DROSOPHILA LYSYL OXIDASE-LIKE PROTEINS

果蝇赖氨酰氧化酶样蛋白

• 批准号: 7381088
• 负责人: Minae Mure
• 金额: $ 15.84万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381088
• 关键词: DROSOPHILA; LYSYL; OXIDASE; LIKE; PROTEINS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. As described in our last report, human lysyl oxidase-like 2 protein (HLOXL2) has been implicated in the metastasis of breast cancer cells and in the deposition of large amounts of collagen fibers. We anticipate therapeutic agents against HLOXL2 will be useful in preventing cancer from spreading and will reduce the recurrence of cancer following surgical removal of breast tumors. In order to develop these drugs, it is important to understand the basic biochemical and physiological functions of HLOXL2 and closely related proteins. To date, few biochemical studies have been reported for HLOXL2. Because it is difficult to work on human proteins (i.e., limited amounts, availability, inability to conduct genetic experiments), we have chosen to work with the fruit fly Drosophila melanogaster proteins. The genome of the fruit fly has two genes (dloxl1 and dloxl2) that encode proteins very similar to HLOXL2. We aim to characterize these fly proteins and use the results to understand the function of human LOXL2 and its role in cancer metastasis. Initially, we determined that the two genes are differentially expressed in different tissue at distinct developmental stages. dloxl1 is expressed robustly in prepupae while dloxl2 is only expressed in adult flies. Tissue specific analyses have been used to demonstrate that the expression of dloxl1 is confined to salivary glands. Surprisingly, we found that dloxl2 was expressed only in adult heads and not in the bodies. To determine the tissue specific expression of dloxl2 in adult heads, we isolated polyA mRNAs from eya2 (eye absent) mutant flies We could not detect dlox2 expression in the eya2 samples, strongly suggesting that dloxl2 is expressed in the eye. We have begun to produce polyclonal antibodies specific for each of the Drosophila LOXL proteins. We have been able to obtain an antibody against a DlOXL2 peptide in the LOX core domain. The antibody recognizes a ~52 kDa band from bacteria that express a GST-tagged DLOXL2 LOX core domain but not from bacteria expressing a similar GST fusion protein from DLOXL1. We are currently testing the antisera on blots of Drosophila tissues. In addition, we are in the process of generating antibodies specific for DLOXL1 using a GST fusion of the entire DLOXL1 coding sequence, because our attempts to use DLOXL1-specific peptides have thus far have not been successful. Our immediate plan is to focus on defining the subcellular localization of DLOXLs by in situ immunostaining, developing loss-of-function-flies that lack DOXL1 to understand the physiological importance of the gene and the biochemical characterization of the native DLOXLs.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。如我们上次报告所述，人赖氨酸氧化酶样蛋白（HLOXL2）与乳腺癌细胞的转移和大量胶原蛋白纤维的沉积有关。我们预计，针对HLOXL2的治疗剂将有助于预防癌症扩散，并在手术切除乳腺肿瘤后会减少癌症的复发。为了开发这些药物，重要的是要了解HLOXL2和密切相关的蛋白质的基本生化和生理功能。迄今为止，Hloxl2的生化研究很少。由于很难处理人类蛋白质（即有限的量，可用性，无法进行遗传实验），因此我们选择与果蝇果蝇Melanogaster蛋白一起使用。果蝇的基因组具有两个基因（dloxl1和dloxl2），它们编码与Hloxl2非常相似的蛋白质。我们旨在表征这些蝇蛋白，并使用结果来了解人Loxl2及其在癌症转移中的作用。最初，我们确定这两个基因在不同的发育阶段在不同组织中差异表达。 dloxl1在预审前稳健表达，而dloxl2仅在成年蝇中表达。组织特定的分析已用于证明Dloxl1的表达仅限于唾液腺。令人惊讶的是，我们发现Dloxl2仅在成年头部而不是在体内表达。为了确定成年头部DLOXL2的组织特异性表达，我们从EYA2（眼睛缺失）突变蝇中分离了Polya mRNA，我们无法检测到EYA2样品中的Dlox2表达，强烈表明dloxl2在眼中表达。我们已经开始产生针对每种果蝇loxl蛋白的多克隆抗体。我们已经能够在LOX核心结构域中获得针对DLOXL2肽的抗体。该抗体从细菌中识别出约52 kDa的带，该细菌表达具有GST标记的DLOXL2 LOX核心结构域，而不是从表达来自Dloxl1的GST融合蛋白的细菌中。我们目前正在果蝇组织的印迹上测试抗血清。此外，我们正在使用整个DLOXL1编码序列的GST融合生成针对Dloxl1特异性的抗体，因为到目前为止，我们尝试使用DLOXL1特异性肽的尝试尚未成功。我们的直接计划是通过原位免疫染色，发展出缺乏DOXL1的功能丧失，以了解基因的生理重要性和天然Dloxls的生化表征，从而定义DLOXL的亚细胞定位。