Nitric Oxide Interaction with Insect Nitrophorins

一氧化氮与昆虫硝化蛋白的相互作用

基本信息

批准号：
8212183
负责人：
FRANCES ANN WALKER
金额：
$ 37.16万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-07-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8212183
关键词：
Affect American Apoproteins Bedbugs Binding Biological Blood Cause of Death Cell Death Cells Chagas Disease Charge Chemical Dynamics Cleaved cell Collaborations Complex Distant Escherichia coli Feces Goals Grant Health Heme Hemeproteins Histamine Histamine Release Human Individual Initiator Codon Insecta Investigation Iron Kinetics Laboratories Latin America Ligand Binding Ligands Lung Measures Metalloproteins Mosses Myocardium N-terminal Names Nitric Oxide Parasites Physicians Platelet aggregation Play Property Prosthesis Protein Dynamics Proteins Recombinant Proteins Recombinants Resolution Rhodnius Rivers Roentgen Rays Role Saliva Salivary Glands Spectrum Analysis Stream Structure Surface Symptoms Thermodynamics Trypanosoma Vascular blood supply Vasodilation Vasodilator Agents Work amino group arginyllysine carboxylate design ferriheme migration mutant nitrophorin novel prevent protein function protein structure response sucking transmission process vector

项目摘要

DESCRIPTION (provided by applicant): The saliva of the blood-sucking insect, Rhodnius prolixus, originally from the Amazon river basin, which carries the parasite Trypanasoma cruzi, the vector of Chagas' disease, contains a suite of heme proteins that we have named Nitrophorins (NPs). The NPs act as vasodilators, anti-platelet aggregation and anti-histaminic agents when injected into the victim. These properties aid the insect in obtaining an ample blood meal, and thus make more likely the transmission of the trypanosome from the insect feces to humans. The trypanasome multiplies intracellularly and is released upon cell death to enter the blood stream and infect distant cells. Chagas' disease slowly weakens the autonomic muscles of the heart, lungs and/or gut and thereby may cause death some 30 or more years later. There is no cure. The North American blood supply is in danger of contamination by the trypanosome. The NPs are novel ferriheme proteins that are reversible carriers of nitric oxide (NO) and tight binders of histamine. They are the only known heme proteins with the heme inside a ?-barrel (lipocalin fold). Our hypothesis is that extreme ruffling of the heme (revealed by high-resolution X-ray crystal structures) is the key to their unusual properties, including stabilization of FeIII and reversible NO binding. Specific aims for the next grant period are: 1) To investigate NP4 and the native terminus-containing constructs of NP1, NP2 and NP3 or their X1A mutants, in which the recombinantly-produced proteins' Met0s are cleaved. The thermodynamics and kinetics of their function, and the roles that individual NP proteins play in making a suite of closely related proteins such a successful strategy for a blood-sucking insect will be studied. 2) To investigate the ps to min dynamics of apo- and holo-NP2 and NP4 in order to understand the fast and slow timescale dynamics of these ?-barrel proteins, how the plasticity of the ?-barrel is affected by adding the prosthetic group and then NO or histamine to the apoprotein, and how the dynamics of the two proteins differ. The slow (5s to min) dynamics of NP2-ligand complexes will be compared to those of NP4 to understand why the rates of NO, but not histamine release are a factor of 40 slower for NP2-D1A than for NP4, and how the dynamics of loop opening and closing are able to control the rate of ligand binding and release. 3) To investigate, by measuring Eos of additional charge mutants and by theoretical calculations, why some buried and surface charge mutants of NP2-D1A affect the Eo of the heme more strongly than others at the same distance from iron. The many charged residues of the NPs make them a useful "laboratory" for probing the effects of charges on the Eo of Fe. Negative charges stabilize the FeIII form of the protein, which is able to release NO with nM Kds, while the FeII form has fM Kds and thus binds NO irreversibly, preventing vasodilation. We envision that the information obtained in this work will allow understanding the roles of the N-terminus, the dynamics of the proteins and their response to both heme and ligand binding, and the roles of the charged residues of the NPs in protein function. These findings will be broadly applicable to other heme proteins and to metalloproteins in general. PUBLIC HEALTH RELEVANCE: The saliva of the blood-sucking insect, Rhodnius prolixus, originally from the Amazon river basin, which carries the parasite Trypanasoma cruzi, the vector of Chagas' disease, contains a suite of heme proteins that we have named Nitrophorins (NPs). The NPs act as vasodilators, anti-platelet aggregation and anti-histaminic agents when injected into the victim. These properties aid the insect in obtaining an ample blood meal, and thus make more likely the transmission of the trypanosome from the insect feces to humans. The information obtained in the work proposed will allow understanding the roles of the N-terminus of the nitrophorins, the dynamics of these proteins and their response to both heme and ligand binding, and the roles of the charged protein residues of the NPs in protein function.

描述（由申请人提供）：吸血昆虫的唾液Rhodnius Prolixus，来自亚马逊河盆地，携带寄生虫锥虫Cruzi（Chagas疾病的媒介），其中包含一套血红素蛋白，我们命名为硝基蛋白（NPS）。注射到受害者时，NP充当血管扩张剂，抗血小板聚集和抗激素药物。这些特性有助于昆虫获得充足的血液粉，因此更有可能使锥虫从昆虫粪便传播到人类。锥虫体在细胞内增加，并在细胞死亡后释放以进入血流并感染远处的细胞。查加斯氏病慢慢地削弱了心脏，肺和/或肠道的自主肌肉，从而大约30多年后可能导致死亡。无法治愈。北美的血液供应有锥虫污染的危险。 NPS是新型的Ferriheme蛋白，是一氧化氮（NO）和组胺紧密粘合剂的可逆载体。它们是唯一已知的血红素蛋白，具有血红素a-Barrel内部（Lipocalin Fold）。我们的假设是，血红素的极端皱纹（由高分辨率X射线晶体结构揭示）是其异常特性的关键，包括FEIII的稳定和可逆的无结合。下一个赠款期的具体目的是：1）研究NP4的NP1，NP2和NP3的含天然末端构建体或其X1A突变体，其中重组产生的蛋白质的Met0s被切割。将研究其功能的热力学和动力学，以及单个NP蛋白在制造密切相关的蛋白质套件中所扮演的角色，以这种成功的吸血昆虫的策略。 2）为了研究pS和holo-np2和np4的最小动力学，以了解这些蛋白的快速和缓慢的时间尺度动力学？ - 桶蛋白，如何通过添加假体组的可塑性来影响？桶的可塑性，然后添加或组胺对pepototian no或组氨基蛋白不存在，以及对两种蛋白质的动力学不同。将NP2配合配合物的慢速（5s至min）动力学与NP4的动力学进行比较，以了解为什么NON的速率（而不是组胺释放的速率）对于NP2-D1A的速度比NP4慢40倍，并且环路打开和关闭的动态如何能够控制配体粘合和释放的速率。 3）通过测量其他电荷突变体的EOS和理论计算，为什么某些埋入NP2-D1A的表面电荷突变体会影响血红素的EO，而与其他距离相同的距离更强烈。 NP的许多充电残留物使其成为探测电荷对FE的影响的有用的“实验室”。负电荷稳定了蛋白质的FEIII形式，该蛋白质能够用NM KD释放NO，而FEII形式具有FM KD，因此不可逆地结合了不可逆地结合，从而阻止了血管舒张。我们设想，这项工作中获得的信息将允许了解N末端的作用，蛋白质的动力学及其对血红素和配体结合的反应以及NP的带电残基在蛋白质功能中的作用。这些发现将广泛适用于其他血红素蛋白和金属蛋白。公共卫生相关性：吸血昆虫的唾液Rhodnius Prolixus，来自亚马逊河盆地，最初带有Chagas疾病的寄生虫锥虫锥虫Cruzi，其中包含一套香肠蛋白，我们命名了硝基蛋白（NPS）。注射到受害者时，NP充当血管扩张剂，抗血小板聚集和抗激素药物。这些特性有助于昆虫获得充足的血液粉，因此更有可能使锥虫从昆虫粪便传播到人类。在提出的工作中获得的信息将允许了解硝基磷酸蛋白N末端的作用，这些蛋白质的动力学及其对血红素和配体结合的反应以及NPS中带电蛋白残基在蛋白质功能中的作用。

项目成果

期刊论文数量（27）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A).

DOI：
10.1007/s00775-013-1063-8
发表时间：
2014-06
期刊：
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
影响因子：
3
作者：
Berry, Robert E.;Muthu, Dhanasekaran;Shokhireva, Tatiana K.;Garrett, Sarah A.;Goren, Allena M.;Zhang, Hongjun;Walker, F. Ann
通讯作者：
Walker, F. Ann

NMR studies of the dynamics of high-spin nitrophorins: comparative studies of NP4 and NP2 at close to physiological pH.

DOI：
10.1021/bi501305a
发表时间：
2015-01-20
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Berry, Robert E.;Muthu, Dhanasekaran;Yang, Fei;Walker, F. Ann
通讯作者：
Walker, F. Ann

1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH.

DOI：
10.1007/s00775-009-0551-3
发表时间：
2009-09
期刊：
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
影响因子：
3
作者：
Yang, Fei;Knipp, Markus;Shokhireva, Tatiana K.;Berry, Robert E.;Zhang, Hongjun;Walker, F. Ann
通讯作者：
Walker, F. Ann

Ligand-induced heme ruffling and bent no geometry in ultra-high-resolution structures of nitrophorin 4.

硝基蛋白 4 的超高分辨率结构中配体诱导的血红素褶皱和弯曲无几何形状。

DOI：
10.1021/bi0109257
发表时间：
2001
期刊：
Biochemistry
影响因子：
2.9
作者：
Roberts,SA;Weichsel,A;Qiu,Y;Shelnutt,JA;Walker,FA;Montfort,WR
通讯作者：
Montfort,WR

Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

DOI：
10.1021/bi5013047
发表时间：
2015-01-20
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Berry, Robert E.;Yang, Fei;Shokhireva, Tatiana K.;Amoia, Angela M.;Garrett, Sarah A.;Goren, Allena M.;Korte, Stephanie R.;Zhang, Hongjun;Weichsel, Andrzej;Montfort, William R.;Walker, F. Ann
通讯作者：
Walker, F. Ann