Epidermal Transglutaminases

表皮转谷氨酰胺酶

• 批准号: 6823073
• 负责人: PETER M STEINERT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6823073
• 关键词: X ray crystallography; calcium channel; congenital ichthyosis; crosslink; enzyme activity; enzyme mechanism; enzyme structure; fibroblasts; human tissue; immunoprecipitation; inclusion body; isozymes; keratinization; keratinocyte; membrane proteins; membrane structure; molecular pathology; myositis; protein glutamine gamma glutamyltransferase; protein structure function; skin; solubility; structural biology; tissue /cell culture; transfection

Transglutaminases (TGases) catalyze the formation of a cross-link between a donor amide group of a protein-bound glutamine residue and an acceptor epsilon-NH2 of a protein-bound lysine residue. This cross-link is an isopeptide bond that cannot be cleaved in vertebrate organisms. The net result therefore is the formation of a permanent, stable, insoluble macromolecular protein complex. In the epidermis and other stratified squamous epithelia, several of the nine known TGase enzymes are expressed. In particular, TGases 1, 2 and 3 cross-link a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying in detail each of these enzymes, and their roles in diseases. Transglutaminase 1 The TGase 1 enzyme in cultured keratinocytes or foreskin epidermal cells is complex since it exists in multiple soluble and membrane-bound full-length as well as proteolytically-processed forms. Most of the enzyme is membrane bound by way of myristate and anchorages on the amino-terminal segment which is unique to the TGase 1 enzyme. The various forms display wide variations in specific activities, but these are difficult to measure because the enzyme is inherently unstable and easily degraded by proteolysis. To address structural and functional questions, we have been successful in expression in baculovirus systems. Previous work from this laboratory has shown that mutations in the TGM1 gene, encoding the TGase 1 enzyme, cause the autosomal recessive disorder lamellar ichthyosis. We examined its molecular basis in a Japanese family and reported two novel TGM1 mutations (R348X, and Y365D). Molecular docking studies revealed that the LI phenotype of this proband can be explained by the prediction that each of the maternally and paternally derived mutations will result in loss of enzyme activity. This study was published. In another investigation of this kind, two self-healing collodion baby siblings with markedly diminished epidermal TGM1 activity we were found to have compound heterozygous TGM1 mutations G278R and D490G. Molecular modeling and biochemical assays of mutant proteins under elevated hydrostatic pressure suggest significantly reduced activity in G278R and a chelation of water molecules in D490G that locks the mutated enzyme in an inactive trans conformation in utero. After birth these water molecules are removed and the enzyme is predicted to isomerize back to a partially active cis form, explaining the dramatic improvement of this skin condition. This study was also published We have continued to perform structural analyses of TGase 3 by X-ray diffraction. In this way, we hope to gain a better understanding of the role of this enzyme in the skin. TGase 3 is expressed in many epithelial cell types, initially as an inactive pro-enzyme, that requires proteolytic activation by specific cleavage. To this end, we developed methods for the large-scale expression and purification of several forms of TGase 3 in the baculovirus system. These include the pro-enzyme, activated enzyme, and the 50 kDa active form. Over the past year, we focussed on regulatory effect of divalent cation binding. Specifically, we solved the structures of three forms of TGM3 in the presence of Ca2+ and/or Mg2+ which provide new insights on the contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty and it has a binding affinity of Kd= 0.3 mM, which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca2+ of the Group 2 family of alkali earth metals, and its occupancy is required for activity. Site three can be occupied by some lanthanides, Ca2+ or Mg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.

转氨酶（TGases）催化蛋白质结合的谷氨酰胺残基的供体酰胺基团和蛋白质结合的赖氨酸残基的受体ε-NH 2之间的交联的形成。这种交联是在脊椎动物生物体中不能被切割的异肽键。因此，最终结果是形成永久的、稳定的、不溶性的大分子蛋白质复合物。在表皮和其他复层鳞状上皮中，表达九种已知的TGase酶中的几种。特别地，TGases 1、2和3交联多种确定的结构蛋白以形成皮质细胞包膜，其是上皮屏障功能的主要组分。我们正在详细研究这些酶中的每一种，以及它们在疾病中的作用。 转氨酶1培养的角质形成细胞或包皮表皮细胞中的TGase 1酶是复杂的，因为它以多种可溶性和膜结合全长以及蛋白水解加工的形式存在。大多数酶通过肉豆蔻酸酯与膜结合，并锚定在TGase 1酶特有的氨基末端片段上。各种形式的比活性显示出广泛的变化，但这些是难以测量的，因为酶本身不稳定，容易被蛋白水解降解。为了解决结构和功能问题，我们已经成功地在杆状病毒系统中表达。本实验室以前的工作表明，编码TGase 1酶的TGM 1基因突变导致常染色体隐性遗传疾病板层状鱼鳞病。我们在一个日本家族中研究了其分子基础，并报告了两个新的TGM 1突变（R348 X和Y365 D）。分子对接研究表明，该先证者的LI表型可以解释为预测，每一个母亲和父亲衍生的突变将导致酶活性的损失。这项研究已经发表。在另一项类似的研究中，我们发现两个自愈的火棉胶婴儿兄弟姐妹的表皮TGM 1活性显著降低，他们具有复合杂合TGM 1突变G278 R和D490 G。分子建模和生物化学测定的突变蛋白质在静水压力升高的建议显着降低活性G278 R和螯合的水分子在D490 G锁定突变的酶在子宫内的非活性反式构象。出生后，这些水分子被去除，预计酶将异构化回到部分活性的顺式形式，这解释了这种皮肤状况的显着改善。这项研究还发表在 我们继续通过X射线衍射对TGase 3进行结构分析。通过这种方式，我们希望更好地了解这种酶在皮肤中的作用。TGase 3在许多上皮细胞类型中表达，最初作为无活性的前酶，需要通过特异性切割进行蛋白水解激活。为此，我们开发了在杆状病毒系统中大规模表达和纯化几种形式的TGase 3的方法。这些包括前酶、活化酶和50 kDa活性形式。在过去的一年里，我们专注于二价阳离子结合的调节作用。具体而言，我们解决了三种形式的TGM 3在Ca 2+和/或Mg 2+的存在下的结构，这为每个Ca 2+离子对活化和活性的贡献提供了新的见解。首先，我们发现位点1中的Ca ~（2+）离子可以很难交换，并且它具有Kd= 0.3mM的结合亲和力，这表明它对酶的稳定性很重要。位点2可以被一些镧系元素占据，但只有碱土金属的第2族的Ca 2+，并且其占据是活性所需的。位点3可以被一些镧系元素、Ca 2+或Mg 2+占据;然而，当Mg 2+存在时，酶是无活性的，并且通道是关闭的。因此，Ca 2+结合在两个网站2和3合作开放的通道。我们推测，操纵的通道开放可以控制细胞内阳离子水平。总之，这些数据对酶的反应机制具有重要意义：通道的打开可能控制活性位点处底物的进入和操纵。