Proteome analysis of matrix vesicles isolated from femurs of chicken embryo

Matrix vesicles (MVs) are extracellular organelles that initiate mineral formation, accumulating inorganic phosphate (P(i)) and calcium leading to the formation of hydroxyapatite (HA) crystals, the main mineral component of bones. MVs are produced during bone formation, as well as during the endochondral calcification of cartilage. MVs are released into the extracellular matrix from osseous cells such as osteoblasts and hypertrophic chondrocytes. In this report, using 1-D SDS-PAGE, in-gel tryptic digestion and an LC-MS-MS/MS protein identification protocol, we characterized the proteome of MVs isolated from chicken embryo (Gallus gallus) bones and cartilage. We identified 126 gene products, including proteins related to the extracellular matrix and ion transport, as well as enzymes, cytoskeletal, and regulatory proteins. Among the proteins recognized for the first time in MVs were aquaporin 1, annexin A1 (AnxA1), AnxA11, glycoprotein HT7, G(i) protein alpha2, and scavenger receptor type B. The pathways for targeting the identified proteins into MVs and their particular functions in the biomineralization process are discussed. Obtaining a knowledge of the functions and roles of these proteins during embryonic mineralization is a prerequisite for the overall understanding of the initial mineral formation mechanisms.     

Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.     

Matrix vesicles originate from apical membrane microvilli of mineralizing osteoblast‐like Saos‐2 cells

In bone, mineralization is tightly regulated by osteoblasts and hypertrophic chondrocytes which release matrix vesicles (MVs) and control extracellular ionic conditions and matrix composition. MVs are the initial sites of hydroxyapatite (HA) mineral formation. Despite growing knowledge about their morphology and function, their biogenesis is not well understood. The purpose of this work was to determine the source of MVs in osteoblast lineage, Saos‐2 cells, and to check whether MVs originated from microvilli. Microvilli were isolated from the apical plasma membrane of Saos‐2 cells. Their morphology, structure, and function were compared with those of MVs. The role of actin network in MV release was investigated by using microfilament perturbing drugs. When examined by electron microscopy MVs and microvillar vesicles were found to exhibit similar morphology with trilaminar membranes and diameters in the same range. Both types of vesicles were able to induce HA formation. Their electrophoretic profiles displayed analogous enrichment in alkaline phosphatase, Na⁺/K⁺ ATPase, and annexins A2 and A6. MVs and microvillar vesicles exhibited almost the same lipid composition with a higher content of cholesterol, sphingomyelin, and phosphatidylserine as compared to plasma membrane. Finally, cytochalasin D, which inhibits actin polymerization, was found to stimulate release of MVs. Our findings were consistent with the hypothesis that MVs originated from cell microvilli and that actin filament disassembly was involved in their biogenesis. J. Cell. Biochem. 106: 127–138, 2009. © 2008 Wiley‐Liss, Inc.     

The roles of annexins and alkaline phosphatase in mineralization process

In this review the roles of specific proteins during the first step of mineralization and nucleation are discussed. Mineralization is initiated inside the extracellular organelles-matrix vesicles (MVs). MVs, containing relatively high concentrations of Ca2+ and inorganic phosphate (Pi), create an optimal environment to induce the formation of hydroxyapatite (HA). Special attention is given to two families of proteins present in MVs, annexins (AnxAs) and tissue-nonspecific alkaline phosphatases (TNAPs). Both families participate in the formation of HA crystals. AnxAs are Ca2+ - and lipid-binding proteins, which are involved in Ca2+ homeostasis in bone cells and in extracellular MVs. AnxAs form calcium ion channels within the membrane of MVs. Although the mechanisms of ion channel formation by AnxAs are not well understood, evidence is provided that acidic pH or GTP contribute to this process. Furthermore, low molecular mass ligands, as vitamin A derivatives, can modulate the activity of MVs by interacting with AnxAs and affecting their expression. AnxAs and other anionic proteins are also involved in the crystal nucleation. The second family of proteins, TNAPs, is associated with Pi homeostasis, and can hydrolyse a variety of phosphate compounds. ATP is released in the extracellular matrix, where it can be hydrolyzed by TNAPs, ATP hydrolases and nucleoside triphosphate (NTP) pyrophosphohydrolases. However, TNAP is probably not responsible for ATP-dependent Ca2+/phosphate complex formation. It can hydrolyse pyrophosphate (PPi), a known inhibitor of HA formation and a byproduct of NTP pyrophosphohydrolases. In this respect, antagonistic activities of TNAPs and NTP pyrophosphohydrolases can regulate the mineralization process.     

Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells

Matrix vesicles (MVs), released by budding from apical microvilli of osteoblasts during bone formation and development, are involved in the initiation of mineralization by promoting the formation of hydroxyapatite in their lumen. To gain additional insights into MV biogenesis and functions, MVs and apical microvilli were co-isolated from mineralizing osteoblast-like Saos-2 cells and their proteomes were characterized using LC-ESI-MS/MS and compared. In total, 282 MV and 451 microvillar proteins were identified. Of those, 262 were common in both preparations, confirming that MVs originate from apical microvilli. The occurrence of vesicular trafficking molecules (e.g. Rab proteins) and of the on-site protein synthetic machinery suggests that cell polarization and apical targeting are required for the incorporation of specific lipids and proteins at the site of MV formation. MV release from microvilli may be driven by actions of actin-severing proteins (gelsolin, cofilin 1) and contractile motor proteins (myosins). In addition to the already known proteins involved in MV-mediated mineralization, new MV residents were detected, such as inorganic pyrophosphatase 1, SLC4A7 sodium bicarbonate cotransporter or sphingomyelin phosphodiesterase 3, providing additional insights into MV functions.     

Staphylococcus aureus produces membrane-derived vesicles that induce host cell death

Gram-negative bacteria produce outer membrane vesicles that play a role in the delivery of virulence factors to host cells. However, little is known about the membrane-derived vesicles (MVs) produced by gram-positive bacteria. The present study examined the production of MVs from Staphylococcus aureus and investigated the delivery of MVs to host cells and subsequent cytotoxicity. Four S. aureus strains tested, two type strains and two clinical isolates, produced spherical nanovesicles during in vitro culture. MVs were also produced during in vivo infection of a clinical S. aureus isolate in a mouse pneumonia model. Proteomic analysis showed that 143 different proteins were identified in the S. aureus-derived MVs. S. aureus MVs were interacted with the plasma membrane of host cells via a cholesterol-rich membrane microdomain and then delivered their component protein A to host cells within 30 min. Intact S. aureus MVs induced apoptosis of HEp-2 cells in a dose-dependent manner, whereas lysed MVs neither delivered their component into the cytosol of host cells nor induced cytotoxicity. In conclusion, this study is the first report that S. aureus MVs are an important vehicle for delivery of bacterial effector molecules to host cells.     

Annexins — Modulators of EGF receptor signalling and trafficking

At the cell surface, activation of the epidermal growth factor (EGF) receptor triggers a complex network of signalling events that regulate a variety of cellular processes. For signal termination, the activated EGF receptor is internalised and targeted to lysosomes for degradation. Microdomain localization at the plasma membrane and endocytic transport of the EGFR is important for the formation of compartment-specific signalling complexes and is regulated by scaffolding and targeting proteins. This includes Ca²⁺-effector proteins, such as calmodulin and annexins (Anx), in particular AnxA1, AnxA2, AnxA6 and as shown recently, AnxA8. Given that these annexins show differences in their expression patterns, subcellular localization and mode of action, they are likely to differentially contribute and cooperate in the fine-tuning of EGFR activity. In support of this hypothesis, current literature suggests these annexins to be involved in different steps that control the endocytic transport and signalling of the EGF receptor. This review summarizes how the coordinated activity of AnxA1, AnxA2, AnxA6 and AnxA8 can contribute to regulate EGF receptor localization and activity.     

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.     

Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization

Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413-420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles, and the initiation of mineralization by these vesicles.     

Role of annexin A6 isoforms in catecholamine secretion by PC12 cells: Distinct influence on calcium response

Noradrenaline and adrenaline are secreted by adrenal medulla chromaffin cells via exocytosis. Exocytosis of catecholamines occurs after cell stimulation with various endogenous activators such as nicotine or after depolarization of the plasma membrane and is regulated by calcium ions. Cytosolic [Ca²⁺] increases in response to cell excitation and triggers a signal‐initiated secretion. Annexins are known to participate in the regulation of membrane dynamics and are also considered to be involved in vesicular trafficking. Some experimental evidence suggests that annexins may participate in Ca²⁺‐regulated catecholamine secretion. In this report the effect of annexin A6 (AnxA6) isoforms 1 and 2 on catecholamine secretion has been described. Overexpression of AnxA6 isoforms and AnxA6 knock‐down in PC12 cells were accompanied by almost complete inhibition or a 20% enhancement of dopamine secretion, respectively. AnxA6‐1 and AnxA6‐2 overexpression reduced Δ[Ca²⁺]_c upon depolarization by 32% and 58%, respectively, while AnxA6 knock‐down increased Δ[Ca²⁺]_c by 44%. The mechanism of AnxA6 action on Ca²⁺ signalling is not well understood. Experimental evidence suggests that two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis in PC12 cells. J. Cell. Biochem. 111: 168–178, 2010. © 2010 Wiley‐Liss, Inc.     

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell–cell cohesion, cell adhesion/spreading onto collagen type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell–cell and/or cell–ECM contacts and anchorage-independent cell proliferation.     

Differentiation of human colon adenocarcinoma cells alters the expression and intracellular localization of annexins A1, A2, and A5

Butyrate induces differentiation and alters cell proliferation in intestinal-epithelial cells by modulation of the expression of several genes. Annexins are a superfamily of ubiquitous proteins characterized by their calcium-dependent ability to bind to biological membranes; their involvement in several physiological processes, such as membrane trafficking, calcium signaling, cell motility, proliferation, and differentiation has been proposed. Thus, we have analyzed changes in annexin A1 (AnxA1), annexin A2 (AnxA2), and annexin A5 (AnxA5) levels and localization in human colon adenocarcinoma cells differentiated by butyrate treatment or by culture in glucose-free inosine-containing medium. The acquired differentiated phenotype increased dipeptidyl peptidase-IV (DPP-IV) expression and alkaline phosphatase (ALP) activity, two well known brush border markers. Butyrate induces cell differentiation and growth arrest in BCS-TC2, BCS-TC2.2, HT-29, and Caco-2 cells, increasing the levels of AnxA1 and AnxA5, whereas AnxA2 decreases except in Caco-2 cells. Inosine-differentiated cells present increased amounts of the three studied annexins, as occurs in spontaneously differentiated Caco-2 cells. AnxA2 down-regulation is not due to proteasome activation and seems to be related to the butyrate-induced cell proliferation arrest; AnxA1 and AnxA5 expression is growth-state independent. AnxA1 and AnxA5 are mainly found in the cytoplasm while AnxA2 is localized underneath the plasma membrane in cell-to-cell contacts. Butyrate induces changes in subcellular localization towards a vesicle-associated pattern. Human colon adenocarcinoma cell differentiation is associated with an up-regulation of AnxA1, AnxA2, and AnxA5 and with a subcellular relocation of these proteins. No correlation between annexin levels and tumorigenicity was found. Up-regulation of AnxA1 could contribute to the reported anti-inflammatory effects of butyrate in colon inflammatory diseases.     

Biophysical aspects of biomineralization

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix (ECM) by promoting the synthesis of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Several lipid and proteins present in the membrane of the MVs mediate the interactions of MVs with the ECM and regulate the initial mineral deposition and posterior propagation. Among the proteins of MV membranes, ion transporters control the availability of phosphate and calcium needed for initial HA deposition. Phosphatases (orphan phosphatase 1, ectonucleotide pyrophosphatase/phosphodiesterase 1 and tissue-nonspecific alkaline phosphatase) play a crucial role in controlling the inorganic pyrophosphate/inorganic phosphate ratio that allows MV-mediated initiation of mineralization. The lipidic microenvironment can help in the nucleation process of first crystals and also plays a crucial physiological role in the function of MV-associated enzymes and transporters (type III sodium-dependent phosphate transporters, annexins and Na⁺/K⁺ ATPase). The whole process is mediated and regulated by the action of several molecules and steps, which make the process complex and highly regulated. Liposomes and proteoliposomes, as models of biological membranes, facilitate the understanding of lipid–protein interactions with emphasis on the properties of physicochemical and biochemical processes. In this review, we discuss the use of proteoliposomes as multiple protein carrier systems intended to mimic the various functions of MVs during the initiation and propagation of mineral growth in the course of biomineralization. We focus on studies applying biophysical tools to characterize the biomimetic models in order to gain an understanding of the importance of lipid–protein and lipid–lipid interfaces throughout the process.     

Proteomics analyses of microvesicles released by Drosophila Kc167 and S2 cells

Distinct types of vesicles are formed in eukaryotic cells that conduct a variable set of functions depending on their origin. One subtype designated circulating microvesicles (MVs) provides a novel form of intercellular communication and recent work suggested the release and uptake of morphogens in vesicles by Drosophila cells. In this study, we have examined cells of the hemocyte-like cell lines Kc167 and S2 and identified secreted vesicles in the culture supernatant. The vesicles were isolated and found to have characteristics comparable to exosomes and plasma membrane MVs released by mammalian cells. In wingless-transfected cells, the full-length protein was detected in the vesicle isolates. Proteomics analyses of the vesicles identified 269 proteins that include various orthologs of marker proteins and proteins with putative functions in vesicle formation and release. Analogous to their mammalian counterparts, the subcellular origin of the vesicular constituents of both cell lines is dominated by membrane-associated and cytosolic proteins with functions that are consistent with their localization in MVs. The analyses revealed a significant overlap of the Kc167 and S2 vesicle proteomes and confirmed a close correlation with non-mammalian and mammalian exosomes.     

Active creatine kinase is present in matrix vesicles isolated from femurs of chicken embryo: Implications for bone mineralization

Proteomic analysis of matrix vesicles (MVs) isolated from 17-day-old chicken embryo femurs revealed the presence of creatine kinase. In this report we identified the enzyme functionally and suggest that the enzyme may participate in the synthesis of ATP from ADP and phosphocreatine within the lumen of these organelles. Then, ATP is converted by nucleotide hydrolyzing enzymes such as Na⁺, K⁺-ATPase, protein kinase C, or alkaline phosphatase to yield inorganic phosphate (P_i), a substrate for mineralization. Alternatively, ATP can be hydrolyzed by a nucleoside triphosphate pyrophosphatase phosphodiesterase 1 producing inorganic pyrophosphate (PP_i), a mineralization inhibitor. In addition, immunochemical evidence indicated that VDAC 2 is present in MVs that may serve as a transporter of nucleotides from the extracellular matrix. We discussed the implications of ATP production and hydrolysis by MVs as regulatory mechanisms for mineralization.     

S100-annexin complexes--biology of conditional association

S100 proteins and annexins both constitute groups of Ca2+-binding proteins, each of which comprises more than 10 members. S100 proteins are small, dimeric, EF-hand-type Ca2+-binding proteins that exert both intracellular and extracellular functions. Within the cells, S100 proteins regulate various reactions, including phosphorylation, in response to changes in the intracellular Ca2+ concentration. Although S100 proteins are known to be associated with many diseases, exact pathological contributions have not been proven in detail. Annexins are non-EF-hand-type Ca2+-binding proteins that exhibit Ca2+-dependent binding to phospholipids and membranes in various tissues. Annexins bring different membranes into proximity and assist them to fuse, and therefore are believed to play a role in membrane trafficking and organization. Several S100 proteins and annexins are known to interact with each other in either a Ca2+-dependent or Ca2+-independent manner, and form complexes that exhibit biological activities. This review focuses on the interaction between S100 proteins and annexins, and the possible biological roles of these complexes. Recent studies have shown that S100-annexin complexes have a role in the differentiation of gonad cells and neurological disorders, such as depression. These complexes regulate the organization of membranes and vesicles, and thereby may participate in the appropriate disposition of membrane-associated proteins, including ion channels and/or receptors.     

Matrix vesicle annexins exhibit proteolipid-like properties. Selective partitioning into lipophilic solvents under acidic conditions

Calcifiable proteolipids present in mineralizing tissues have been postulated to enhance apatite deposition by structuring membrane phosphatidylserine molecules into a conformation conducive to mineral formation. To examine whether proteolipid-like molecules are present in mineralizable matrix vesicles (MV), the vesicles were first extracted with chloroform/methanol (2:1, v/v), and then with chloroform/methanol/HCl (200:100:1, v/v) and the organic-soluble proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Protein fractions were analyzed by Coomassie Blue staining and by immunoblot analysis of electrophoretically transferred MV protein with antisera to the 33- and 36-kDa annexins. We found that several MV proteins selectively partitioned into the lipophilic milieu under acidic conditions; however, very little protein did so at neutral pH. The principal organic-soluble MV proteins had molecular masses of 14, 33, and 36 kDa, with lesser bands at 28, 30, and 68 kDa. Immunological analyses revealed that the 33- and 36-kDa proteins were the MV annexins; the 14-kDa protein appeared to be hemoglobin, based on NH2-terminal sequencing. Our findings indicate that under acidic conditions the 33- and 36-kDa MV annexins undergo a conformational change which imparts a marked increase in the hydrophobicity of the proteins. While these observations reveal that the annexins possess proteolipid-like properties, radiolabeling and immunoprecipitation studies using [3H]myristic acid in chondrocyte cultures indicate that the MV annexins are not myristylated. Amino-terminal sequence analysis of the peptides generated by site-specific cleavage of the 33- and the 36-kDa MV annexins at tryptophan residues indicate that the 33 kDa is highly homologous to anchorin CII, a protein known to bind type II collagen, while the 36-kDa protein shares close homology with endonexin II, a tyrosine kinase substrate.     

运用BioID技术筛选S100A7互作蛋白*

目的: 通过邻近生物素鉴定(BioID)技术筛选S100A7互作蛋白并进行验证。方法: 采用邻近生物素BioID和质谱相结合的方法,筛选S100A7互作蛋白。通过免疫荧光和免疫共沉淀方法,对相互作蛋白进行验证。结果: 与对照组比对,通过BioID技术共获得94个可能与 S100A7 相互作用的候选蛋白质。选取 Annexin A2(AnxA2)蛋白在 HEK293 细胞中进行了验证,结果表明S100A7 与 AnxA2 存在直接的相互作用,且二者共定位于细胞质和细胞膜。结论: 可将BioID技术作为互作蛋白筛选的一项新技术,通过该技术发现S100A7和AnxA2存在相互作用。     

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P₂ and other acidic phospholipids in a Ca²⁺-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P₂, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P₂ clustering are also capable of forming interconnections between PI(4,5)P₂-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P₂ and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P₂-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.