Chunliang Xie

Proteomic analysis of the venom from the scorpion Mesobuthus martensii.

UNLABELLED The scorpion Mesobuthus martensii is the most populous species in eastern Asian countries, and several toxic components have been identified from their venoms. Nevertheless, a complete proteomic profile of the venom of M. martensii is still not available. In this study, the venom of M. martensii was analyzed by comprehensive proteomic approaches. 153 fractions were isolated from the M. martensii venom by 2-DE, SDS-PAGE and RP-HPLC. The ESI-Q-TOF MS results of all fractions were used to search the scorpion genomic and transcriptomic databases. Totally, 227 non-redundant protein sequences were unambiguously identified, composed of 134 previously known and 93 previously unknown proteins. Among 134 previously known proteins, 115 proteins were firstly confirmed from the M. martensii crude venom and 19 toxins were confirmed once again, involving 43 typical toxins, 7 atypical toxins, 12 venom enzymes and 72 cell associated proteins. In typical toxins, 7 novel-toxin sequences were identified, including 3 Na(+)-channel toxins, 3K(+)-channel toxins and 1 no-annotation toxin. These results increased 230% (115/50) venom components compared with previous studies from the M. martensii venom, especially 50% (24/48) typical toxins. Additionally, a mass fingerprint obtained by MALDI-TOF MS indicated that the scorpion venom contained more than 200 different molecular mass components. BIOLOGICAL SIGNIFICANCE This work firstly gave a systematic investigation of the M. martensii venom by combined proteomics strategy coupled with genomics and transcriptomics. A large number of protein components were unambiguously identified from the venom of M. martensii, most of which were confirmed for the first time. We also contributed 7 novel-toxin sequences and 93 protein sequences previously unknown to be part of the venom, for which we assigned potential biological functions. Besides, we obtained a mass fingerprint of the M. martensii venom. Together, our study not only provides the most comprehensive catalog of the molecular diversity of the M. martensii venom at the proteomic level, but also enriches the composition information of scorpion venom.

Proteomic Screen for Multiprotein Complexes in Synaptic Plasma Membrane from Rat Hippocampus by Blue Native Gel Electrophoresis and Tandem Mass Spectrometry

Neuronal synapses are specialized sites for information exchange between neurons. Many diseases, such as addiction and mood disorders, likely result from altered expression of synaptic proteins, or altered formation of synaptic complexes involved in neurotransmission or neuroplasticity. A detailed description of native multiprotein complexes in synaptic plasma membranes (PM) is therefore essential for understanding biological mechanisms and disease processes. For the first time in this study, two-dimensional Blue Native/SDS-PAGE electrophoresis, combined with tandem mass spectrometry, was used to screen multiprotein complexes in synaptic plasma membranes from rat hippocampus. As a result, 514 unique proteins were identified, of which 36% were integral membrane proteins. In addition, 19 potentially novel and known heterooligomeric multiprotein complexes were found, such as the SNARE and ATPase complexes. A potentially novel protein complex, involving syntaxin, synapsin I and Na+/K+ ATPase alpha-1, was further confirmed by co-immunoprecipitation and immunofluorescence staining. As demonstrated here, Blue Native-PAGE is a powerful tool for the separation of hydrophobic membrane proteins. The combination of Blue Native-PAGE and mass spectrometry could systematically identify multiprotein complexes.

A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes.

To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM‐associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one‐dimensional (1‐D) SDS–PAGE or two‐dimensional (2‐D) benzyldimethyl‐n‐hexadecylammonium chloride (BAC)/SDS–PAGE before nano‐Liquid Chromatography‐Electrospray Ionization—tandem mass spectrometry analysis (LC‐ESI‐MS/MS). A total of 613 non‐redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane‐associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction‐specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1‐D SDS–PAGE and 2‐D 16‐BAC/SDS–PAGE on the separation of integral membrane proteins. J. Cell. Biochem. 104: 965–984, 2008.

An in Vivo Membrane Density Perturbation Strategy for Identification of Liver Sinusoidal Surface Proteome Accessible from the Vasculature

Liver sinusoidal endothelial cells (LSEC), the predominant nonparenchyma cells in liver, play critical roles in many important physiological and pathological processes by virtue of their unique location at the blood-tissue interface. To uncover the protein composition of LSEC plasma membrane (PM) comprehensively and give implications for the tissue microenvironment heterogeneity, we have developed an in vivo modified membrane density perturbation method for purification of the PM fraction. The proteins were separated and identified by SDS-PAGE combined with LC-MS/MS (GeLC-MS/MS). A total of 837 nonredundant proteins were identified, including a number of proteins previously reported to be localized to the PM of LSEC, as well as others not described. A diversity of membrane proteins involved in signaling, traffic, transporting and adhesion functions were identified. Our results demonstrated that the in vivo membrane density perturbation was an effective strategy to purify LSEC PM.

Evaluation of two cell surface modification methods for proteomic analysis of plasma membrane from isolated mouse hepatocytes.

The hepatocyte is a highly polarized cell with a heterogeneous distribution of plasma-membrane (PM) proteins. To reduce the complexity of the proteome of liver tissue and give a comprehensive profile of hepatocyte PM, two PM purification methods based on cell surface modification, named the biotin-avidin (BA) and cationic silica-polyanion (CSP) strategies were evaluated and compared with the traditional cell fractionation method to prepare highly enriched PM from freshly isolated C57 mouse hepatocytes. Employing different principles for PM modification, both methods were effective in the isolation of general and purified PM fraction. The CSP strategy showed better yield for the PM purification from freshly isolated hepatocytes. 189 non-redundant proteins were identified, including 49 from the BA method and 185 from CSP strategy. Many known and novel PM-associated proteins were also found. Our evaluation here should give implications for PM preparation from other freshly isolated tissue-derived cells. The hepatocyte PM proteins identified here should be taken as a references for the PM-related functional and diseases research.

Development of cationic colloidal silica-coated magnetic nanospheres for highly selective and rapid enrichment of plasma membrane fractions for proteomics analysis.

PM (plasma membrane) proteins play critical roles in many biological processes and are often used as molecular targets for drug discovery. In PM proteome research, fast and highly selective methods for PM preparation are highly desirable for efficient PM protein identification. In the present study, an improved PM isolation technique involving coating intact cells with synthesized cationic silica‐coated magnetic nanospheres was developed and applied to the proteomic analysis of the PM from human erythroleukaemia K562 cells. Western blotting characterization and protein identification of the prepared PM indicated that the PM enrichment method using the prepared magnetic nanospheres is a fast and inexpensive strategy with high specificity. Our results demonstrate the potential of these cationic silica‐coated magnetic nanospheres for high‐throughput identification of PM proteins from cells.

Blue native/SDS-PAGE combined with iTRAQ analysis reveals advanced glycation end-product-induced changes of synaptosome proteins in C57 BL/6 mice†

Evidence shows that administration of high‐level D‐galactose induces the production of advanced glycation end‐products (AGEs) that have been implicated in the development of diabetic complications such as neuropathy. The deterioration of learning and memory during neuropathy might be associated with the altered expression of proteins in synapse. To evaluate AGE‐induced protein network alterations in synapse, blue native/SDS‐PAGE and iTRAQ proteomic methods were used to screen for differentially expressed synaptic proteins of cerebral cortex in D‐galactose‐induced C57 BL/6 mice. In total, the expression level of 84 proteins is changed during AGE accumulation. The significantly differentially expressed proteins mainly participate in neurotransmission, energy metabolism and signal transduction pathway, suggesting that energy metabolism is damaged and neurotransmission is attenuated in synapse. The results of in vivo activities of malondialdehyde and superoxide dismutase suggested that AGE accumulation in the brain leads to the generation of reactive oxygen species. Therefore, elucidating the differentially expressed proteins underlying the AGE accumulation will open a new window to the mechanism of learning and memory impairments in neuropathy.

Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism

Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca(2+)-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes.

Localization of Rab3A-binding site on C2A domain of synaptotagmin I to reveal its regulatory mechanism

Synaptotagmin I (Syt I) functions in the regulation of neurotransmitter release and multiple other cellular processes through its C2 domain binding to other molecules. Our previous study demonstrated that Rab3A, a small GTP-binding protein, is a new interacting partner of Syt I and could bind to both of the C2 domains; the polylysine motif in C2B is a key site for Rab3A binding, but the binding site on C2A is not clear. In order to localize Rab3-binding site on C2A and reveal the relevant regulatory mechanism, in the present study we investigated the interaction between recombinant Rab3A and various C2A mutants. The results showed that a key Rab3A-binding site on C2A is located at R199K200 in the flexible loop 2 of the domain, and the site does not overlap with most of the known functional sites or residues. It was speculated that the interaction between Rab3A and C2A is not simply based on electrostatic force, and Rab3A regulates C2A-mediated vesicle-presynaptic membrane fusion mainly through affecting the C2A binding to phospholipids in the presynaptic membrane. These results have contributed to the comprehension of action mechanism of Rab3 and synaptotagmin in the regulation of synaptic vesicle exocytosis.

Synaptotagmin I delays the fast inactivation of Kv1.4 channel through interaction with its N-terminus

BackgroundThe voltage-gated potassium channel Kv1.4 is an important A-type potassium channel and modulates the excitability of neurons in central nervous system. Analysis of the interaction between Kv1.4 and its interacting proteins is helpful to elucidate the function and mechanism of the channel.ResultsIn the present research, synaptotagmin I was for the first time demonstrated to be an interacting protein of Kv1.4 and its interaction with Kv1.4 channel did not require the mediation of other synaptic proteins. Using patch-clamp technique, synaptotagmin I was found to delay the inactivation of Kv1.4 in HEK293T cells in a Ca2+-dependent manner, and this interaction was proven to have specificity. Mutagenesis experiments indicated that synaptotagmin I interacted with the N-terminus of Kv1.4 and thus delayed its N-type fast inactivation.ConclusionThese data suggest that synaptotagmin I is an interacting protein of Kv1.4 channel and, as a negative modulator, may play an important role in regulating neuronal excitability and synaptic efficacy.