STRUCTURAL FEATURES OF KERATIN AND RELATED INTERMEDIATE FILAMENTS

中间丝（IF）是真核细胞骨架中普遍存在的成分。它们由至少6种不同类型组成，其中数量最多、最复杂的是广泛表达于上皮细胞的I型和II型角蛋白。我们不仅对人类皮肤角蛋白IF的结构、功能和表达及其在角变性疾病中的作用感兴趣，而且对其他细胞类型的相关IF感兴趣，以了解它们在生物学中的作用。正在进行的结构研究虽然角蛋白在许多遗传疾病中的作用现在已经被很好地理解，但进一步的结构研究对于制定合理的治疗方法是必要的。我们已经启动了两种类型的结构/功能研究。首先，我们开发了与1A开头或2B棒状结构域段结束相对应的合成肽。其中一些合成肽已被注射到活细胞中，以探索它们的动态行为。大多数作为破坏所有类型IF的非常特定的试剂，但不干扰微管或微丝的组装状态。然而，它们确实影响细胞骨架的超分子组织，从而表明细胞骨架的所有三个组成部分在细胞中协同工作。另一方面，我们发现H1肽，一个仅在II型角蛋白链中特异性发现的序列，是上皮中角蛋白IF组织的一种非常特异性的毒素。在同时表达角蛋白干扰素和蛋白干扰素的培养细胞中，只有角蛋白干扰素的组织被破坏。在第二种实验方法中，我们合成了一系列与角蛋白链序列相对应的肽，这些肽与组装细丝中重要的重叠区域有关，用于生物物理结构研究、溶液核磁共振和x射线晶体学。我们现在已经确定，从III型蛋白vimentin衍生的2B肽显示简单的二聚化，基本上是单分散在溶液中，具有> - 90%的a-螺旋，并形成适合x射线衍射分析的晶体。因此，获取原子级分辨率结构信息的尝试将继续进行。同样，我们将继续构建适用于x射线晶体学的1A棒结构域片段的肽结构。我们之前已经发现角蛋白1链2B棒状结构域段的Glu106Asp取代导致中度重度表皮松解性角化过度。这促使我们重新评估离子相互作用在IF中角蛋白链稳定性中的作用。我们注意到有三对潜在的带电残基占据七重体的e-g位置，这些残基在所有IF链类型中都是精确保守的。因此，使用I/II型角蛋白5/14范式，我们在细菌中表达了大量突变链，这些突变链在1A、1B和2B棒结构域段的各种带电残基位置上进行了替换。在实验中，我们对体外阴性染色后形成的IF进行了目测检查，通过尿素溶解实验评估了盘曲线圈分子的稳定性，并对富含a-螺旋的蛋白水解片段进行了分离和表征，以检查分子排列。此外，我们将GFP偶联到角蛋白14链上，并探索了在体内活细胞中替换对KIF组装和组织的影响。我们已经发现，Glu106的位置是形成双链盘绕线圈所绝对需要的。此外，我们的数据表明，为了稳定A22的排列模式，Glu106的位置也是绝对必需的，但Asp106的替换未能形成这种排列模式。这可能为这种疾病提供分子上的解释。使用类似的技术，我们已经探索了角蛋白14链1A棒结构域段Arg10的位置。我们的数据表明，虽然它不是形成双链螺旋状分子所必需的，但它对于A11排列模式的稳定是必不可少的。正在进行的交联研究在探索角化细胞包膜的结构和组织时，我们已经发现了大量涉及角蛋白链和各种其他包膜蛋白之间交联的肽。值得注意的是，绝大多数交联涉及位于II型角蛋白K1， K5或K6头部结构域V1区域的非常特定且精确保守的赖氨酸残基。有趣的是，我们之前发现了一例非表皮松解性掌跖角化病，其中K1链的这种保守赖氨酸残基被异亮氨酸取代。在电子显微镜下对患者组织进行详细的超微结构分析，发现表皮上部颗粒细胞的细胞外周和角蛋白IF细胞骨架之间存在严重的不协调，这种不协调接近于角化细胞包膜的形成。因此，我们认为这种残留物在细胞骨架的结构组织中起着至关重要的作用，包括在终末分化的表皮和其他相关的层状鳞状上皮中形成角化的细胞包膜。失去这种关键的组织模式会导致表皮屏障功能的减弱。未来的研究将包括尝试清除赖氨酸残基，以创建该疾病的小鼠模型，并进一步研究细胞骨骼-角质细胞包膜协调与屏障功能之间的联系。此外，我们还发现了其他交联，这些交联表明角蛋白IF通过一系列相关的中间丝相关蛋白间接附着在桥粒的桥粒蛋白成分上。进一步的工作将旨在了解这些明显联系的复杂性及其在疾病中的作用。我们已经通过详细的交联实验证明，表皮角蛋白分子对以A11、A22和A12三种基本模式排列。当同化成中频时，同一轴列的分子对采用第四种称为ACN的模式，其中一个分子的末端与相邻分子的开始重叠约1nm。有趣的是，几乎所有已知的角变性突变/替换都位于这个重叠窗口。我们还发现，III型中频分子采用相同的4种基本模式，但前3种模式的排列略有偏移。这充分解释了为什么III型和I/II型角蛋白链在体内或体外不能也不能聚集。然而，先前的研究表明，基于x射线衍射数据，头发/羊毛角蛋白IF的分子排列应该是不同的。为了开始解决毛发角蛋白IF如何以及为什么不同的问题，我们在细菌中表达并纯化了全长代表性的I型和II型小鼠毛发角蛋白链。最近，我们已经确定了它们在体外组装成天然型IF的最佳条件，现在将使用这些条件进行额外的交联研究，以确定它们的分子排列。在某些重要的方面，头发角蛋白IF链如何排列的问题类似于发育中的神经细胞。在神经元发育过程中，干细胞首先表达VI型IF蛋白巢蛋白。后期表达III型波形蛋白。随着神经元分化的进行，IV型a-连接蛋白链表达，随后是其他神经丝三重体蛋白。通过交联实验，我们已经证明a-间连接蛋白分子和a-间连接蛋白-静脉蛋白异二聚体分子在IF中确实具有相同的尺寸和相同的分子排列。这些数据充分解释了动态交换/替换过程如何在发展和分化过程中发生。综上所述，这些数据表明，至少有两种不同的方式可以将分子打包，即表皮I/II型角蛋白的IF和III/IV型IF。我们对头发角蛋白IF的新实验将试图确定头发角蛋白IF分子是否以与表皮角蛋白IF分子相同的方式排列，还是以第三种不同的方式排列。寻找高分子量IF相关蛋白当III型vimentin IF在体外通过组装和拆卸循环时，某些高分子量蛋白总是共循环。我们从BHK-21成纤维细胞中纯化了其中一个，通过氨基酸测序，现在已经表明它实际上是VI型巢蛋白。利用RT-PCR和RACE方法测定了仓鼠巢蛋白的全长序列，结果表明，仓鼠巢蛋白具有一个很长的羧基末端尾巴，其中有1750个氨基酸，分布在近11个残基重复序列中。巢蛋白可以在体外形成同型二聚体，但更倾向于与波形蛋白形成异源二聚体。虽然nestin本身不能组装成IF，但它可以与vimentin组装，前提是nestin的含量不超过25%左右。这些数据表明，巢蛋白是一种高分子量蛋白，在细胞内波形蛋白IF的超分子组织中起着重要作用。进一步的研究将探讨巢蛋白在细胞中已知的IF动态行为中的作用。同样，在角化细胞中，单个角蛋白IF以松散排列的束或张力原纤维的形式存在。正在进行的实验旨在解决角蛋白IF是否由类似的组织蛋白捆绑在一起的问题。

Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryote cells. They consist of at least 6 different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested in not only the structure, function and expression of keratin IF of human skin and their roles in keratinopathy diseases, but also of the related IF of other cell types in order to understand their roles in biology. Ongoing structural studies While the roles of the keratins in many genetic diseases are now well understood, further structural studies are necessary to develop rational approaches to therapy. We have initiated two types of structural/functional studies. In the first, we have developed synthetic peptides corresponding to the beginning of the 1A or end of the 2B rod domain segments. Several of these synthetic peptides have been injected into living cells to explore their dynamic behavior. Most function as very specific reagents for the disruption of all types of IF, but do not interfere with the state of assembly of microtubules or microfilaments. However, they do affect the supramolecular organization of them, thus indicating that all three components of the cytoskeleton function cooperatively in cells. On the other hand, we have discovered that an H1 peptide, a sequence which is specifically found only in type II keratin chains, is a very specific poison for the organization of keratin IF in epithelia. In those cultured cells which express both keratin IF and vimentin IF, only the keratin IF organization is disrupted. In a second experimental approach, we have synthesized a series of peptides of sequence corresponding to keratin chains which are involved in the important overlap regions in assembled filaments, for biophysical structural studies, solution nmr, and for X-ray crystallography. We have now determined that 2B peptides derived from the type III protein vimentin show simple dimerization, are essentially monodisperse in solution, and possess >90% a-helix, and form crystals that are suitable for X-ray diffraction analyses. Thus attempts to obtain atomic-level resolution structural information will continue. Similarly, we shall continue to make peptide constructs of the 1A rod domain segment that will be suitable for X-ray crystallography. The role of ionic interactions in IF structure We have previously discovered that a Glu106Asp substitution in the 2B rod domain segment of the keratin 1 chain causes a moderately severe form of epidermolytic hyperkeratosis. This has prompted us to re-evaluate the role of ionic interactions in the stability of the keratin chains in IF. We note that there are three potential pairs of charged residues which occupy e-g positions of the heptad repeat that have been precisely conserved in all IF chain types. Accordingly, using the type I/II keratin 5/14 paradigm, we have expressed in bacteria a large number of mutant chains bearing substitutions at a variety of charged residue positions in each of the 1A, 1B and 2B rod domain segments. For assays, we have used visual examination of IF formed in vitro following negative staining, coiled-coil molecule stabilities as assessed by urea dissolution experiments, and isolation and characterization of a-helix-enriched proteolytic fragments to examine molecular alignments. In addition, we have coupled GFP to the keratin 14 chain and explored the consequences of substitutions on KIF assembly and organization in living cells in vivo. We have found that the Glu106 position is absolutely required for the formation of a two-chain coiled-coil. Moreover, our data suggest that the Glu106 position is also absolutely required to stabilize the A22 mode of alignment, but the Asp106 substitution fails to form this alignment mode. This may provide a molecular explanation for the disease. Using similar techniques, we have explored the Arg10 position of the 1A rod domain segment of the keratin 14 chain. Our data show that while it is not essential for the formation of a two-chain coiled-coil molecule, it is essential for stabilization of the A11 mode of alignment. Ongoing crosslinking studies In explorations of the structure and organization of the cornified cell envelope, we havediscovered a large number of peptides involving crosslinks between a keratin chain and a variety of other envelope proteins. Notably, the vast majority of crosslinks involve a very specific and precisely conserved lysine residue located in the V1 region of the head domain of the type II keratins K1, K5 or K6. Interestingly, we have previously identified a case of non-epidermolytic palmaplantar keratoderma in which this conserved lysine residue of the K1 chain was substituted by isoleucine. Detailed ultrastructural analyses of the patient tissue at the level of the electron microscope revealed a severe discoordination between the cell periphery and the keratin IF cytoskeleton in the upper granular cells of the epidermis, proximal to the formation of the cornified cell envelope. Therefore, we believe this residue is critically involved in the structural organization of the cytoskeleton with the cornified cell envelope in terminally differentiated epidermis, and otherrelated stratified squamous epithelia. Loss of this critical mode of organization results in a diminished barrier function for the epidermis. Future studies will involve attempts to ablate this lysine residue to create a mouse model for this disease and to further study the connection between cytoskeletal-cornified cell envelope coordination and barrier function. In addition, we have identified other crosslinks which reveal that the keratin IF are attached to the desmoplakin component of desmosomes indirectly through a series of related intermediate filament associated proteins. Further work will be directed to understand the complexity of these apparent connections and role in disease. The organization of molecules in various IF types We have previously demonstrated by detailed crosslinking experiments that pairs of epidermal keratin molecules are aligned in three basic modes termed A11, A22 and A12. When assimilated into IF, pairs of molecules in the same axial row adopt a fourth mode termed ACN, in which the end of one molecule overlaps the beginning of the adjacent molecule by about 1 nm. Interestingly, almost all known keratinopathy mutations/substitutions reside in this overlap window. We also have shown that the molecules of type III IF adopt the same basic 4 modes, but the alignments of the former three are slightly offset. This adequately explains why type III and types I/II keratin chains cannot and do not coassemble in vivo or in vitro. However, previous studies have indicated that the molecular alignments in hair/wool keratin IF should be different, based on X-ray diffraction data. In order to begin to address the question of how and why hair keratin IF are different, we have expressed in and purified from bacteria full length representative type I and II mouse hair keratin chains. Recently, we have determined optimal conditions for their assembly into native-type IF in vitro, and will now use these for additional crosslinking studies to determine their molecular alignments. In certain important ways, this issue of how hair keratin IF chains are aligned resembles the situation in developing neuronal cells. During neuronal development, stem cells first express the type VI IF protein nestin. Later type III vimentin is expressed. As neuronal differentiation proceeds, the type IV a-internexin chain is expressed, followed eventually by the other neurofilament triplet proteins. By crosslinking experiments, we have shown that both a- internexin molecules and a-internexin-vimentin heterodimer molecules do indeed have the same dimensions and adopt the same molecular alignments in IF. Such data adequately explain how dynamic exchange/replacement processes may occur during development and differentiation. Together, these data establish that there are at least two different ways in which molecules may be packed as in the IF of the epidermal type I/II keratins and type III/IV IF. Our new experiments with the hair keratin IF will attempt to determine whether hair keratin IF molecules are aligned the same way as epidermal keratin IF molecules, or in a third different way.Searches for high molecular weight IF associated proteins When type III vimentin IF are passaged through cycles of assembly and disassembly in vitro, certain high molecular weight proteins always co-cycle. We have purified one of these from BHK-21 fibroblasts, and by amino acid sequencing, have now shown that it is in fact type VI nestin. By use of RT-PCR and RACE methods, we have determined the full length sequence of hamster nestin and showed that it possesses a very long carboxy-terminal tail of 1750 amino acids configured in quasi-11 residue repeats. The nestin can form homodimers in vitro, but prefers to form heterodimers with vimentin. While nestin does not assemble into IF by itself, it cancoassemble with vimentin, providing the amount of nestin does not exceed about 25%. These data establish that nestin is a high molecular weight protein which plays an important role in the supramolecular organization of vimentin IF in cells. Further studies will investigate the role of nestin in the known dynamic behavior of IF in cells. Likewise, in keratinocytes, individual keratin IF exist as loosely-arranged bundles or tonofibrils. Ongoing experiments are designed to address the question as to whether keratin IF are bundled together by an analogous organizing protein.