Báo cáo khoa học: Induction of raft-like domains by a myristoylated NAP-22 peptide and its Tyr mutant

Induction of raft-like domains by a myristoylated NAP-22 peptide and its Tyr mutant

Tác giả: Raquel F. Epand, Brian G. Sayer, and Richard M. Epand

Lĩnh vực: Biochemistry and Biomedical Sciences, Chemistry

Nội dung tài liệu: Nghiên cứu này khám phá cách peptide NAP-22, một phần của protein thần kinh, tương tác với màng tế bào, đặc biệt là vai trò của cholesterol và phosphatidylinositol (4,5) diphosphate (PtdIns(4,5)P2) trong việc hình thành các miền trên màng. Sử dụng phương pháp nhiệt lượng quét vi sai (DSC), các nhà nghiên cứu đã chỉ ra rằng peptide NAP-22, khi có mặt cholesterol, không chỉ tạo ra các miền giàu PtdIns(4,5)P2 mà còn dẫn đến sự hình thành các miền nghèo cholesterol. Sự thay thế axit amin tyrosine (Tyr) bằng leucine (Leu) trong peptide đã làm giảm khả năng này, mặc dù vẫn có thể cô lập PtdIns(4,5)P2. Các kỹ thuật như cộng hưởng từ hạt nhân (NMR) cũng được áp dụng để làm sáng tỏ sự tương tác giữa peptide và màng lipid. Kết quả cho thấy cholesterol ảnh hưởng đến cách peptide gắn vào màng, với sự hiện diện của cholesterol làm thay đổi bản chất tương tác này. Những phát hiện này có ý nghĩa quan trọng trong việc hiểu cơ chế mà NAP-22 tác động đến việc tái tổ chức khung xương actin trong tế bào thần kinh.

Mục lục chi tiết:

• Induction of raft-like domains by a myristoylated NAP-22 peptide and its Tyr mutant
• Keywords
• cholesterol; domains; differential scanning calorimetry; MAS/NMR; phosphatidylinositol (4,5) diphosphate
• Correspondence
• (Received 10 December 2004, revised 2 February 2005, accepted 14 February 2005)
• doi:10.1111/j.1742-4658.2005.04612.x
• The N-terminally myristoylated, 19-amino acid peptide, corresponding to the amino terminus of the neuronal protein NAP-22 (NAP-22 peptide) is a naturally occurring peptide that had been shown by fluorescence to cause the sequestering of a Bodipy-labeled PtdIns(4,5)P2 in a cholesterol-dependent manner. The present work, using differential scanning calorimetry (DSC), extends the observation that formation of a PtdIns(4,5)P2-rich domain is cholesterol dependent and shows that it also leads to the formation of a cholesterol-depleted domain. The PtdIns(4,5)P2 used in the present work is extracted from natural sources and does not contain any label and has the native acyl chain composition. Peptide-induced formation of a cholesterol-depleted domain is abolished when the sole aromatic amino acid, Tyr11 is replaced with a Leu. Despite this, the modified peptide can still sequester PtdIns(4,5)P2 into domains, probably because of the presence of a cluster of cationic residues in the peptide. Cholesterol and PtdIns(4,5)P2 also modulate the insertion of the peptide into the bilayer as revealed by H NOESY MAS/NMR. The intensity of cross peaks between the aromatic protons of the Tyr residue and the protons of the lipid indicate that in the presence of cholesterol there is a change in the nature of the interaction of the peptide with the membrane. These results have important implications for the mechanism by which NAP-22 affects actin reorganization in neurons.
• NAP-22 is a 22-kDa protein found in neurons that is important for neuronal sprouting and plasticity [1]. In addition to the intact 22-kDa protein, significant amounts of N-terminal myristoylated fragments of this protein are also found in many tissues [2]. A protein with a high sequence homology to NAP-22 and probably with very similar properties, cortical cytoskeleton-associated protein (CAP)-23, was first identified by Widmer and Caroni [3]. Myristoylated proteins are commonly found in cholesterol-rich domains in membranes [4,5]. Full length NAP-22 partitions into the low density, detergent-insoluble fraction of neuronal membranes [6], suggesting its presence in neuronal rafts. Support for this comes from fluorescence microscopy studies using both intact biological membranes [7,8] as well as model membranes [9]. The protein binds to liposomes of phosphatidylcholine only when the bilayer contains high mol fractions of cholesterol [10,11].
• Several proteins with cationic clusters, including CAP-23 as well as the MARCKS protein and GAP-43, accumulate in rafts, colocalizing with PtdIns(4,5)P2 [8].
• Abbreviations
• AHcal, calorimetric enthalpy; Bodipy-TMR-PI(4,5)P2, BODIPY TMR-X C6-phosphatidylinositol 4,5-diphosphate; CAP-23, cortical cytoskeleton-associated protein (a protein expressed in chicken having a high degree of homology to NAP-22); DP, direct polarization; DSC, differential scanning calorimetry; LUV, large unilamellar vesicle; MAS, magic angle spinning; NAP-22 peptide, the myristoylated amino terminal 19 amino acids of NAP-22 (myristoyl-GGKLSKKKKGYNVNDEKAK-amide); NAP-22, neuronal axonal membrane protein, also referred to as brain acid soluble protein 1 (BASP1 protein), a 22 kDa myristoylated protein; PC, phosphatidylcholine; PO, 1-palmitoyl-2-oleoyl; PtdIns(4,5)P2, L-a-phosphatidylinositol-4,5-bisphosphate from porcine brain; SO, 1-stearoyl-2-oleoyl; Tm, transition temperature.
• 1792
• FEBS Journal 272 (2005) 1792-1803© 2005 FEBS
• Cholesterol-dependent lipid rearrangement
• R. F. Epand et al.
• The importance of electrostatic interactions in the sequestering of PtdIns(4,5)P2 by proteins with a cationic domain has been demonstrated [12]. We have also demonstrated the loss of ability of the NAP-22 peptide to sequester Bodipy-labeled PtdIns(4,5)P2 in the presence of high salt concentration [13]. In that work we also demonstrate specificity of the NAP-22-peptide for Bodipy-labeled PtdIns(4,5)P2 compared with Bodipy-labeled PtdIns(3,5)P2 [13]. In addition, using total internal reflectance fluorescence microscopy, we have shown that the sequestering of Bodipy-labeled PtdIns(4,5)P2 into domains can be a cholesterol-dependent phenomenon [13]. This was demonstrated using a myristoylated N-terminal peptide of NAP-22, Myristoyl-GGKLSKKKKGYNVNDEKAK-amide. It is known that in vivo, in addition to the intact NAP-22 protein, a significant amount of myristoylated N-terminal fragments of this protein are also present [2], indicating that the myristoylated N-terminal peptide of NAP-22, such as that used in this work, is also found physiologically. In the present work we demonstrate that not only does cholesterol affect the ability of the NAP-22-peptide to induce the formation of PtdIns(4,5)P2 domains, but it also causes the rearrangement of cholesterol leading to the formation of cholesterol-depleted domains. We also test the role of the aromatic amino acid residue of the peptide in these phenomena. In addition we show that cholesterol also affects the arrangement of the peptide in the bilayer. The present study uses PtdIns(4,5)P2 from porcine brain, a natural form that has long acyl chains enriched in arachidonic acid, and it also does not contain any fluorescent probes. Although PtdIns(4,5)P2 from natural sources is highly enriched in arachidonoyl groups that should not interact well with liquid ordered domains of rafts, this lipid nevertheless is found in raft domains of biological membranes [14].
• Results
• Differential scanning calorimetry (DSC)
• We determined the phase transitions of SOPC and mixtures of this lipid with one or more of the following components: cholesterol, PtdIns(4,5)P2 and NAP-22-peptide, using differential scanning calorimetry (DSC). For each sample, six consecutive DSC scans were run, three heating scans and three cooling scans at a scan rate of 2 °C·min¯¹. Sequential heating and cooling scans were reproducible. In the absence of cholesterol a prominent transition is observed in the region 0-10 °C, corresponding to the chain melting transition of SOPC. This transition is better resolved in cooling than in heating scans, since in some cases the heating scans, initiated at 0 °C, had not reached a steady-state baseline in the temperature range of the transition. The transition of POPC would have been even more difficult to measure, although POPC was used for the NMR experiments (see below) because the NMR results could be more directly compared with our earlier observations on other systems and to avoid any artefacts that may result from storing peptide-lipid mixtures that could attain the gel phase. Nevertheless, we would expect that these two lipids, SOPC and POPC, that differ only by two CH2 groups on one of the acyl chains, would interact almost identically with peptides. One of the three cooling scans is presented for samples of different compositions (Fig. 1A). In the presence of 40 mol% cholesterol, the chain melting transition of SOPC is broadened and the enthalpy lowered (Fig. 1B). We also studied the role of the sole aromatic amino acid, Tyr, of the NAP-22-peptide by replacing it with Leu. The temperatures and enthalpies for the phospholipid chain melting transition are shown (Table 1). The temperature of the transition is shifted slightly among the different samples and is lowered by the presence of peptide. This is probably a result of the peptide partitioning more favorably into the liquid-crystalline phase than into the gel phase. In addition, the enthalpy of this transition in the presence of cholesterol, PtdIns(4,5)P2 and the NAP-22-peptide is increased almost threefold. This indicates that cholesterol has been depleted from a domain of the membrane that can now undergo a more cooperative and endothermic transition, more like that of the pure phospholipid. Estimates of the transition enthalpy of mixtures containing cholesterol have a higher error because of the low temperature and broadness of the transition. In addition to the phospholipid transition, some samples also exhibit a transition corresponding to the polymorphic transition of anhydrous cholesterol crystals, which appears in the cooling scans at 21 °C. The enthalpy and temperature of this transition was estimated from both cooling and heating scans where this transition occurs at 38 °C. The temperature difference between the heating and cooling curves is characteristic of this transition and is caused by the slow rate of interconversion of two forms of anhydrous cholesterol crystals [15]. The polymorphic transition of anhydrous cholesterol crystals is most clearly seen by DSC in heating scans. We present examples of heating scans of either SOPC/cholesterol (60: 40) or SOPC/cholesterol (50:50) containing either 10 or 20 mol% of the NAP-22 peptide or the Y11L NAP-22 (Fig. 2). The transition enthalpies of these peaks, obtained from the areas of the peaks, provide an estimate of the amount of crystalline cholesterol (Table 2). Pure anhydrous
• 1793
• FEBS Journal 272 (2005) 1792-1803© 2005 FEBS
• Table 1. DSC Transition of SOPC. Transitions observed in cooling scans at 2°-min¯¹ of SOPC with additional components listed in the first three columns. When cholesterol is present it is at a 6: 4 molar ratio of SOPC:cholesterol Ptdlns (4,5)P2 is at 0.2% of total lipids, while NAP-22-peptide is 10 mol% of total lipids when present.
• Additional components
• Cholesterol
• Ptdlns (4,5)P2
• Peptide
• Tm (°C)
• ΔΗ (kcal-mol-¹)
• None
• –
• –
• None
• 6
• 4.0
• +
• –
• None
• 4.8
• 4.6
• +
• +
• None
• 1.7
• 4
• +
• +
• NAP-22 peptide
• 1.7
• 3.2
• –
• –
• None
• 6
• 0.35
• +
• –
• NAP-22 peptide
• Broad Transition
• –
• +
• +
• NAP-22 peptide
• 0.7
• 0.36
• +
• +
• NAP-22 peptide
• 1.6
• 0.85
• +
• +
• Y11L mutant
• No transition observed
• +
• +
• Y11L mutant
• 0.8
• 0.37
• Fig. 1. DSC cooling scans. (A) SOPC alone (curve 1) and SOPC with 0.2 mol% PtdIns(4,5)P2 added (curve 2); 0.2 mol% PtdIns(4,5)P2 and 10 mol% NAP-22-peptide added (curve 3); 10 mol% NAP-22-peptide added (curve 4). (B) SOPC/cholesterol 60: 40 with 0.2 mol% PtdIns(4,5)P2 and 10 mol% NAP-22-peptide added (curve 1); SOPC/cholesterol 60:40 with 10 mol% NAP-22- peptide added (curve 2); SOPC/cholesterol 60:40 with 0.2 mol% PtdIns(4,5)P2 added (curve 3); SOPC/cholesterol 60:40 (curve 4); SOPC/cholesterol 60:40 with 10 mol% mutant Y11L-NAP-22-pep- tide added (curve 5) and SOPC/cholesterol 60:40 with 0.2 mol% PtdIns(4,5)P2 and 10 mol% mutant Y11L-NAP-22-peptide added (curve 6); Scan rate 2°-min-1.
• A
• B
• Temperature (°C)
• Temperature (°C)
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• FEBS Journal 272 (2005) 1792-1803© 2005 FEBS
• Cholesterol-dependent lipid rearrangement
• R. F. Epand et al.
• Fig. 2. DSC heating scans. (A) NAP-22 peptide. (B) Y11L-NAP-22- peptide. Curve 1, SOPC/cholesterol 60: 40 with 10 mol% peptide; Curve 2, SOPC:cholesterol 60: 40 with 20 mol% peptide; Curve 3, SOPC/cholesterol 50:50 with 10 mol% peptide; Curve 4, SOPC/cholesterol 50:50 with 20 mol% peptide. Scan rate 2°-min-1.
• Excess Heat Capacity (cal/°C/mole)
• Excess Heat Capacity (cal/°C/mol)
• A
• B
• Temperature (°C)
• Temperature (°C)
• Table 2. DSC transition of anhydrous cholesterol crystallites. Tran- sitions observed in heating scans at 2°-min¯¹ of SOPC with 40 or 50 mol% cholesterol, as well as with added peptide.
• % Cholesterol
• Peptide
• ΔΗ (cal-mol cholesterol-¹)
• 40
• 10% NAP-22 peptide
• 20
• 40
• 20% NAP-22 peptide
• 33
• 40
• 10% Y11L mutant peptide
• 0
• 40
• 20% Y11L mutant peptide
• 12
• 50
• 10% NAP-22 peptide
• 74
• 50
• 20% NAP-22 peptide
• 130
• 50
• 10% Y11L mutant peptide
• 97
• 50
• 20% Y11L mutant peptide
• 110
• cholesterol crystals have an enthalpy of 910 cal·mol¯¹ [16]. In some cases the height of the transition peak is not proportional to the area because the peaks differ in their breadth (cooperativity). At SOPC/cholesterol (60: 40) it is clear that