DETERMINATION OF INTERACTIONS BETWEEN RIBONUCLEASE 1 AND CHEMICAL LIGANDS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。对该分项目和该分项目首席调查员的主要支持可能是由其他来源提供的，包括国家卫生研究院的其他来源。列出的子项目总成本可能代表子项目使用的中心基础设施的估计数量，而不是NCRR拨款提供给子项目或子项目工作人员的直接资金。 Onconase是一种来自北豹蛙的核糖核酸酶(RNase)，已显示出作为选择性化疗药物的前景，目前正在进行恶性间皮瘤(1，2)的IIIb期临床试验。核糖核酸酶A超家族的这个成员切割tRNA，通过细胞凋亡导致细胞死亡(3，4)。然而，已经观察到使用Onconase治疗会引起一些肾毒性(5)。胰腺核糖核酸酶是一种小的高度阳离子的蛋白质，在各种物种中普遍存在，其功能是降解RNA，用于消化、基因调节和病毒免疫。这一领域的药物设计研究已经发现了该蛋白质家族中的其他核糖核酸酶作为潜在的癌症疗法的用途(6，7)。人类核糖核酸酶(RNase1)和牛核糖核酸酶(RNaseA)作为化疗药物正在研究中，因为变异体选择性地针对癌细胞，同时像Onconase(7)那样引起毒性较小的肾脏影响。核糖核酸酶进入细胞的机制包括：与细胞膜结合、内化、移位到内体外、逃避抑制物结合，最后RNA裂解导致细胞死亡(8)。我们实验室以前的研究已经观察了不同表面群的细胞之间的核糖核酸酶的内化，但没有明确的数据表明核糖核酸酶变体如何结合到癌细胞的表面并内化(9)。然而，核糖核酸酶的细胞毒性与总体阳离子(10，11)和最近正电荷的特殊分布(8)相关。因此，结合和内化与细胞外膜上带负电荷的分子有关。与正常细胞相比，癌细胞膜上成分的变化可能会选择性地将核糖核酸酶靶向癌细胞(7，12)，增加了在动物模型和人类试验中观察到的令人鼓舞的治疗指数(13，14)。癌细胞表面经常表现为糖胺多糖图谱(15)、磷脂组成(16，17)和神经节苷脂阵列(18)的变化。感兴趣的特定残留物包括硫酸乙酰肝素、带电荷的糖基如唾液酸和脂类如磷脂酰丝氨酸，因为最近的研究通过可能的治疗靶向表明了这一重要性(19)。 我们推测，核糖核酸酶与细胞表面这些特定残基的结合可能是蛋白质内化的一个因素，从而影响细胞毒性的效率以及肿瘤细胞系的选择性。最近利用核磁共振(20)确定了人核糖核酸酶1的结构。最近的其他论文也使用核磁共振作为一种方法来研究蛋白质残基之间的特定相互作用(21)。为了确定RNase1与细胞质成分的相互作用，我们希望用15N同位素类似地标记RNase1，并滴定细胞膜成分，包括但不限于磷脂酰丝氨酸、硫酸乙酰肝素和唾液酸。然后，我们将查看在600 MHz核磁共振光谱仪上记录的2D NOESY(混合时间=ms)谱，正如求解RNase1结构的方法所描述的那样(20)。将光谱的位移与PDB(注册号2K11)中解析的结构进行比较，将表明RNase1与质膜组分之间的特定相互作用的位置和广泛的结合亲和力。这些结果将确定RNase1是如何与细胞表面成分结合的，并可能阐明内化的机制。然后，我们可以使用15N标记的RNase A和细胞毒性变体重复检测，以观察细胞膜相互作用和RNase细胞毒性之间的可能相关性。此外，还可以进一步研究广泛的细胞膜成分。这可能允许发展具有更高内部化的核糖核酸酶，因为增加了对癌细胞上过度表达的细胞标记物的结合或靶向。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Onconase, a ribonuclease (RNase) from Northern Leopard Frogs, has shown promise as a selective chemotherapeutic and is currently undergoing Phase IIIb clinical trials for malignant mesothelioma (1,2). This member of the RNase A superfamily of ribonucleases cleaves tRNA leading to cell death via apoptosis (3,4). However, treatment with Onconase has been observed to induce some renal toxicity (5). Pancreatic RNases are small highly cationic proteins found ubiquitously across species and function to degrade RNA for digestion, gene regulation, and viral immunity. Drug design research in this area has uncovered the use of other ribonucleases in this protein family as potential cancer therapeutics (6,7). Human ribonuclease (RNase 1) and the bovine homologue (RNase A) are under inquiry as chemotherapeutics as variants selectively target cancer cells while inducing less toxic renal effects as seen with Onconase (7). The mechanism of RNase into the cell consists of the steps: binding to the cell membrane, internalization, translocation out of the endosome, escaping inhibitor binding, and finally RNA cleavage leading to cell death (8). Previous research in our lab has looked at internalization of RNases among cells of varying surface groups, yet there is no definitive data that indicates how RNase variants bind onto the surface of cancerous cells and internalize (9). However, RNase cytotoxicity has been correlated to overall cationicity (10,11) and more recently the specific distribution of positive charges (8). As such binding and internalization has been linked to the negatively charged molecules on the outer membrane of the cell. Variation of the composition on cancer cell membranes compared to normal cells might target ribonucleases selectively to cancer cells (7, 12), adding to the encouraging therapeutic index observed in animal models and human trials (13,14). The surface of cancer cells frequently display changes in glycosaminoglycan profile (15), phospholipid composition (16,17) and ganglioside array (18). Specific residues of interest include heparan sulfates, charged sugar groups such as sialic acid, and lipids such as phosphotidylserine as recent research has indicated this importance by possible therapeutic targeting (19). We hypothesize that ribonuclease binding to these specific residues on the cell surface may be a factor of protein internalization and thus affect efficiency of cytotoxicity as well as selectivity of cancerous cell lines. The structure of human RNase 1 was recently determined using NMR (20). Other recent papers have also used NMR as a method to look at specific interactions between residues of proteins (21). In order to determine RNase interactions with cell plasma components, we want to similarly label RNase 1 with 15N isotope and titrate cell membrane components including but not limited to phosphatidylserine, heparan sulfate, and sialic acid. We will then look at 2D NOESY (mixing time = ms) spectra recorded on the 600 MHz NMR spectrometer as described by the methods that solved the RNase1 structure (20). Comparison of the shifts in the spectra to the structure solved in the PDB (accession number 2K11) will indicate the location and a broad degree of binding affinity of specific interactions between the RNase 1 and plasma membrane components. The results will establish how RNase1 is bound to the cell surface components and may elucidate the mechanism of internalization. We can then repeat examination using the 15N labeled RNase A and cytotoxic variants to observe a possible correlation between cell membrane interactions and RNase cytotoxicity. Also a broad range of cell membrane components can be further investigated. This may allow for developing of RNases with higher internalization due to increased binding or targeting towards cell markers that are over-expressed on cancerous cells.