EPIDERMAL TRANSGLUTAMINASES

表皮谷氨酰胺转胺酶

• 批准号: 6431736
• 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/6431736
• 关键词: active sites; crosslink; enzyme activity; enzyme mechanism; genetic promoter element; human tissue; isozymes; keratinocyte; lysine; membrane proteins; membrane structure; protein glutamine gamma glutamyltransferase; protein structure function; skin; solubility; tissue /cell culture

Transglutaminases (TGases) catalyze the formation of a crosslink between a donor amide group of a protein-bound glutamine residue and an acceptor e-NH2 of a protein-bound lysine residue. This crosslink is an isopeptide bond that cannot be cleaved in eukaryote organisms. The net result therefore is the formation of a permanent, stable, insoluble macromolecular protein complex. In the epidermis and other stratified squamous epithelia, at least three different TGase enzymes, TGases 1, 2 and 3, are expressed. They crosslink a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying each of these enzymes in detail. 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. The partitioning between the cytosol and membranes is controlled by differential acylation by myristate and palmitate of a cluster of cysteine residues located on a membrane anchorage 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 this problem, we have developed methods for its expression in the baculovirus system in which it is postsynthetically modified essentially the same ways as in keratinocytes, but as it is not proteolyzed it is more stable. In this way, we are able to produce microg amounts of enzyme which can be stored for use indefinitely. 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 have expressed in baculovirus some of the known mutations. Most result in a product of no or only very low activity. However, two others result in a product of >10-fold activity than the wildtype enzyme. Examination of their structures using the coordinates of the available structure of the related factor XIIIa TGase enzyme, we anticipate that these two mutations result in an unusually stabilized protein. In order to test this idea, we next expressed these same forms with an attached histidine tag at the carboxy terminus, and used them for transfection experiments into cultured keratinocytes. By specific immuno-precipitations with the his-tag antibody, we found that these mutant proteins were not proteolytically processed into highly active forms in vivo. These data suggest that lamellar ichthyosis disease may be caused by either insufficient enzyme activity, or an enzyme form that is not appropriately postsynthetically modified and cannot be utilized by the cell. Ongoing work is directed toward an understanding of the mechanism by which the TGase 1 enzyme is anchored to membranes and how this affects its substrate specificity toward certain known substrates including loricrin, small proline rich proteins, involucrin and various members of the plakin family including envoplakin and desmoplakin. In addition, available data suggest the 90 residue membrane anchorage segment controls TGase 1 activity. We will attempt to express this fragment in bacteria/baculovirus systems in order to study its structure and functional properties. Transglutaminase 2Our main focus of this enzyme is to obtain atomic resolution structural information by X-ray diffraction of crystals. To date, we have developed methods for the large scale preparation of the enzyme in baculovirus. Ongoing work will attempt to purify active forms of the enzyme in order to initiate crystallization trials. Transglutaminase 3The TGase 3 enzyme is expressed in many epithelial cell types, initially as an inactive pro-enzyme, that requires proteolytic activation by specific cleavage. In addition, data from this laboratory have shown that it is the preferred enzyme for crosslinking in vivo of several important substrates involved in barrier or other functions, including loricrin, small proline rich proteins, and trichohyalin. The proximal promoter region of the TGM 3 gene is located within the first 126 bp above the transcription start site, and consists of an Sp1 motif modulated by adjacent ets-like motifs. These are sufficient to confer epithelial-specific expression. This region also contains a calcium responsive element. In addition, we have found evidence for a single-stranded DNA binding protein that may serve as a negative controlling element. Studies on this promoter will continue. Activatable pro-TGase 3 enzyme has been expressed in large quantities in baculovirus. Preliminary work has demonstrated the formation of small crystals, from which we have generated structural information at the 3.5 A level using the Brookhaven synchronton X-ray facility. We are now developing procedures for the growth of larger crystals that may be suitable for complete structural ascertainment. We have found that labeling during synthesis in baculovirus with selenomethionine may enhance the X-ray signals and resolution. These studies will continue. Expression of TGases in non-epithelial tissues: roles in degenerative diseasesBy RT-PCR methods, we have found that TGases 1 and 3 are widely expressed in a variety of non-epithelial tissues, including in particular, various tissues within the brain, connective tissues, fibroblasts and muscle. Heretofore, these tissues were thought to express only the cytosolic TGase 2 enzyme. The expression of these enzymes has been confirmed by both immunoprecipitation reactions with specific antibodies, and by indirect immunofluorescence methods. Moreover, mRNA and enzyme levels of TGases 1 and 3 are upregulated in pathological conditions, including Alzheimer?s Disease (AD) and sporadic inclusion body myositis (SIBM). We found co-localization of the TGase enzyme antigens with the inclusion bodies/senile plaques of both diseases. In addition, we isolated insoluble proteins from tissue slices from these AD and SIBM which contained substantial amounts of isopeptide crosslink formed by TGases, suggesting a direct causative link. Also, in the case of SIBM, we performed sequencing analyses to demonstrate the presence of significant amounts of b-amyloid protein crosslinked to itself, myosin and desmin muscle proteins. Together, these data demonstrate that elevated levels of TGases 1 and/or 3 correlate with disease pathogenesis and contribute directly to the formation of insoluble crosslinked bodies that interfere with normal cellular function and thus degenerative disease. Depending on availability of adequate amounts of tissue, further work will be directed toward analysis of the forms of the TGase 1 and 3 enzymes and more detailed sequencing analyses. We anticipate that such studies may provide valuable new insights into both disease etiology as well as methods to control the degenerative diseases processes. Finally, we have found that when fibroblasts are irritated by low levels of a reagent such as SDS, they greatly elevate the expression of the TGase 1 enzyme, which begins to crosslink various cytoskeletal proteins including vimentin and actin. Preliminary data suggest this does not promote apoptosis. However, this system may serve as an experimental model of contact dermatitis.

转谷氨酰胺酶 (TGase) 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 e-NH2 之间形成交联。这种交联是一种异肽键，在真核生物中不能被裂解。因此，最终结果是形成永久、稳定、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，至少表达三种不同的TGase酶，即TGase 1、2和3。它们交联各种确定的结构蛋白以形成角质化细胞包膜，这是上皮屏障功能的主要组成部分。我们正在详细研究每种酶。转谷氨酰胺酶 1 培养的角质形成细胞或包皮表皮细胞中的 TGase 1 酶很复杂，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。胞质溶胶和膜之间的分配是通过肉豆蔻酸和棕榈酸对位于膜锚定氨基末端片段上的半胱氨酸残基簇的差异酰化来控制的，这是 TGase 1 酶所特有的。各种形式的比活性存在很大差异，但这些很难测量，因为酶本质上不稳定并且容易被蛋白水解降解。为了解决这个问题，我们开发了其在杆状病毒系统中表达的方法，其中它以与角质形成细胞中基本相同的方式进行合成后修饰，但由于它不被蛋白水解，因此更稳定。通过这种方式，我们能够生产微克量的酶，并可以无限期地储存使用。该实验室之前的工作表明，编码 TGase 1 酶的 TGM1 基因突变会导致常染色体隐性遗传病层状鱼鳞病。我们已经在杆状病毒中表达了一些已知的突变。大多数会产生没有活性或活性非常低的产物。然而，另外两种酶产生的产物的活性是野生型酶的 10 倍以上。使用相关因子 XIIIa TGase 酶的可用结构坐标检查其结构，我们预计这两个突变会产生异常稳定的蛋白质。为了测试这个想法，我们接下来表达了这些相同的形式，并在羧基末端附加了组氨酸标签，并将它们用于培养的角质形成细胞的转染实验。通过使用组氨酸标签抗体进行特异性免疫沉淀，我们发现这些突变蛋白在体内并未被蛋白水解加工成高活性形式。这些数据表明，层状鱼鳞病可能是由于酶活性不足或酶形式未经过适当的合成后修饰而无法被细胞利用引起的。正在进行的工作旨在了解 TGase 1 酶锚定在膜上的机制，以及这如何影响其对某些已知底物的底物特异性，包括兜甲蛋白、富含脯氨酸的小蛋白、外皮蛋白和 plakin 家族的各种成员，包括 envoplakin 和 desmoplakin。此外，现有数据表明 90 个残基的膜锚定片段控制 TGase 1 活性。我们将尝试在细菌/杆状病毒系统中表达该片段，以研究其结构和功能特性。转谷氨酰胺酶2我们对该酶的主要关注点是通过晶体的X射线衍射获得原子分辨率的结构信息。迄今为止，我们已经开发出大规模制备杆状病毒酶的方法。正在进行的工作将尝试纯化酶的活性形式，以启动结晶试验。转谷氨酰胺酶 3 TGase 3 酶在许多上皮细胞类型中表达，最初作为无活性的酶原，需要通过特定切割进行蛋白水解激活。此外，该实验室的数据表明，它是参与屏障或其他功能的几种重要底物体内交联的首选酶，包括兜甲蛋白、富含脯氨酸的小蛋白和毛玻璃蛋白。 TGM 3 基因的近端启动子区域位于转录起始位点上方的前 126 bp 内，由受相邻 ets 样基序调节的 Sp1 基序组成。这些足以赋予上皮特异性表达。该区域还含有钙反应元素。此外，我们还发现了单链 DNA 结合蛋白可作为负控制元件的证据。对该启动子的研究将继续进行。可激活的前 TGase 3 酶已在杆状病毒中大量表达。初步工作已经证明了小晶体的形成，我们使用布鲁克海文同步子 X 射线设备从中生成了 3.5 A 级的结构信息。我们现在正在开发可能适合完整结构确定的较大晶体的生长程序。我们发现，在杆状病毒合成过程中用硒代蛋氨酸进行标记可以增强 X 射线信号和分辨率。这些研究将会继续下去。 TGase在非上皮组织中的表达：在退行性疾病中的作用通过RT-PCR方法，我们发现TGase 1和3在多种非上皮组织中广泛表达，特别包括脑内的各种组织、结缔组织、成纤维细胞和肌肉。迄今为止，这些组织被认为仅表达胞质TGase 2酶。这些酶的表达已通过特异性抗体的免疫沉淀反应和间接免疫荧光方法得到证实。此外，TGases 1 和 3 的 mRNA 和酶水平在病理条件下上调，包括阿尔茨海默病 (AD) 和散发性包涵体肌炎 (SIBM)。我们发现 TGase 酶抗原与两种疾病的包涵体/老年斑共定位。此外，我们从这些 AD 和 SIBM 的组织切片中分离出不溶性蛋白质，其中含有大量由 TGase 形成的异肽交联，表明存在直接因果关系。此外，就 SIBM 而言，我们进行了测序分析，以证明存在大量与其自身交联的 b-淀粉样蛋白、肌球蛋白和结蛋白肌肉蛋白。总之，这些数据表明，TGase 1 和/或 3 水平升高与疾病发病机制相关，并直接导致不溶性交联体的形成，从而干扰正常细胞功能，从而干扰退行性疾病。根据是否有足够数量的组织，进一步的工作将针对 TGase 1 和 3 酶的形式分析以及更详细的测序分析。我们预计此类研究可能会为疾病病因学以及控制退行性疾病过程的方法提供有价值的新见解。最后，我们发现，当成纤维细胞受到低水平试剂（例如 SDS）刺激时，它们会大大提高 TGase 1 酶的表达，该酶开始交联各种细胞骨架蛋白，包括波形蛋白和肌动蛋白。初步数据表明这不会促进细胞凋亡。然而，该系统可以作为接触性皮炎的实验模型。