DROSOPHILA LYSYL OXIDASE-LIKE PROTEINS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。正如我们在上一篇报道中所描述的，人赖氨酸氧化酶样2蛋白（HLOXL2）与乳腺癌细胞的转移和大量胶原纤维的沉积有关。我们预计针对HLOXL2的治疗药物将有助于防止癌症扩散，并减少乳房肿瘤手术切除后的癌症复发。为了开发这些药物，了解HLOXL2及其密切相关蛋白的基本生化和生理功能至关重要。迄今为止，很少有关于HLOXL2的生化研究报道。由于很难对人类蛋白质进行研究（即，数量有限，可用性有限，无法进行基因实验），我们选择了对果蝇的蛋白质进行研究。果蝇的基因组有两个基因（dloxl1和dloxl2），它们编码的蛋白质与HLOXL2非常相似。我们的目标是对这些果蝇蛋白进行表征，并利用这些结果来了解人类LOXL2的功能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发育阶段的不同组织中有差异表达。cloxl1在预蛹中表达强烈，而cloxl2仅在成虫中表达。组织特异性分析已被用来证明，表达的dloxl1局限于唾液腺。令人惊讶的是，我们发现dloxl2只在成人的头部而不是在身体中表达。为了确定dloxl2在成人头部的组织特异性表达，我们从eya2（眼缺失）突变蝇中分离了多a mrna。我们在eya2样本中未检测到dlox2的表达，这强烈表明dloxl2在眼睛中表达。我们已经开始生产针对每种果蝇LOXL蛋白的多克隆抗体。我们已经能够在LOX核心区域获得针对DlOXL2肽的抗体。该抗体识别来自表达GST标记的DLOXL2 LOX核心结构域的细菌的~52 kDa条带，而不识别来自表达类似GST融合蛋白的DLOXL1的细菌的条带。我们目前正在果蝇组织的斑点上测试抗血清。此外，我们正在使用整个DLOXL1编码序列的GST融合来生成DLOXL1特异性抗体，因为我们使用DLOXL1特异性肽的尝试到目前为止还没有成功。我们的近期计划是通过原位免疫染色来确定DLOXLs的亚细胞定位，培养缺乏DOXL1的功能丧失果蝇，以了解该基因的生理重要性和天然DLOXLs的生化特性。

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.