The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L. We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice. This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice.

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Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons.

Coral reef ecosystems are metabolically founded on the mutualism between corals and photosynthetic dinoflagellates of the genus Symbiodinium. The glass anemone Aiptasia sp. has become a tractable model for this symbiosis, and recent advances in genetic information have enabled the use of mass spectrometry-based proteomics in this model. We utilized label-free liquid chromatography electrospray-ionization tandem mass spectrometry to analyze the effects of symbiosis on the proteomes of symbiotic and aposymbiotic Aiptasia. We identified and obtained relative quantification of more than 3,300 proteins in 1,578 protein clusters, with 81 protein clusters showing significantly different expression between symbiotic states. Symbiotic anemones showed significantly higher expression of proteins involved in lipid storage and transport, nitrogen transport and cycling, intracellular trafficking, endocytosis and inorganic carbon transport. These changes reflect shifts in host metabolism and nutrient reserves due to increased nutritional exchange with the symbionts, as well as mechanisms for supplying inorganic nutrients to the algae. Aposymbiotic anemones exhibited increased expression of multiple systems responsible for mediating reactive oxygen stress, suggesting that the host derives direct or indirect protection from oxidative stress while in symbiosis. Aposymbiotic anemones also increased their expression of an array of proteases and chitinases, indicating a metabolic shift from autotrophy to heterotrophy. These results provide a comprehensive Aiptasia proteome with more direct relative quantification of protein abundance than transcriptomic methods. The extension of "omics" techniques to this model system will allow more powerful studies of coral physiology, ecosystem function, and the effects of biotic and abiotic stress on the coral-dinoflagellate mutualism.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/cmi.12564

Symbiosis induces widespread changes in the proteome of the model cnidarian Aiptasia.

Nonalcoholic fatty liver disease (NAFLD) is a major health issue throughout the world. However, no validated treatments for NAFLD are currently available. In-depth studies have demonstrated the efficacy of (-)-epigallocatechin-3-gallate (EGCG), a main bioactive chemical extracted from green tea, in treating NAFLD. EGCG exhibits multi-pronged preventive and therapeutic activities, including promoting lipid and glucose metabolism, anti-lipid peroxidation and anti-inflammation activities, anti-fibrosis, and anti-NAFLD related tumor, thus contributing to the mitigation of NAFLD occurrence and progression. The objectives of this paper are to review and discuss the currently known targets, signaling pathways and roles of EGCG that interfere with NAFLD pathogenesis, then providing additional experimental evidence and the foundation for the further studies and clinical applications of EGCG in the prevention and treatment of NAFLD.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/mnfr.201700483

Potential Biological Effects of (-)-Epigallocatechin-3-gallate on the Treatment of Nonalcoholic Fatty Liver Disease

Filter-aided sample preparation is a proteomic technique for the preparation and on column proteolysis of proteins. Recently an enhanced FASP protocol was developed that uses deoxycholic acid (DCA) and that reportedly enhances trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). Although there was no difference in trypsin activity, 14 099 and 13 414 peptides, describing 1723 and 1793 protein groups, from Escherichia coli K12 were identified using FASP and eFASP, respectively. Characterization of the physicochemical properties of identified peptides showed no significant differences other than eFASP extracting slightly more basic peptides. At the protein level, both methods extracted essentially the same number of hydrophobic transmembrane helix-containing proteins as well as proteins associated with the cytoplasm or the c...

Comparative reevaluation of FASP and enhanced FASP methods by LC-MS/MS.

Natural alkaloid and polyphenol compounds targeting lipid metabolism: Treatment implications in metabolic diseases.

In current shotgun-proteomics-based biological discovery, the identification of membrane proteins is a challenge. This is especially true for integral membrane proteins due to their highly hydrophobic nature and low abundance. Thus, much effort has been directed at sample preparation strategies such as use of detergents, chaotropes, and organic solvents. We previously described a sample preparation method for shotgun membrane proteomics, the sodium deoxycholate assisted method, which cleverly circumvents many of the challenges associated with traditional sample preparation methods. However, the method is associated with significant sample loss due to the slightly weaker extraction/solubilization ability of sodium deoxycholate when it is used at relatively low concentrations such as 1%. Hence, we present an enhanced sodium deoxycholate sample preparation strategy that first uses a high concentration of sodium deoxycholate (5%) to lyse membranes and extract/solubilize hydrophobic membrane proteins, and then dilutes the detergent to 1% for a more efficient digestion. We then applied the improved method to shotgun analysis of proteins from rat liver membrane enriched fraction. Compared with other representative sample preparation strategies including our previous sodium deoxycholate assisted method, the enhanced sodium deoxycholate method exhibited superior sensitivity, coverage, and reliability for the identification of membrane proteins particularly those with high hydrophobicity and/or multiple transmembrane domains.

Improvement of a sample preparation method assisted by sodium deoxycholate for mass‐spectrometry‐based shotgun membrane proteomics

Background
(−)-Epigallocatechin-3-gallate (EGCG), the most abundant catechin found in green tea, effectively reduces body weight and tissue and blood lipid accumulation. To explore the mechanism by which EGCG inhibits cellular lipid accumulation in free fatty acid (FFA) induced HepG2 cell culture, we investigated the proteome change of FFA-induced HepG2 cells exposed to EGCG using two-dimensional gel electrophoresis and mass spectrometry.

/pdf/proteomic-analysis-of-the-inhibitory-effect-of-cnnvxjt7dz.pdf

Proteomic analysis of the inhibitory effect of epigallocatechin gallate on lipid accumulation in human HepG2 cells

SDS and sodium deoxycholate (SDC) as two representative detergents have been widely used in LC-MS/MS-based shotgun analysis of membrane proteomes. However, some inherent disadvantages limit their applications such as interference with MS analysis or their weak ability to disrupt membranes. To address this, the combinative application of SDS and SDC was developed and evaluated in our study, which comprehensively used the strong ability of SDS to lyse membranes and solubilize hydrophobic membrane proteins, and the high efficiencies of an optimized acetone precipitation method and SDC in sample clean-up, protein recovery, and redissolution and digestion of precipitated proteins. The comparative study using a rat-liver-membrane-enriched sample showed that, compared with other three commonly used methods including the filter-aided sample preparation strategy, the combinative method not only increased the identified number of total proteins, membrane proteins, and integral membrane proteins by an average of 19.8, 23.9, and 24.8%, respectively, but also led to the identification of the highest number of matching peptides. All these results demonstrate that the method yielded better recovery and reliability in the identification of the proteins especially highly hydrophobic integral membrane proteins than the other three methods, and thereby has more potential in shotgun membrane proteomics.

Evaluation of the combinative application of SDS and sodium deoxycholate to the LC–MS-based shotgun analysis of membrane proteomes

Shotgun analysis of membrane proteomes by an improved SDS-assisted sample preparation method coupled with liquid chromatography-tandem mass spectrometry.

Yujun Yan

Papers

Improvement of a sample preparation method assisted by sodium deoxycholate for mass‐spectrometry‐based shotgun membrane proteomics

Proteomic analysis of the inhibitory effect of epigallocatechin gallate on lipid accumulation in human HepG2 cells

Evaluation of the combinative application of SDS and sodium deoxycholate to the LC–MS-based shotgun analysis of membrane proteomes

Shotgun analysis of membrane proteomes by an improved SDS-assisted sample preparation method coupled with liquid chromatography-tandem mass spectrometry.