Extracellular Matrix and Stress Substrates: the Role of Prolidase

我们想要一个动物模型的增殖酶缺乏症，反映表型在人类与这种遗传代谢紊乱。人类疾病的特点是伤口愈合不良，下肢溃疡，骨骼异常和智力迟钝。然而，这些在人类身上的发现并不一致。谱系中描述了具有相同生化表型的兄弟姐妹可能具有不同的临床表现。为了进行这些研究，我们与蒙大拿州大瀑布市麦克劳克林医学研究所的特蕾莎·冈恩博士建立了合作关系。她描述了一种毛色异常的小鼠品系（dal/dal）。她培育了一群纯合子dal/dal小鼠，然后在编码脯氨酸酶的pepd基因中发现了一个4 bp的缺失。这些动物的表型包括肥厚性心肌病。我们建立了这些小鼠的一个群体，由于背景菌株不确定，我们获得了用C3H繁殖dal/dal的杂合子，并获得了F2只动物，进行了红细胞增殖酶测试，以确定WT和dal/dal。然后用产仔配偶作为我们研究的对照。执行了两项方案。首先，对伤口进行5毫米穿刺活检。对照伤口用载药处理，而试验伤口用各种一氧化氮剂（ProliNO）制剂处理，该制剂释放脯氨酸和一氧化氮。我们发现使用的ProliNO水平是有毒的，因为它会抑制伤口愈合。重要的是，WT和dal/dal小鼠之间没有明显差异。我们尝试的另一个方案是两阶段皮肤肿瘤发生模型。再次，我们发现WT和daql/dal在乳头状瘤的产生上没有差异。因此，我们必须得出结论，在这些条件下，增殖酶活性不是一个限制因素。脯氨酸酶是脯氨酸营养和从内源性蛋白质中回收脯氨酸所必需的酶，但脯氨酸可以由谷氨酸或鸟氨酸内源性合成。我们的假设是，在各种应激条件下，内源性脯氨酸的生物合成将被上调，以补偿无法从营养蛋白或从内源性蛋白质降解中恢复的蛋白质中获得脯氨酸。我们将通过比较两个时期来检验这一假设：最大生长期（6-8周龄）和成熟期（5个月龄）。他们还将吃两种不同的饮食，一种是含有鱼粉（常规饮食），其中含有一些游离脯氨酸，另一种是只含有植物产品的可选饮食。

We desired an animal model for prolidase deficiency which reflected the phenotype in humans with this inherited metabolic disorder. The human disorder is characterized by poor wound healing with ulcers on the lower extremities, bony abnormalities and mental retardation. These findings in humans, however, are not consistent. Pedigrees have been described in which siblings with identical biochemical phenotype may have variable clinical manifestations. To pursue these studies, we established a collaboration with Dr. Teresa Gunn of the McLaughlin Medical Research Institute in Great Falls, Montana. She has described a mouse strain (dal/dal) which have abnormalities in coat color. Breeding a colony of homozygous dal/dal mice, she then identified a 4-bp deletion in the pepd gene encoding prolidase. The phenotype in these animals included hypertrophic cardiomyopathy. We established a colony of these mice and since the backgrouind strain was indeterminate, we obtained heterozygotes breeding dal/dal with C3H and obtained F2 animals which were tested for red cell prolidase to determine WT and dal/dal. Litter mates were then used as controls for our studies. Two protocols were carried out. First, wounds were inflicted with a 5 mm punch biopsy. Control wounds were treated with vehicle whereas test wounds were treated with various preparations of a nitric oxide agent (ProliNO) which released proline as well as nitric oxide. We found that the level of ProliNO used was toxic in that it inhibited wound healing. Importantly, there was no observable difference between WT and dal/dal mice. Another protocol we tried is the two-stage skin tumorigenesis model. Again, we found no difference between WT and daql/dal in their production of papillomas. Thus, we had to conclude that under these conditions, prolidaase activity was not a limiting factor. Prolidase is necessary for both proline nutrition and for proline recycling from endogenous protein, However, proline can be endogenously synthesized from either glutamate or ornithine. Our hypothesis is that under the various stress conditions, endogenous proline biosynthesis would be upregulated to compensate for inability to obtain proline from nutritional proteins or from protein recovered from degradation of endogenous proteins. We will test this hypothesis by comparing two periods a period of maximal growth (age 6-8 weeks) and at maturity (age 5 months). They will also be on two different diets, one containing fish meal (regular diet) which contains some free proline and an optional diet containing only plant products.