former condition. The calorimetric data obtained are further discussed in relation to the elastic bending energy of the membranes and membrane-peptide interaction.

A putative polypeptide N-acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis

Background: WBSCR17 is a potential polypeptide N-acetylgalactosaminyltransferase with unknown function. Results: WBSCR17, induced with N-acetylglucosamine, regulated O-glycosylation, lamellipodium formation, and macropinocytosis. Conclusion: Mucin-type O-glycosylation may be involved in lamellipodium formation and membrane trafficking through macropinocytosis. Significance: The data suggest that mucin-type O-glycosylation modulates the dynamic membrane transport of the cell and may be involved in the control of nutrient uptake. We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.