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Biomolecular Recognition and Self-Assembly: from DNA-DNA Interactions to Connective Tissue Pathologies

Sergey Leikin, PhD, Head, Section on Physical Biochemistry
Nydea Aviles, BS, Postbaccalaureate Fellow
Wynee Lee, BS, Postbaccalaureate Fellow
Se Jin Han, BS, Postbaccalaureate Fellow
Elena N. Makareeva, PhD, Postdoctoral Fellow
Edward L. Mertz, PhD, Staff Scientist
Juan Carlos Vera, BS, Postbaccalaureate Fellow

In living organisms, recognition and self-assembly reactions are some of the most fundamental molecular processes responsible for, among others, folding, interactions, and aggregation of proteins and nucleic acids. We laid the foundation for our work by studying basic principles relating the structural organization of helical macromolecules, specifically DNA and collagen, to their interactions and biological function. We continue to build our understanding of DNA-DNA interactions, particularly recognition and pairing between intact, double-stranded DNAs with homologous sequences. However, we have shifted the major focus of our research to collagen and other extracellular matrix proteins and proteoglycans. We investigate their role in cancer, fibrosis, osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplasias, osteoporosis, and other diseases involving connective tissues. Together with NICHD and extramural clinical scientists, we strive to improve our knowledge of the molecular mechanisms contributing to these diseases. We hope to use the resultant knowledge for diagnostics, characterization, and treatment, bringing our expertise in physical biochemistry and theory to clinical research and practice.

Structural stability of type I collagen triple helix and its role in osteogenesis imperfecta (OI)

Type I collagen is a triple-helical protein that forms the stable matrix of bone, skin, and other tissues. We discovered, however, that, at body temperature, the equilibrium state of collagen as well as its procollagen precursor is a random coil rather than the triple helix. Even in a crowded environment mimicking the endoplasmic reticulum (ER), procollagen triple helix folding occurs only below 35°C. Cells use specialized chaperones to fold procollagen within the ER. After secretion from cells and cleavage of N- and C-propeptides, collagen helices assemble into fibrils, in which they are protected from complete unfolding. Nevertheless, local unfolding/refolding of the triple helix occurs even in fibrils and plays an important physiological role.

All known mutations responsible for severe forms of OI affect the structure and/or folding of type I collagen. The majority of these mutations are substitutions of a single glycine in the obligatory (Gly-X-Y)n sequence of the triple helix, where the X and Y positions are often occupied by Pro and Hyp residues, respectively. By disrupting the stability and folding of the triple helix, Gly substitutions result in abnormalities in collagen production, secretion, and fibril formation. Our studies revealed that the effect of a Gly substitution on collagen stability is determined by the structural region within which the substitution is located rather than by the type of substituting residue. From analysis of published studies of different collagen-mimetic peptides and our own studies of collagens from OI patients with over 50 different Gly substitutions, we found evidence for several distinct structural regions within the triple helix (Makareeva et al.). For instance, the first 85-90 amino acids at the N-terminal end of the triple helix form an “N-anchor” region with higher than average triple helix stability. Gly substitutions within this region disrupt the whole N-anchor, preventing normal cleavage of the adjacent N-propeptide. Incorporation of collagen with uncleaved N-propeptides into fibrils leads to a distinctive OI/EDS phenotype with hypextensibility and joint laxity more characteristic of the Ehlers-Danlos syndrome (EDS). Disruptions of a stable “C-anchor” region at the other end of the molecule delay initiation of the triple helix folding, potentially explaining predominantly lethal outcomes of α1(I) Gly substitutions within this region.

During the last year, we focused on a large region surrounding the mammalian collagenase cleavage site, in which all known substitutions of α(I) glycine residues are lethal. We found that this region contains two low-stability, flexible sub-regions separated by a "clamp" with higher triple helix stability. Disruptions of this clamp by Gly substitutions result in triple helix unwinding propagating through many adjacent Gly-X-Y triplets, dramatically increasing the susceptibility to cleavage by collagenases. Such extensive disruptions may be an important factor in increased disease severity.

Translational studies of patients with novel/unusual OI mutations

In addition to Gly substitutions, we characterized several novel types of mutations that disrupt the structure and folding of type I collagen and result in severe OI. These studies began with observations of unusual mutations and/or phenotypes in OI patients by our intramural and extramural clinical collaborators. In particular, we assisted clinical scientists from the Bone and Extracellular Matrix Branch (BEMB) at NICHD in discovery and characterization of two new types of severe recessive OI associated with deficiencies in the cartilage-associated protein (CRTAP) and prolyl-3-hydrohylase (P3H1). We demonstrated that CRTAP and P3H1 are essential for normal folding of type I procollagen in vivo. The lack of these proteins accelerates synthesis of type I procollagen chains but delays folding of these chains, causing overhydroxylation and overglycosylation of Lys residues and accumulation of up to five times the normal amount of collagen within the cells. The excessive collagen accumulation in the ER likely leads to ER stress, contributing to the severe/lethal outcome in the patients.

During the past year, we assisted clinical scientists from the University of Washington in investigating several OI cases with Cys and Leu substituting for Arg-780 in the α1(I) chain. Substitutions at the X or Y position in the Gly-X-Y repeat within the triple helix, mostly of Cys for Arg, were recently identified in individuals with OI, osteopenia, arterial rupture, EDS, and Caffey disease. Given that non-glycine substitutions were not considered detrimental for triple helix folding and structure, the molecular pathophysiology of these conditions remained unclear. In 2007, we reported a change in the triple helix stability caused by a Cys substitution for an Y–position Arg-888 in patients with a combination of OI and EDS symptoms. Similar stability changes in Cys and Leu substitutions for the Y-position Arg-780 suggested that triple helix destabilization was caused by the loss of an Y-Arg rather than by the gain of a Cys residue not normally present in the triple helix. The Cys gain in the X- or Y- positions might be responsible for the arterial rupture and EDS phenotypes. Indeed, we observed formation of aberrant disulfide bonds between the Cys residues in molecules with two mutant α1(I) chains. These bonds resulted in kinking, register shift, and abnormal N-propeptide cleavage from the triple helix; the latter effect is known to be involved in EDS.

Murine OI models

Murine models offer unique opportunities for systematic studies of the molecular mechanisms and development of treatment strategies for OI. We work with all three existing murine OI models, including the oim mouse with nonfunctional α2(I) chains, the Brtl mouse with a knock-in G349C substitution in the α1(I) chain, and a mouse with a knock-in G610C substitution in the α2(I) chain (Daley et al.). Our studies revealed decreased stability of type I collagen in G610C animals, increased stability in oim animals, and abnormal collagen-collagen interactions in both of these models. However, in Brtl animals, which exhibit a more severe phenotype, we found no significant abnormalities in the stability or interactions of type I collagen. Instead, we discovered selective retention and intracellular degradation of molecules with a single mutant chain, resulting in ER stress and malfunction of osteoblasts. Based on our advances in high-definition infra-red and Raman microspectroscopic imaging technology, we found similar changes in the extracellular bone matrix in all these models despite very different defects in collagen biosynthesis. We observed less regular organization of the matrix, higher mineral content, and lower collagen content than in matched normal controls. Given the nature of mechanical stresses in bone, the latter abnormalities are likely to increase bone fragility, contributing to or determining the disease phenotype. At least in these three models, disease severity may be related to osteoblast malfunction, caused by abnormal folding and secretion of mutant collagen molecules, rather than to abnormal interactions of secreted molecules in the extracellular matrix. However, more studies are needed to verify our hypothesis.

In the past year, we focused on testing gene and cell therapy strategies for OI treatment in the Brtl mouse model. One potential approach would be to degrade the mRNA transcribed from the mutant collagen allele by introducing a gene encoding the corresponding ribozyme. BEMB/NICHD scientists generated transgenic animals, in which such a ribozyme was expressed in osteoblasts. However, our analysis of bones from these animals did not reveal a significant reduction in the content of mutant collagen molecules in the bone matrix. We also found no substantial improvements in collagen content or bone matrix mineralization, suggesting that the achieved ribozyme expression was not sufficient to degrade a significant fraction of mRNA from the mutant allele. In our opinion, the cell therapy approach is showing more promise so far. Our colleagues from Pavia University in Italy performed intrauterine transplantation of bone marrow stromal cells, which reduced perinatal lethality and significantly improved the bone strength in Brtl mice. However, it remains unclear how 1-2% engraftment of the donor cells achieved in these animals could result in the observed improvements. Generally, low donor cell engraftment is considered to be one of the major challenges in stem cell therapy. Our study revealed that the donor cells, constituting just 2% of osteoblasts, were responsible for synthesis of about 20% of bone matrix as well as dramatic improvements in the matrix composition and mineralization, explaining the observed outcomes and raising hopes for future clinical applications of this strategy (Panaroni et al.).

Collagenase-resistant isoform of type I collagen in cancer and fibrosis

The normal isoform of type I collagen is a heterotrimer of two α1(I) and one α2(I) chains. However, homotrimers of three α1(I) chains were found in carcinomas and fibrotic tissues as well as in rare forms of OI and EDS associated with α2(I) chain deficiency. Our studies revealed less favorable interactions between the homotrimers, partial segregation of the homo- and heterotrimers within fibrils (Han et al.), and increased stiffness of homotrimer-rich fibrils. More recently, we found the homotrimers to be at least 5-10 times more resistant to cleavage by mammalian collagenases (MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14) than the heterotrimers. We demonstrated that increased stability of the homotrimer hinders unwinding of the triple helix at the MMP cleavage site, preventing proper positioning of unwound chains within the enzyme catalytic cleft.

We hypothesized that synthesis of collagenase-resistant fibrils may play an important role in cancer invasion. Massive production of collagenases is a hallmark of invasive cancers. Synthesis of collagenase-resistant fibrils may enable cancer cells to clear the invasion path by degrading the host tissue matrix and collagen produced by recruited fibroblasts, while protecting the matrix necessary for their own proliferation and migration. Our measurements during the last year supported this hypothesis. We tested nine human cell lines from four types of metastatic cancers, which all produced 20-40% homotrimeric type I collagen in culture and an even higher fraction of homotrimers in xenograft tumors in mice. We found that the more rigid homotrimeric type I collagen matrix supported faster proliferation and migration of cancer cells in culture. Analysis of xenograft tumor collagen revealed that the homotrimers were produced in vivo only by the engrafted cancer cells—not by normal mesenchymal cells or fibroblasts recruited into the tumors.

In addition to their presence in carcinomas, type I collagen homotrimers were reported in a variety of fibrotic tissues. We developed and validated a specific assay for the type I homotrimers, which revealed that previous, less specific assays might produce unreliable results for highly cross-linked tissue collagens. So far, our studies based on this new assay did not confirm the presence of the homotrimers in mesenchymal tissue fibrosis, e.g., scleroderma. However, we found a large fraction of the homotrimers in non-mesenchymal fibrosis, e.g., in various animal models of glomerular sclerosis. At least in the case of glomerular sclerosis, our data suggest that the source of the pathology may indeed be homotrimer synthesis by mesangial cells, resistance of the homotrimer fibers to collagenases, and the resulting accumulation of these fibers. Currently, we are investigating the regulation of the homotrimer synthesis by various cells and developing approaches to selectively target the homotrimers for potential diagnostic and treatment applications in cancer and fibrosis.

High-definition infrared and Raman micro-spectroscopy of tissues: cartilage pathology in diastrophic dysplasia

Label-free micro-spectroscopic infrared and Raman imaging of tissues and cell cultures provides important information about chemical composition, organization, and biological reactions inaccessible by more traditional histological techniques. However, applications of these promising technologies were severely restricted by the limited accuracy of micro-spectroscopic imaging of soft, hydrated specimens under physiological conditions. In the past several years, we resolved this problem by designing special specimen chambers with precise thermo-mechanical stabilization, reducing the light path instabilities and improving the spectral reproducibility by over two orders of magnitude.

In addition to the studies of bone mineralization discussed above, we applied this newly developed high-definition (HD) technology to an analysis of cartilage pathology in a mouse model of dyastrophic dysplasia (DTD). Undersulfation of cartilage proteoglycans caused by mutations in the SLC26A2 sulfate/chloride antiporter is believed to be responsible for severe progressive cartilage degradation and skeletal deformities in DTD patients. In DTD mice, the net sulfation is only slightly reduced at birth and normalizes with age. However, DTD articular cartilage progressively degrades with age, and bones develop abnormally. Our HD-infrared imaging revealed strong chondroitin undersulfation only within narrow regions of growth plate and articular surface but normal sulfation elsewhere in articular cartilage from newborn DTD animals. The undersulfation disrupted a thin layer of well-oriented collagen fibers at the articular surface, a layer that is normally present in newborn mice. The malformation of this layer, which protects cartilage from mechanical damage and synovial enzymes, may explain the progressive degradation of cartilage with animal age despite normalization of chondroitin sulfation. In the growth plate, the undersulfation also resulted in disorganization of collagen fibers, potentially contributing to abnormal bone growth and osteoporosis in DTD mice. Apparently, proteoglycan undersulfation affects collagen fiber formation and/or chondrocyte functions, but the mechanisms underlying these effects remain unclear.

Theory and measurements of sequence-dependent DNA-DNA interactions

For years, interactions between double stranded (duplex) DNA molecules were presumed to be independent of the DNA structure and base pair sequence because the nucleotides are buried inside the double helix and shielded by the highly charged sugar-phosphate backbone. However, accumulating experimental evidence suggested that this intuitive concept might be wrong, i.e., sequence-dependent structure of the sugar phosphate backbone might be important for DNA-DNA interactions (Wyveen et al.). To account for this structure, over the last decade we have been developing a theory of electrostatic interactions between macromolecules with helical patterns of surface charges. This theory suggested explanations for the observed counter-ion specificity of DNA condensation, changes in DNA structure upon aggregation, and multiple packing arrangements observed in DNA aggregates, for example.

Most importantly, our theory predicted that electrostatic DNA-DNA interactions might result in direct recognition of sequence homology and pairing between 100-bp or longer DNA duplexes. Pairing of intact, homologous DNA duplexes was postulated to precede double strand breaks and subsequent events in homologous recombination of DNA in vivo, but the mechanism of the pairing remained unknown. Our recent in vitro experiments confirmed pairing of homologous DNA sequences in a protein-free environment within liquid-crystalline aggregates, in which the density of DNA is comparable to that found within the cell nucleus. The extent of the pairing was consistent with our theoretical expectations. Currently, we are refining the theory, e.g., by more accurately accounting for thermal fluctuations, DNA bending, and imperfections in DNA structure. At the same time, we are conducting more detailed experimental studies, specifically to test various theoretical predictions for the molecular mechanism of the observed pairing.

Additional Funding

NIH Director’s Challenge Award, 2008

Publications

Makareeva E, Mertz EL, Kuznetsova NV, Sutter MB, DeRidder AM, Cabral WA, Barnes AM, McBride DJ, Marini JC, Leikin S. Structural heterogeneity of type I collagen triple helix and its role in osteogenesis imperfecta. J Biol Chem 2008 283:4787-4798.
Daley E, Streeten EA, Sorkin JD, Kuznetsova N, Shapses SA, Carleton SM, Shuldiner AR, Marini JC, Phillips CL, Goldstein SA, Leikin S, McBride DJ. Variable bone fragility associated with an Amish COL1A2 variant and a knock-in mouse model. J Bone Miner Res 2009 [E-pub ahead of print].
Panaroni C, Gioia R, Lupi A, Besio R, Goldstein SA, Kreider J, Leikin S, Vera JC, Mertz EL, Perilli E, Baruffaldi F, Villa I, Farina A, Casasco M, Cetta G, Rossi A, Frattini A, Marini JC, Vezzoni P, Forlino A. In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knock-in murine model for classical, dominant osteogenesis imperfecta. Blood 2009 114:459-468.
Han S, McBride DJ, Losert W, Leikin S. Segregation of type I collagen homo- and heterotrimers in fibrils. J Mol Biol 2008 383:122-132.
Wynveen A, Lee DJ, Kornyshev AA, Leikin S. Helical coherence of DNA in crystals and solution. Nucleic Acids Res 2008 36:5540-5541.

Collaborators

Geoff S. Baldwin, PhD, Imperial College, London, UK
Aileen M. Barnes, MS, Bone and Extracellular Matrix Branch, NICHD, Bethesda, MD
Nicholas J. Brooks, PhD, Imperial College, London, UK
Peter H. Byers, MD, University of Washington, Seattle, WA
Wayne A. Cabral, AB, Bone and Extracellular Matrix Branch, NICHD, Bethesda, MD
Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
Alexei A. Kornyshev, PhD, Imperial College, London, UK
Dominic J. Lee, PhD, Imperial College, London, UK
Wolfgang Losert, PhD, University of Maryland, College Park, MD
Anna Lupi, PhD, Università degli Studi di Pavia, Pavia, Italy
Joan C. Marini, MD, PhD, Bone and Extracellular Matrix Branch, NICHD, Bethesda, MD
Daniel J. McBride, Jr., MD, PhD, University of Maryland School of Medicine, Baltimore, MD
Hideaki Nagase, PhD, Imperial College, London, UK
James M. Pace, PhD, University of Washington, Seattle, WA
Charlotte L. Phillips, PhD, University of Missouri, Columbia, MO
Antonio Rossi, PhD, Università degli Studi di Pavia, Pavia, Italy
John M. Seddon, PhD, Imperial College, London, UK
Ruggero Tenni, PhD, Università degli Studi di Pavia, Pavia, Italy
Thomas E. Uveges, PhD, Bone and Extracellular Matrix Branch, NICHD, Bethesda, MD
Robert Visse, PhD, Imperial College, London, UK
Aaron Wynveen, PhD, Imperial College, London, UK

Contact

For more information, email leikins@mail.nih.gov or visit physbiochem.nichd.nih.gov.

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