次壶腹腺丝蛋白功能模块的研究

蜘蛛丝是天然的生物材料,具有潜在的巨大应用价值。研究蜘蛛丝蛋白质的结构与功能,有助于破解蜘蛛丝蛋白质的成丝机理,为制备优良材料学性能的仿生蜘蛛丝纤维提供理论依据。以MiSp蜘蛛丝的重复区和C端非重复区蛋白多肽为研究对象,在不同pH值和离子条件下,在体外研究其二级结构与成丝的关系。CD图谱显示:表达纯化的重组蜘蛛丝蛋白R1R2在pH 7.5、6.5和5.5时二级结构相似,均为无规则卷曲,而R1R2CT则主要呈现为α螺旋构象;扫描电镜结果表明:在以上3种pH条件下,只有pH 5.5时R1R2和R1R2CT才形成重组丝纤维,R1R2CT纤维形态较平整,类似于天然蛛丝纤维形态,而R1R2丝纤维则呈条带状,表面粗糙。另外,氯化钠不利于形成形态平整的丝纤维。该成果为研究蛛丝蛋白的成丝机理奠定基础,也为制备仿生蛛丝蛋白纤维提供理论依据。     

重组蜘蛛丝蛋白MiSp NT结构域在不同pH环境下的Trp荧光光谱及圆二色谱分析

为明确蛛丝蛋白NT结构域的生物学功能,首次研究了MiSp NT结构域在不同pH条件下的二聚化动力学及二级结构特性. 基于蛛丝蛋白MiSp全长编码基因,克隆并分别在BL21(DE3)和Rosetta-gami 2(DE3)中重组表达MiSp NT结构域: BL21(DE3)表达水平较高,约35 mg/L LB培养基,表达产物需经短暂超声和2 mol/L尿素促溶;Rosetta-gami 2(DE3)表达产物可溶性较好,但表达水平仅为BL21 (DE3) 一半左右. 融合蛋白经凝血酶介导的2次Ni-NTA纯化,纯度达到95%以上. Trp 荧光光谱分析表明,MiSpNT在pH为7.0左右时开始二聚化,pH5.7时二聚体为主要构象,pH_T约为6.6. MiSp NT在pH 7.5,6.5和5.5条件下的CD图谱相似,均主要为α-helix,表明NT二聚化与二级结构水平的变化没有直接联系. 本研究为进一步探索蛛丝蛋白NT结构域的结构和功能以及成丝机理提供线索.     

大腹园蛛管状腺丝重组蛋白的克隆表达及纺丝

TuSp1蛋白(tubuliform spidroin 1)是管状腺丝(tubuliform silkfiber)的主要组成成分。管状腺丝作为蛛丝卵袋的外层包卵丝,其结构具有很好的耐腐蚀性和良好的力学性能。目前国内外对大腹园蛛TuSp1蛋白的研究很少,仅有一条基因序列的报道。本课题首次构建含大腹园蛛N端非重复结构域、重复单元以及C端非重复结构域的重组管状腺丝蛋白TuSp1 NT-Rp-CT,并经湿法纺丝获得重组蛋白丝纤维。重组蛋白液圆二色谱分析结果显示,pH由7.0降低到5.5的过程中,始终保持稳定的α-螺旋构象;重组蛋白丝纤维的傅里叶变换红外光谱结果显示,丝纤维中主要二级结构为β-折叠及β-转角;经扫描电镜观察发现,冻干的絮状重组蛋白能自组装成丝纤维,且表面光滑纤细;湿纺后的重组蛋白丝纤维直径较粗,但表面较平整均匀,具有类似天然管状腺丝的形态特征,这些为TuSp1蛋白的成丝机理及仿生纺丝研究提供了理论依据。     

重组杂合蛛丝蛋白MiSpNT-PySpRp-MiSpCT的二级结构表征

重组蛛丝纤维作为一种性能优异的生物材料,具有良好的生物相容性,在生物医学工程领域具有极大的潜在应用价值。已有研究表明,重组蛛丝蛋白可用作血管、神经导管及药物载体等,但其生物学功能仍有待研究。本研究以大腹园蛛基因组为模板,设计特异性引物,通过PCR扩增获得大腹园蛛梨状腺丝（piriform spidroin: PySp）一个完整重复区（Rp）编码序列;此Rp模块与MiSpNT/CT模块重组,构建微小型杂合蛛丝蛋白MiSpNT-PySpRp-MiSpCT,成功在大肠杆菌BL21中高效表达,借助8 mol/L尿素裂解缓冲液进行变性纯化,得到纯度较高的杂合蛛丝蛋白MiSpNT-PySpRp-MiSpCT,产量约100 mg/L。CD图谱显示,MiSpNT-PySpRp-MiSpCT蛋白质溶液主要以α-螺旋和无规卷曲形式存在,随着溶液pH值降低,部分α-螺旋向β-折叠转变;红外光谱显示,在自然成丝及冻干过程中,部分α-螺旋转化为β-折叠,符合天然蛛丝蛋白成丝过程的二级结构变化特征。本研究结果为今后获得具有天然蛛丝纤维优异性能的人工重组蛛丝纤维材料提供一种新的可能。     

蛋白质内含子介导蛛丝功能化平台的建立

为建立高效快捷的蛛丝功能化修饰平台,蛋白质内含子的反式剪接技术被首次应用于重组蛛丝的功能化修饰。在体外通过Ssp Dna B的反式剪接作用,在蛋白质水平上将12 k Da泛素相关修饰蛋白(SUMO)与蛛丝蛋白(W2CT)连接形成功能化蛛丝蛋白SUMOW2CT。修饰后SUMOW2CT与W2CT均能形成纳米至微米级的丝纤维,但SUMOW2CT自动成丝速度明显下降且产量约为W2CT的一半。与W2CT丝纤维(W)相似,SUMOW2CT丝纤维(UW)不具有超收缩能力和对2%SDS不耐受,但机械性能低于W2CT丝纤维。功能化蛋白SUMOW2CT形成的丝纤维中SUMO蛋白仍保持着正确三维结构,可被SUMO蛋白酶酶切。外源功能化蛋白质虽在一定程度上降低了丝的形成速度和机械性能,但修饰上的功能化蛋白仍保持着生物活性,表明断裂蛋白质内含子介导的蛛丝修饰平台成功建立,也为蛛丝的功能化修饰和应用奠定了坚实的技术基础。     

蛛丝蛋白MiSp重组模块R1R2CT表达及其纤维化动力学研究

为探索蛛丝蛋白模块单元在成丝过程中的作用,研究了大腹园蛛Mi Sp R1R2CT功能模块在不同p H条件下的纤维化动力学特性。大腹园蛛Mi Sp蛋白的R1R2CT功能模块与硫氧还蛋白融合,并在BL21(DE3)中进行表达,表达量约为15 mg/L LB培养基。R1R2CT蛋白在p H 7.5和6.5时较为稳定,在22 h之内不发生纤维化;当p H值降至5.5时,R1R2CT在前2 h内快速纤维化,3~5 h趋势较为平缓;5 h之后R1R2CT蛋白再次出现Th T信号的快速增长,7 h后保持缓慢增长至22 h。R1R2CT的增长速率及幅度高于单独的CT(C-terminal)蛋白,而单独的R1R2在p H 7.5、6.5和5.5时均保持稳定,表明重复区依赖CT模块的快速纤维化模式。已有研究表明,CT在重复区纤维化过程中起晶核作用,该成果也从反面证实CT的晶核理论。     

大腹园蛛次壶腹腺丝的表达

基于大腹园蛛次壶腹腺丝Minor Ampullate Spidroin全长编码基因最新报道,研究了该基因的表达。利用PCR扩增该基因重复区一段长1 348 bp的片段P1,融合his-tag标签,构建酵母表达载体,在毕赤酵母菌GS115进行表达。同时构建大肠杆菌表达载体,在大肠杆菌BL21(DE3)中进行表达。SDS-PAGE和Western blotting检测结果表明,P1在两种表达系统中均可实现表达。研究结果显示:P1在GS115中的表达经优化后产量、产率有较大提高,且远高于BL21(DE3)中的表达,相应的纯化效率GS115也远高于对照BL21(DE3)的表达。研究表明酵母表达系统更适合重复度高、且富含Gly/Ala的天然蛛丝蛋白基因的表达,为表达全长天然MiSp编码序列提供前期实验基础,也为大规模蛛丝蛋白的重组表达建立了平台。     

硫醇基团的氧化还原过程对人正常朊蛋白聚集和纤维化特征的影响

本研究旨在通过体外对纯化的原核重组人正常朊蛋白硫醇基团的氧化还原过程,探究二硫键的改变对其生化特性的影响。蛋白沉淀实验显示重组正常人朊病毒蛋白经过硫醇基团的氧化还原过程明显增加了其聚集性;硫磺素T(Thioflavin T ,ThT)实验测定发现,经过硫醇基团的氧化还原过程重组PrP蛋白的纤维形成增多;圆二色谱（Circular Dichroism, CD）测定显示,处理后的重组PrP蛋白二级结构发生改变,其β-折叠结构比例显著增多;蛋白酶K消化实验也进一步显示硫醇基团的氧化还原后PrP的蛋白酶K抵抗能力有所增加。这些结果提示二硫键的形成可明显地改变PrP的二级结构,促进朊蛋白聚集和成纤维过程。     

三种类型蜘蛛丝的结构及生物学功能

利用付里叶变换红外光谱仪(FFIR)对棒络新妇(Nephila clavata)、悦目金蛛(Argiope amoena)的大壶状腺丝(拖丝)、悦目金蛛的捕丝(粘性螺旋丝)和卵袋丝这3种不同类型蜘蛛丝的二级结构进行了测试研究。结果表明：蜘蛛丝同时包含无规则卷曲、α-螺旋和β-折叠构象;对这3种蛛丝的红外光谱进行比较表明同一蜘蛛的不同类型蛛丝所含的这3种二级结构的比例不同,这种不同组成的二级结构就赋予了蜘蛛丝不同的特性,这种特性又与其不同的功能相适应。此外,还用扫描电镜(SEM)和光学显微镜对悦目金蛛和小悦目金蛛(A．minuta)的拖丝和捕丝做了形态结构观察。蜘蛛丝这种天然动物蛋白纤维所具有的特殊的形态结构、蛋白质二级结构与其特殊的性能和生物学功能是高度一致的。     

蜘蛛丝蛋白研究进展

由于蜘蛛丝蛋白分子高度重复的一级结构、特殊的溶解特性和分子折叠行为以及具有形成非凡力学特性丝纤维的能力而引人注目。本文从蛛丝蛋白基因、天然蛛丝形成过程、蛛丝蛋白的基因工程生产及蛛丝蛋白的应用前景等几个方面着重介绍了近20年来对蛛丝蛋白的研究进展。围绕蛛丝蛋白展开的研究将有助于揭示蛋白质一级结构、蛋白质分子折叠与蛋白质大分子特性之间的内在联系。     

The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases.

Using homology search, structure prediction, and structural characterization methods we show that the C-terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled-related proteins, and (4) type I procollagen C-proteinase enhancer proteins (PCOLCEs) are homologous with the N-terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR module) fulfill diverse biological roles ranging from axon guidance, regulation of Wnt signaling, to the control of the activity of metalloproteases. With the exception of TIMPs, it is not known at present what role the NTR modules play in these processes. In view of the fact that the NTR modules of TIMPs are involved in the inhibition of matrixin-type metalloproteases and that the NTR module of PCOLCEs is involved in the control of the activity of the astacin-type metalloprotease BMP1, it seems possible that interaction with metzincins could be a shared property of NTR modules and could be critical for the biological roles of the host proteins.     

NMR structure of the netrin-like domain (NTR) of human type I procollagen C-proteinase enhancer defines structural consensus of NTR domains and assesses potential proteinase inhibitory activity and ligand binding

Procollagen C-proteinase enhancer (PCOLCE) proteins are extracellular matrix proteins that enhance the activities of procollagen C-proteinases by binding to the C-propeptide of procollagen I. PCOLCE proteins are built of three structural modules, consisting of two CUB domains followed by a C-terminal netrin-like (NTR) domain. While the enhancement of proteinase activity can be ascribed solely to the CUB domains, sequence homology of the NTR domain with tissue inhibitors of metalloproteinases suggest proteinase inhibitory activity for the NTR domain. Here we present the three-dimensional structure of the NTR domain of human PCOLCE1 as the first example of a structural domain with the canonical features of an NTR module. The structure rules out a binding mode to metalloproteinases similar to that of tissue inhibitors of metalloproteinases but suggests possible inhibitory function toward specific serine proteinases. Sequence conservation between 13 PCOLCE proteins from different organisms suggests a conserved binding surface for other protein partners.     

Electron Transfer Pathways and Dynamics of Chloroplast NADPH-dependent Thioredoxin Reductase C (NTRC)

NADPH-dependent thioredoxin reductases (NTRs) contain a flavin cofactor and a disulfide as redox-active groups. The catalytic mechanism of standard NTR involves a large conformational change between two configurations. Oxygenic photosynthetic organisms possess a plastid-localized NTR, called NTRC, with a thioredoxin module fused at the C terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs) and thus is involved in the protection against oxidative stress, among other functions. Although the mechanism of electron transfer of canonical NTRs is well established, it is not yet known in NTRC. By employing stopped-flow spectroscopy, we have carried out a comparative kinetic study of the electron transfer reactions involving NTRC, the truncated NTR module of NTRC, and NTRB, a canonical plant NTR. Whereas the three NTRs maintain the conformational change associated with the reductive cycle of catalysis, NTRC intramolecular electron transfer to the thioredoxin module presents two kinetic components (k_ET of ∼2 and 0.1 s⁻¹), indicating the occurrence of additional dynamic motions. Moreover, the dynamic features associated with the electron transfer to the thioredoxin module are altered in the presence of 2-Cys Prx. NTRC shows structural constraints that may locate the thioredoxin module in positions with different efficiencies for electron transfer, the presence of 2-Cys Prx shifting the conformational equilibrium of the thioredoxin module to a specific position, which is not the most efficient.     

Neurotensin regulates DARPP-32 thr34 phosphorylation in neostriatal neurons by activation of dopamine D1-type receptors

Neurotensin modulates dopaminergic transmission in the nigrostriatal system. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cAMP-dependent protein kinase, resulting in its conversion into a potent inhibitor of protein phosphatase-1 (PP 1). Here, we examined the effect of neurotensin on DARPP-32 Thr34 phosphorylation using mouse neostriatal slices. Neurotensin stimulated DARPP-32 Thr34 phosphorylation by 4-7-fold with a K(0.5) of approximately 50 nM. The effect of neurotensin was antagonized by a combined neurotensin receptor type-1 (NTR1)/type-2 (NTR2) antagonist, SR142948. It was not antagonized by a NTR1 antagonist, SR48692 or by a NTR2 antagonist, levocabastine; neither was it antagonized by the two combined. Pretreatment with TTX or cobalt abolished the effect of neurotensin. The effect of neurotensin was antagonized by a dopamine D1 antagonist, SCH23390, and by ionotropic glutamate receptor antagonists, MK801 and CNQX. These results indicate that neurotensin stimulates the release of dopamine from nigrostriatal presynaptic terminals in an NMDA receptor- and AMPA receptor-dependent manner, leading to the increase in DARPP-32 Thr34 phosphorylation. Neurotensin stimulated the phosphorylation of Ser845 of the AMPA receptor GluR1 subunit in wild-type mice but not in DARPP-32 knockout mice. Thus, neurotensin, by stimulating the release of dopamine, activates the dopamine D1-receptor/cAMP/PKA/DARPP-32/PP 1 cascade.     

Neurotensin negatively modulates Akt activity in neurotensin receptor-1-transfected AV12 cells

Neurotensin (NT) regulates a variety of biological processes primarily through interaction with neurotensin receptor-1 (NTR1), a heterotrimeric G-protein-coupled receptor (GPCR). Stimulation of NTR1 has been linked to activation of multiple signaling transduction pathways via specific coupling to G(q), G(i/o), or G(s), in various cell systems. However, the function of NT/NTR1 in the regulation of the Akt pathway remains unknown. Here, we report that activation of NTR1 by NT inhibits Akt activity as determined by the dephosphorylation of Akt at both Ser473 and Thr308 in AV12 cells constitutively expressing human NTR1 (NTR1/AV12). The inactivation of Akt by NT was rapid and dose-dependent. This effect of NT was completely blocked by the specific NTR1 antagonist, (S)-(+)-[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)-carbonylamino] cyclohexylacetic acid (SR 48527), but unaffected by the less active enantiomer ((R)-(-)-[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)-carbonylamino] cyclohexylacetic acid (SR 49711)), indicating the stereospecificity of NTR1 in the negative regulation of Akt. In addition, NT prevented insulin- and epidermal growth factor (EGF)-mediated Akt activation. Our results provide insight into the role of NT in the modulation of Akt signaling and the potential physiological significance of Akt regulation by NT.     

Dynamic characterisation of the netrin-like domain of human type 1 procollagen C-proteinase enhancer and comparison to the N-terminal domain of tissue inhibitor of metalloproteinases (TIMP)

The backbone mobility of the C-terminal domain of procollagen C-proteinase enhancer (NTR PCOLCE1), part of a connective tissue glycoprotein, was determined using 15N NMR spectroscopy. NTR PCOLCE1 has been shown to be a netrin-like domain and adopts an OB-fold such as that found in the N-terminal domain of tissue inhibitors of metalloproteinases-1 (N-TIMP-1), N-TIMP-2, the laminin-binding domain of agrin and the C-terminal domain of complement protein C5. NMR relaxation dynamics of NTR PCOLCE1 highlight conformational flexibility in the N-terminus, strand A and the proximal CD loop. This region in N-TIMP is known to be essential for inhibitory activity against the matrix metalloproteinases and suggests that this region is of equal importance for NTR PCOLCE1, although the specific functional activity of the NTR PCOLCE1 domain is still unknown. Dynamics observed within the structural core of NTR PCOLCE1 that are not observed in N-TIMP molecules suggest that although the two domains have a similar architecture, the NTR PCOLCE1 domain will show different thermodynamic properties on binding and hence the target molecule could be somewhat different from that observed for the TIMPs. ModelFree order parameters show that NTR PCOLCE1 has more flexibility than both N-TIMP-1 and N-TIMP-2.     

The p75NTR extracellular domain: A potential molecule regulating the solubility and removal of amyloid-��

Senile plaque composed of amyloid-beta (Aβ) in the brain is one of the hallmarks of Alzheimer disease (AD). Removal of Aβ from the brain is the most important therapeutic strategy for AD. The solubility of Aβ is critical for its endocytosis, transcytosis and removal from the brain. Our recent study has found that the extracellular domain of p75NTR, the neurotrophin receptor, plays an important role in the solubility of Aβ and might be one of the endogenous mechanisms in the regulation of Aβ plaque formation. The physiologically shedded extracellular domain of p75NTR is able to inhibit Aβ aggregation and diasggregate preformed Aβ fibrils, while the full p75NTR expressed on neurites, endothelial cells and smooth muscle cells in blood-brain barrier (BBB) might initiate Aβ endocytosis and degradation, and/or remove Aβ from the brain via BBB. Understanding the roles of p75NTR in the solubility and clearance of Aβ may allow targetting p75NTR as a unique opportunity to develop therapeutic drugs for the prevention and treatment of AD.Key words: Alzheimer disease, amyloid-β, p75NTR, extracellular domain, blood-brain barrier, clearanceSenile plaque in the brain is one of the hallmarks of Alzheimer disease (AD). The main component of the senile plaques is amyloid-beta (Aβ), which is a metabolic product of amyloid precursor protein (APP). The steady-state level of Aβ in the normal brain is maintained by the balance between its production and clearance. However in the AD brain this balance is broken due to either over-production of Aβ or a reduction in Aβ clearance;^1,2 thus Aβ accumulates in the brain and forms amyloid plaques which cause dementia and neurodegeneration in patients. Based on the Aβ hypothesis proposed a decade ago, Aβ plays a causal and pivotal role in the development of AD.³ Therefore, removal of Aβ from the brain is the most important therapeutic strategy for AD.⁴ To reach this goal, it is essential to understand how the Aβ metabolism is regulated in the AD brain. Despite the dramatic progress has been made in the understanding of how Aβ is produced from APP, the mechanisms of Aβ aggregation, metabolism and clearance from the brain remain unclear so far. Only 5% of AD (familial cases) is due to the over-production of Aβ because of mutations in the APP gene or in the APP processing enzymes, while the majority (95%) of so-called sporadic or late-onset AD (LOAD) are likely caused by dysfunctions in Aβ solubility or aggregation, endocytosis, degradation, transcytosis and removal.The solubility of Aβ is critical for its endocytosis, transcytosis and removal from the brain. In AD patients, one of the most consistent biomarkers found so far is the reduction of Aβ level in the cerebral spinal fluid (CSF).⁵ This is the most convincing evidence that there is a reduction in the solubility of Aβ and an increase in the Aβ aggregation and beta-sheet formation in AD patients. In sporadic cases of AD, the polymorphism of the Aβ-binding protein ApoE4 is highly associated with AD. It is known that other variants of ApoE proteins have a higher binding affinity to Aβ than ApoE4. It is likely that ApoE protein plays a critical role in the solubility of Aβ.⁶ The reduction in the Aβ binding ability of ApoE4 may reduce the solubility of Aβ. Thus ApoE protein may act on Aβ keeping it soluble and preventing its aggregation, and ApoE4 variant may reduce Aβ solubility and increase aggregation in the brain. It is not fully understood at this time, what other proteins that might regulate Aβ solubility and prevent its aggregation. Understanding the endogenous mechanism of suppressing Aβ aggregation and enhancing its removal will help to target Aβ for developing disease-modifying drugs.The neurotrophin receptor p75NTR may be such the protein which plays critical roles in the Aβ solubility and prevents Aβ aggregation and deposition in the brain. During the investigation into the functions of p75NTR in the development of AD in a recent study, we have found that the extracellular domain of p75NTR regulates the deposition of Aβ in a mouse model of AD.⁷ In p75NTR gene-knockout APPswe/PS1dE9 mice, soluble Aβ which reflects the steady-state level of Aβ production, is reduced in the brain. The serum Aβ level, which is associated with the level of soluble Aβ in the brain, is also reduced in p75NTR-knockout animals. In comparison, we found that p75NTR knockout increases the insoluble Aβ as reflected by the increased amyloid plaques and formic acid-extracted Aβ levels. Our results indicate that p75NTR may play critical roles in the solubility of Aβ in the brain of AD mice. To test the hypothesis, we have made recombinant extracellular domain-fused with human immunoglobulin Fc fragment and tested its effects in the solubility of Aβ in vitro. We have found that the recombinant extracellular domain of p75NTR has a very strong effect on the solubility of Aβ. It reduces Aβ oligomerization and fibrillization, solubilizes fibrilized Aβ. Most interestingly, when injected into the hippocampus of AD mice, it reduces the number and size of Aβ plaques. Thus, we have clearly demonstrated that the extracellular domain plays an important role in the solubility of Aβ and might be one of endogenous mechanisms in the regulation of Aβ plaque formation in patients.How might p75NTR play a role in the Aβ plaque formation or deposition in AD brain? One of the features of the Aβ pathology is that Aβ exclusively deposits in the neocortex, hippocampus and vessel walls. These areas are also the projection area of p75NTR positive fibers. The close anatomical association between p75NTR expression and Aβ deposition strongly suggests that p75NTR is involved in the initiation and development of Aβ deposition in the brain. Interestingly, we have observed there is a spatial relationship between p75NTR fibers and Aβ plaques in the brain of AD mice. We have found that p75NTR positive neurites locates in the center of compact senile plaques, while p75NTR negative degenerative neurites locate in the outer region of Aβ plaques.⁷ This phenomenon suggests that p75NTR positive neurodegenerative fibers may play a seeding role to initiate the Aβ aggregation and plaque formation. It is known that Aβ can bind to the extracellular domain of p75NTR.⁸ Normally, Aβ-bound p75NTR is likely endocytosed and degraded in the lysosomes, but the degenerated neurites may be abnormal in the endocytosis of the Aβ-p75NTR complex. Thus Aβ which binds to p75NTR on the cell surface may act as seeds to initiate the cascade of Aβ aggregation and beta-sheet formation.On the other hand, the extracellular domain of p75NTR after enzymatic shedding may play a different role than the membranous p75NTR. It is known that the p75NTR extracellular domain is physiologically shedded by TACE to generate a soluble and diffusible factor.⁹ The physiological function of the shedded diffusible extracellular domain of p75NTR remains unknown. During the aging process and during the development of AD, p75NTR expression is upregulated,^10,11 and presumably the production of the diffusible extracellar domain of p75NTR is also increased. The increased extracellular domain of the p75NTR is likely a critical factor to maintain the solubility of Aβ, acting in concert with ApoE and other Aβ-binding proteins such as low-density lipoprotein receptor-related protein-1 (LRP1).¹² Indeed, in our animal experiment, we have found that knockout of p75NTR significantly increases the insoluble Aβ, even though the production of Aβ is reduced in p75NTR knockout neurons.⁷ Our data have provided strong evidence that the brain Aβ deposition and amyloid plaque formation may be mainly due to the decreased Aβ solubility or decreased Aβ clearance.The solubility of Aβ goes hand in hand with the clearance of Aβ because only the solubilized Aβ can be endocytosed and degraded in lysosomes by neurons, microglia and astrocytes, and be transported from the brain to the blood for degradation and clearance.⁴ Whether p75NTR plays any roles in the endocytosis of Aβ and degradation is unclear. Our data of increased Aβ deposition in the brain of p75NTR knockout mice may also be explained by the decreased endocytosis of Aβ via p75NTR. It is likely that p75NTR may play a role in Aβ endocytosis, as p75NTR is a receptor of Aβ and mediates its toxicity in neurons.⁸ p75NTR ligands can trigger a clathrin-dependent endocytosis of both p75NTR and its ligands.¹³ If p75NTR normally mediates Aβ endocytosis, the knockout of p75NTR would reduce the removal of Aβ by endocytosis and lead to the increased deposition in the brain.Normally, p75NTR is also expressed in endothelial cells and smooth muscle cells of blood vessels, vessel-innervating sympathetic and sensory neurons and choroid plexus in the brain.^14,15 This raises the possibility that p75NTR within blood vessels may play a role in transport and trafficking of Aβ from the brain to the blood. It is well known that LRP1 plays important roles in the transport and transcytosis of Aβ and clears Aβ from the brain.¹² LRP1 on the cell-surface of the blood-brain barrier (BBB) can bind, transcytose and transport Aβ from the brain to the blood. p75NTR expressed on the BBB may play similar roles in the removal of Aβ in a similar manner to the Aβ binding proteins, LRP1 and G-glycoprotein, expressed on the BBB. Future studies should test roles of p75NTR in the endocytosis, transcytosis and clearance of Aβ by different cells such as neurons, endothelial cells and smooth muscle cells. The levels of shedded diffusible p75NTR in the brain and blood of AD patients should be determined as a biomarker and correlated with Aβ levels or Aβ plaques to reveal its potential roles in the solubility and clearance of Aβ in AD patients.In summary, our studies have provided strong evidence that p75NTR is an essential molecule to keep the solubility of Aβ during development of AD. We speculate that p75NTR might also play many vital roles in removing Aβ from brain (Fig. 1). However, it is still far from certain how p75NTR regulates the solubility of Aβ and suppresses its deposition in the AD brain. Understanding the roles of p75NTR in the solubility and clearance of Aβ may help using p75NTR as a target to develop therapeutic drugs for the prevention and treatment of AD.Open in a separate windowFigure 1Schematic diagram depicting functions of p75NTR in Aβ solubility and clearance. p75NTR locates on the neurites, epithelial cells and smooth muscle cells of the blood-brain barrier (BBB). Binding of Aβ to p75NTR on neurites may initiate the endocytosis of Aβ and its degradation in the neurons. Shedding of p75NTR from the cell membrane releases the soluble extracellular domain (p75NTR-ECD), which is capable of inhibiting Aβ aggregation and disaggregating preformed Aβ fibrils. p75NTR at BBB might be able to transport Aβ from the brain to blood.     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants

CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.     

Therapeutic Efficacy Evaluation of 177Lu-DOTA-NT and 177Lu-DOTA-SR48692 in Murine RS-1 Hepatoma

Neurotensin (NT) is a peptide with biological affinity to neurotensin receptors (NTR), while SR48692 is a non-peptide molecule with competitive/inhibitory activity to the same receptors. This paper aims to bring a scientific contribution to elucidate the paradigm concerning the capture of NT agonist or antagonist (SR48692) by tumor cells, depending on the competition between NT and SR48692 for identical receptors. For this reason, we have tested the therapeutic efficacy of a single dose of both ¹⁷⁷Lu-DOTA-NT and ¹⁷⁷Lu-DOTA-SR48692 administered to positive NTR malignant hepatoma bearing rats. Additionally, in order to evaluate the competition between NT and SR we have studied under similar circumstances, the therapeutic effects of the following combinations: ¹⁷⁷Lu-DOTA-NT/DOTA-SR48692 and ¹⁷⁷Lu-DOTA-SR48692/DOTA-NT. Male Wistar rats inoculated with RS1 hepatoma cells were divided in four treatment groups and one control group and treated intraperitoneally with a dose of 74-GBq (specific activity of 2 Ci/mg) per compound. At different time after compounds administration, five animals from each group were sacrificed, and removed several specimens: blood, tumor, liver, pancreas, spleen, kidney, bone marrow and small intestine. The radiobiological effects of these different regimens were evaluated by biochemistry (thiols, malonaldialdehyde and total antioxidant status) and flow cytometry (DNA ploidy, cell proliferation status, proliferative index). Treatment with the aforementioned compounds resulted in the tumor regression and the increased density of cells in G1 corresponding to a decrease of S and G2 that indicate the arrest in G1. Redox parameters recorded a proportional increase subsequently to radiotherapy induction. Our data evidenced in vivo a therapeutic potential of the two radiolabeled compounds in radionuclide therapy of murine RS-1 hepatoma. In addition, the combination between the radiolabeled compound and its unlabeled counterpart may become a promising strategy to improve the therapeutic effects.