Characterization of a Novel Lysophospholipase

描述（由申请人提供）：大量研究表明磷脂和溶血磷脂作为脂质分裂原，通过改变各种细胞信号通路影响细胞增殖和存活。细胞内磷脂和溶血磷脂的水平受到各自的脂肪酶的严格调节，因此，这些酶的表达和活性对细胞稳态至关重要。我们已经克隆了一个编码一种新的假定溶血磷脂酶的cDNA。鉴于其预测分子质量为34 kDa，我们将其命名为LPL34 （lysophospholipase 34）。我们的初步结果表明，LPL34 mRNA在正常结肠黏膜中高表达，但在已建立的结肠癌细胞系中不表达或表达减少。对原发结肠肿瘤及其匹配的正常组织的分析也显示，LPL34在4/4的肿瘤组织中缺失或表达减少。与这些结果一致的是，在缺乏内源性LPL34的癌细胞中，外源性LPL34的表达会导致生长抑制。我们已经提出进一步研究LPL34在消化道疾病如结肠癌中的作用。我们将分析大量新鲜冷冻和石蜡包埋的组织标本，以在mRNA和蛋白水平上表达LPL34。我们还将研究LPL34状态与临床病理特征之间的相关性。为了研究lfl34介导的生长抑制的分子基础，我们将研究LPL34对PI3-K/Akt和erk依赖性有丝分裂和生存信号通路的影响。为了确定脂肪酶活性是否是LPL34介导的关键生长抑制作用，将采用定点诱变方法突变保守的脂肪酶基序“GHSMG”，并研究突变的LPL34对细胞生长和有丝分裂信号通路的影响。这些都是探索性/发育性研究，根据结论，将为我们提供足够的新数据和试剂，为未来深入研究这种新型溶血磷脂酶的分子作用机制及其在消化系统疾病中的作用奠定基础。

DESCRIPTION (provided by applicant): Numerous studies have demonstrated that phospholipids and lysophospholipids act as lipid mitogens that affect cell proliferation and survival by altering various cell signaling pathways. Intracellular levels of phospholipids and lysophospholips are tightly regulated by their respective lipases and thus, the expression and activity of these enzymes is critical for cellular homeostasis. We have cloned a cDNA that encodes a novel putative lysophospohlipase. In view of its predicted molecular mass of 34 kDa, we have named it LPL34 (lysophospholipase 34). Our preliminary results indicate that the LPL34 mRNA is highly expressed in normal colon mucosa but absent or reduced in established colon cancer cell lines. Analysis of primary colon tumors and their matching normal tissues also revealed that the expression of LPL34 is absent or reduced in 4/4 tumor tissues. Consistent with these results, expression of exogenous LPL34 in cancer cells lacking endogenous LPL34 results in growth inhibition. We have proposed studies to further examine the role of LPL34 in digestive diseases such as colon cancer. We will analyze a larger pool of fresh-frozen and paraffin-embedded tissue specimens for the expression of LPL34 at both mRNA and protein levels. We will also investigate a correlation between LPL34 status and clinicopathological features. To investigate the molecular basis of LFL34-mediated growth suppression, we will study the effect of LPL34 on PI3-K/Akt and ERK-dependent mitogenic and survival signaling pathways. To determine whether lipase activity is critical LPL34-mediated growth suppressive effect, site-directed mutagenesis approach will be used to mutate the conserved lipase motif 'GHSMG' and effects of mutated LPL34 on cell growth and mitogenic signaling pathways will be investigated. These are exploratory /developmental studies that, upon conclusion, will provide us with sufficient new data and reagents that will form the basis of future in-depth studies investigating the molecular mechanism(s) of action of this novel lysophospholipase in general and its role in the digestive diseases in particular.