Real-time NMR investigations of triple-helix folding and collagen folding diseases

Folding of the collagen triple helix provides an opportunity to look at multichain molecular assembly. This triple helix also offers unique advantages for the study of folding because the process is very slow compared to globular proteins, and the kinetics of folding can be obtained in real time by NMR. Studies on triple-helical peptides illustrate the ability to observe kinetic folding intermediates directly and the ability to propose detailed mechanisms of folding through the use of real-time NMR methods. Defective collagen folding has been implicated in various connective tissue diseases and the capacity of NMR to look at the folding of specific sites provides a tool for obtaining information about altered folding mechanisms. Comparison of folding in peptides that model normal and diseased collagens could shed light on the molecular perturbation and the etiology of disease.     

Positional preferences of ionizable residues in Gly-X-Y triplets of the collagen triple-helix

Collagens contain a high amount of charged residues involved in triple-helix stability, fibril formation, and ligand binding. The contribution of charged residues to stability was analyzed utilizing a host-guest peptide system with a single Gly-X-Y triplet embedded within Ac(Gly-Pro-Hyp)3-Gly-X-Y-(Gly-Pro-Hyp)4-Gly-Gly-NH2. The ionizable residues Arg, Lys, Glu, and Asp were incorporated into the X position of Gly-X-Hyp; in the Y position of Gly-Pro-Y; or as pairs of oppositely charged residues occupying X and Y positions. The Gly-X-Hyp peptides had similar thermal stabilities, only marginally less stable than Gly-Pro-Hyp, whereas Gly-Pro-Y peptides showed a wide thermal stability range (Tm = 30-45 degrees C). The stability of peptides with oppositely charged residues in the X and Y positions appears to reflect simple additivity of the individual residues, except when X is occupied by a basic residue and Y = Asp. The side chains of Glu, Lys, and Arg have the potential to form hydrogen bonds with available peptide backbone carbonyl groups within the triple-helix, whereas the shorter Asp side chain does not. This may relate to the unique involvement of Asp residues in energetically favorable ion pair formation. These studies clarify the dependence of triple-helix stability on the identity, position, and ionization state of charged residues.     

The collagen fibril: the almost crystalline structure

Blood vessels from aged animals and humans have impaired relaxation and cAMP production to beta-adrenergic stimulation. Direct activators of adenylyl cyclase (AC) such as forskolin are not affected. We hypothesized that analogous findings would occur in membrane preparations. Aortic media membrane preparations from Fischer 344 rats of four age groups (6 weeks to 24 months) were studied. Basal AC activity increased significantly with age. Forskolin-stimulated activity compared to basal tended to be greater in the 6-week and 6-month preparations compared to the 12- and 24-month preparations. AC activity was assessed in the presence of the G protein activators (GTP, GppNHp, NaF). There was no age-related decrease in responsiveness. The receptor agonists isoproterenol (beta-adrenergic), and PGE-1 (prostaglandin), were studied. There was no significant age-related change in responsiveness over basal activity to either of these agonists. There was a slight, but significant increase in the isoproterenol responsiveness over GTP responsiveness in the 6-week-old animals which also approached significance in the 6-month-old animals, but was not seen in the 12- and 24-month-old animals. These data suggest that using a membrane system to assess age-related changes in beta-adrenergic responsiveness in vascular smooth muscle does not retain the robust differences seen in whole vessels.     

Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N-1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure

A 36-month-old girl had a 3-week history of proptosis of the right eye. Computed tomography showed an ill-defined homogeneous mass filling the intraconal space. Histopathologic examination and immunohistochemistry findings of an incisional biopsy specimen were consistent with malignant undifferentiated tumor with rhabdoid features. Despite chemotherapy (a combination of vincristine sulfate and dactinomycin) and radiotherapy, massive orbital recurrence occurred 6 months later and orbital exenteration was performed. The recurrent tumor was composed entirely of pleomorphic epithelial cells with prominent nucleoli and many filamentous cytoplasmic inclusions. Immunohistochemical staining showed positive immunoreactivity for vimentin, cytokeratin, and epithelial membrane antigen, and negative immunoreactivity for muscle-specific antigen, melanoma, neural, and histiocytic markers. Electron microscopy excluded myogenic differentiation and showed that the filamentous cytoplasmic inclusions were composed of whorls of intermediate filaments. Aggressive chemotherapy with a combination of vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide phosphate was continued after exenteration. At 17 months' follow-up, orbital debulking surgery with externalization of the maxillary sinus was performed because of massive tumor recurrence in the right orbit and growth into the maxillary sinus. The child died 23 months after initial diagnosis from tumor invasion into the central nervous system. Extrarenal rhabdoid tumor is a rare orbital mass that carries a poor prognosis.     

Hydration structure of a collagen peptide

BACKGROUND: The collagen triple helix is a unique protein motif defined by the supercoiling of three polypeptide chains in a polyproline II conformation. It is a major domain of all collagen proteins and is also reported to exist in proteins with host defense function and in several membrane proteins. The triple-helical domain has distinctive properties. Collagen requires a high proportion of the post-translationally modified imino acid 4-hydroxyproline and water to stabilize its conformation and assembly. The crystal structure of a collagen-like peptide determined to 1.85 Angstrum showed that these two features may be related. RESULTS: A detailed analysis of the hydration structure of the collagen-like peptide is presented. The water molecules around the carbonyl and hydroxyprolyl groups show distinctive geometries. There are repetitive patterns of water bridges that link oxygen atoms within a single peptide chain, between different chains and between different triple helices. Overall, the water molecules are organized in a semi-clathrate-like structure that surrounds and interconnects triple helices in the crystal lattice. Hydroxyprolyl groups play a crucial role in the assembly. CONCLUSIONS: The roles of hydroxyproline and hydration are strongly interrelated in the structure of the collagen triple helix. The specific, repetitive water bridges observed in this structure buttress the triple-helical conformation. The extensively ordered hydration structure offers a good model for the interpretation of the experimental results on collagen stability and assembly.     

Model structure of decorin and implications for collagen fibrillogenesis

The three-dimensional structure of human decorin, a secreted proteoglycan involved in the regulation of collagen fibrillogenesis and cellular growth, has been modeled based on the crystal structure of the porcine ribonuclease inhibitor. Both proteins contain leucine-rich repeats and share 18% identical residues. This model structure of decorin has an arch shape with the single glycosaminoglycan chain and the three N-linked oligosaccharides located on the same side of the molecule. Decorin was modeled as binding to a polar sequence of collagen type I found in the d band. The inner concave surface is the appropriate size and shape to accommodate only one collagen triple helix of approximately 3 nm in length. The binding of one collagen triple helix to decorin is proposed to play a major role in the formation of the staggered arrangement of collagen molecules within the microfibrils by preventing lateral fusion of collagen molecules.     

新型胶原支架材料的结构特征与性能

为了克服猪脱细胞真皮基质作为组织工程支架材料渗透性差、降解速度过慢、免疫原性较强等缺点,采用多种化学、生物与物理综合方法处理猪皮制备了一种新型天然胶原支架材料,通过光学显微镜、扫描电镜观察以及体外降解时间、透水汽性、拉伸强度、孔隙率、收缩温度等的测定,对其性能进行了研究.实验结果显示:支架中的成纤维细胞、脂肪细胞及组织纤维间质完全去除,胶原纤维得到了松散,并维持其原有的天然三维网络多孔结构;该材料透水汽性处于3000g·m^-2·d^-1左右,适合创面恢复;体外降解时间处于25~50h之间,并可根据需要调整工艺条件控制降解时间;拉伸强度介于10.20~11.50MPa之间,具有良好的拉伸强度;收缩温度介于70~85℃之间.上述结果表明该材料已解决了猪脱细胞真皮基质渗透性差、降解速度过慢的缺点,并且其透气性和拉伸强度高、降解性优良且可控,符合组织工程支架材料的要求.     

Crystal structure analysis of collagen model peptide (Pro-pro-Gly)10

Single crystals of (Pro-Pro-Gly)10 were grown by the hanging drop method. The crystals diffracted to a resolution of 1.8 A. In the crystals the polypeptides form triple helices that aggregate end-to-end mediated by the solvent molecules, with the basic repeat being 20 A along the helical axis. Analysis of the 20 A structure of (Pro-Pro-Gly)10 using data up to a resolution of 1.9 A revealed that the overall structure is in accordance with the 7/2 model proposed for collagen. The three strands are held together by the (Gly) N-H O (Pro-X) hydrogen bond interactions, and additional stability is provided by the (Pro-Y) Calpha -H O (Pro-X) hydrogen bonding interactions.     

The human genome--chromosome 10 and the collagen genes

In relation to locuses of the 10th chromosome at present the following are in the focus of interest: tumours of endocrine glands, medullary carcinoma of the thyroid gland (MTC) and multiple endocrine neoplasias (MEN). It seems that the unifying basis is the oncogene RET, responsible for the development of Hirschsprung's disease HSCR. The authors mentions also metabolically important locuses for choline acetyltransferase (CHAT), uriporphyrinogen synthase (UROS) and methyl guanine methyltransferase (MGMT). A special paragraph is devoted to a list of collagenous genes COL1-COL18 and diseases associated with them.     

Type VIII collagen

Type VIII collagen is a product of endothelial cells, keratinocytes, mast cells, microvascular endothelial cells and some tumour cells. It is also present in a variety of extracellular matrices as diverse as sclera, skin and glomerulus. Type VIII molecules have a proposed chain composition of [alpha 1(VIII)2 alpha 2(VIII)]. While the function of collagen type VIII is uncertain recent work has highlighted the importance of this collagen in the vasculature. Particularly significant may be its up-regulation in smooth muscle cell migration and potential role in maintaining the smooth muscle cell phenotype. It is interesting to speculate that this collagen may provide a substratum for a variety of cells and facilitate movement of endothelial cells in angiogenesis, smooth muscle cells in intimal invasion and myofibroblasts in fibrotic conditions.     

The importance of collagen in the biological value of meat

Rat experiments showed that increases in the levels of collagen from 6.1 to 15.5% of the total content of meat proteins enhanced the growth and weight indices of animals. The differences in the biological value of the above quantity of collagen for man and rats were attributable to different physiological requirements for the quality of protein. Therefore, the findings in man are in agreement with those in rats.     

The polarization microscopy characteristics of collagen in dura mater transplants

In order to establish the polarizing microscopical characteristics of collagen in preserved dura mater transplants and in controls, a study was performed. Attempt was made for an intrpretation of velocity of collagen rehydratation by means of data of total anisotropy. The results show a faster rehydratation of collagen fibrils in control (unpreserved) dura mater. Changes in the velocity of rehydratation of collagen fibrils in lyophilized dura mater were connected with structural changes during lyophilization. The most appropriate period of time for rehydratation of lyophilized dura mater transplant was determined from the results of the polarizing microscopical study.     

The cognitive structure of a social structure.

"A paired-associate learning experiment was performed in which men's names were the stimuli and the labels freshman, sophomore, junior, and senior were the responses… . Ss learned most rapidly to apply the labels freshman and senior correctly, a result that was interpreted as end-anchoring. The errors… Ss made during learning were patterned in such a way as to demonstrate generalization gradients for each label. On the supposition that the frequency of confusions of labels was inversely related to the psychological distance between them, it was determined that psychologically the labels fell on a single dimension, and that the distance from freshman to sophomore was largest… from sophomore to junior, shortest… from junior to senior of intermediate size." (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The role of type III collagen in spontaneous cervical arterial dissections

OBJECTIVE: To determine the efficacy of lymphadenectomy after nephroureterectomy in patients with transitional cell carcinoma (TCC) of the upper urinary tract. PATIENTS AND METHODS: Between January 1986 and December 1995, 72 patients (mean age 67 years, range 45-82) underwent nephroureterectomy for primary TCC of the upper urinary tract. In 35 patients, a lymphadenectomy was also performed. The clinicopathological data were analysed retrospectively, focusing on the significance of lymphadenectomy. RESULTS: Lymph vessel invasion was found in 28 patients and its incidence was closely correlated with both tumour grade and pathological stage. Of the 35 patients who underwent lymphadenectomy, lymph node metastases were found in 13 patients, all of whom had lymph vessel invasion. There was no significant difference in the survival rate between patients with and without lymphadenectomy; however, among the 44 patients with no lymph vessel invasion, the survival rate of those with lymphadenectomy was significantly higher than in those without (P<0.05). CONCLUSION: Lymphadenectomy may provide a therapeutic advantage in patients with upper urinary tract TCC and no lymph vessel invasion. However, patients with lymph vessel invasion seem to have systemic disease; therefore, aggressive systemic adjuvant therapies rather than regional lymphadenectomy should be applied in these patients.     

The dynamics of collagen deposition in liver damaged by CCl4

The rates of synthesis and catabolism of collagen are determined in the livers of normal rats and of rats treated with carbon tetrachloride. Several experimental conditions are applied with the intention of investigating the dynamic processes separately, in order to determine the relevant values. The deposition of collagen in liver after damage by CCl4 is a consequence of the reduction of turnover with a simultaneous increase in collagen-production. Together with the increase in synthesis, which seems to be a consequence of a numerical increase in cells, there is a reduction in catabolism of collagen: the more severe the damage, the more marked is the decrease of specific catabolism. Collagen-deposition in the liver is thus the result of accumulation. The results indicate the possibility that collagen-catabolism is effected by enzymes produced by adjacent parenchymal cells, so that their destruction enhances the accumulation of collagen material. The findings mentioned above are discussed in the context of the general criteria of scar-formation and the repair of damaged tissues.     

The structure of alpha-keratin

Known structural principles (close packing, maximum hydrogen bonding, the tendency of like groups to be surrounded in like manner, and the approximate constancy of interatomic distances and bond angles) are used, with meridional and equatorial x-ray data, to deduce and check the structure pattern for alpha-keratin. Internally hydrogen-bonded polypeptide helices are grouped into "3-stacks", in which each chain is rotated and shifted vertically a distance equal to the helix pitch (5.15 A, average), relative to the other two. This shift accounts simply for the meridional x-ray reflection at this spacing. The 3-stack structure repeats after three turns, except for differences in the R groups and a slight twist, required to give satisfaction of both intrachain and interchain forces. The 3-stacks are grouped into 9-stacks and these into 27-stacks (all twisting), giving a crystallographic unit containing 81 chains, with the chain axes spaced approximately like those of close-packed cylinders. When the twisting reaches the limit of stability for good interchain contacting and cross-linking, the residue/turn ratio in each chain helix shifts to another, with twisting in the opposite direction. The twisting reversal mechanism keeps all the helix axes approximately straight, parallel, and in a close-packed arrangement. Interchain distances and orientations are suitable for cystine cross-linking. The dimensions of the 27-stacks agree well with estimates of the "effective radius" of microfibrils. X-ray measurements of spacing changes during fiber extension are explained as due to alternation of zones with much cross-linking and zones with few cross-links.