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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
Lynn Felts, BS, Predoctoral Fellow (Graduate Student)
Aaron Konopko, BA, Postbaccalaureate Fellow
Wynee Lee, BS, Postbaccalaureate Fellow
Elena N. Makareeva, PhD, Postdoctoral Fellow
Edward L. Mertz, PhD, Staff Scientist

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 other 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 folds 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.

Most 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 various 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. 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 hyperextensibility 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 past two years, 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. This large-scale triple helix destabilization may be particularly detrimental for procollagen folding, resulting in severe ER stress and malfunction of osteoblasts. Combined with other studies of the role of ER stress and osteoblast malfunction in OI, our findings might explain the lethal phenotype of α1(I) and increased severity of α2(I) Gly substitutions in this region.

Translational studies of patients with novel or unusual OI mutations

In addition to Gly substitutions, we characterized several novel types of mutations that disrupt the structure and/or folding of type I collagen and result in severe OI. In particular, we assisted clinical scientists from the Bone and Extracellular Matrix Branch (BEMB) at NICHD and clinical scientists from the University of Washington in investigating several OI cases with substitutions of Arg in the Y-position in the Gly-X-Y repeat within the triple helix. Specifically, we studied Arg-780 substitutions to Leu and Cys and an Arg-888 substitution to Cys in the α1(I) chain. These studies revealed that the loss of Y-position Arg residues is detrimental for procollagen triple helix folding, because it lowers the triple helix stability and prevents binding of the molecular chaperone HSP47 at the substitution site. Deficient triple helix folding in these mutants may cause ER stress response and malfunction of osteoblasts, resulting in OI. The gain of a Cys residue, normally not present in the triple helix, may be responsible for the arterial rupture and EDS phenotypes in patients with Arg to Cys substitutions. For instance, 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.

During the past several years, we also assisted BEMB scientists in the discovery and characterization of three new forms of severe recessive OI associated with deficiencies in the cartilage-associated protein (CRTAP), prolyl-3-hydrohylase (P3H1) and cyclophilin B (CYPB). It was suggested that these molecules form a stable three-protein complex essential for procollagen folding in ER. We demonstrated that the lack of CRTAP and P3H1 accelerates the synthesis but delays folding of type I procollagen 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 severity of OI outcome.

During the past year, we focused on OI caused by a CYPB deficiency, in which no CYPB was translated by the patient's cells (Barnes et al.). Surprisingly, our studies suggested that CYPB might not be essential for CRTAP/P3H1 complex formation or procollagen folding. A normal procollagen folding rate in the absence of this most potent ER peptidylprolyl isomerase indicated that proline peptide bond isomerization might not be limiting the folding rate, contrary to the popular paradigm. Instead, our measurements suggested that CYPB deficiency might cause OI by altering procollagen trafficking from ER to Golgi. Further studies of this deficiency are currently under way.

Murine models of bone disorders

Murine models offer unique opportunities for systematic studies of molecular mechanisms of bone disorders. We work with all three existing murine OI models, including the oim mouse, which has nonfunctional α2(I) chains, the Brtl mouse which has 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 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 osteoblast ER stress and malfunction. Based on our advances in high-definition 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 also investigated bone pathology in the caudal vertebrae of mice with bone tumors caused by defects in protein kinase A, a crucial protein in cAMP signaling (Tsang et al.). We found that these tumors caused periosteal deposition of immature cortical bone, in which collagen matrix and mineral organization were intermediate between those expected in woven and lamellar bones. Accelerated matrix formation and deficient mineralization were reminiscent of the McCune–Albright syndrome, which is caused by deficient cAMP signaling associated with defects in the guanine binding protein Gs-α.

In addition to fundamental studies, mouse models also provide unique opportunities for developing novel treatments. In the last two years, we participated in 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 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 so far showing more promise. 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 was 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.

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, 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 (Makareeva et al.). 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 (Han et al.).

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 supported this hypothesis (Makareeva et al.). 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 have been 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 (Makareeva et al.). Analysis of normal and fibrotic tissues from different patients (e.g., scleroderma, uterine fibroids, and pheochromocytomas) and mouse models (e.g., glomerular sclerosis and endocrine tumors) suggested that the homotrimers are produced by undifferentiated, dedifferentiated, or transformed cells but not by normal or activated collagen-producing mesenchymal cells. 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 had been severely restricted by the limited accuracy of micro-spectroscopic imaging of soft, hydrated specimens under physiological conditions. Over 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 epiphyseal 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. To account for this structure, we have, over the past decade, been developing a theory of electrostatic interactions between macromolecules with helical patterns of surface charges. The theory suggested, for example, explanations for the observed counter-ion specificity of DNA condensation, changes in DNA structure upon aggregation, and multiple packing arrangements observed in DNA aggregates.

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.

Publications

Barnes AM, Carter EM, Cabral WA, Weis M, Chang W, Makareeva E, Leikin S, Rotimi CN, Eyre DR, Raggio CL, Marini JC. Lack of cyclophilin B in osteogenesis imperfecta with normal collagen folding. N Engl J Med. 2010;362:521-528.
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. 2010;25:247-261.
Han S, Makareeva E, Kuznetsova NV, DeRidder AM, Sutter MB, Losert W, Phillips CL, Visse R, Nagase H, Leikin S. Molecular mechanism of type I collagen homotrimer resistance to mammalian collagenases. J Biol Chem. 2010;285:22276-22281.
Makareeva E, Han S, Vera JC, Sackett DL, Holmbeck K, Phillips CL, Visse R, Nagase H, Leikin S. Carcinomas contain a matrix metalloproteinase-resistant isoform of type I collagen exerting selective support to invasion. Cancer Res. 2010;70:4366-4374.
Tsang KM, Starost MF, Nesterova M, Boikos SA, Watkins T, Almeida MQ, Harran M, Li A, Collins MT, Cheadle C, Mertz EL, Leikin S, Kirschner LS, Robey P, Stratakis CA. Alternate protein kinase A activity identifies a unique population of stromal cells in adult bone. Proc Natl Acad Sci USA. 2010;107:8683-8688.

Collaborators

Peter H. Byers, MD, University of Washington, Seattle, WA
Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
Kenn Holmbeck, PhD, Craniofacial and Skeletal Diseases Branch, NIDCR, Bethesda, MD
Alexei A. Kornyshev, PhD, Imperial College, London, UK
Phyllis C. Leppert, MD, PhD, Duke University School of Medicine, Durham, NC
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
Karel Pacak, MD, PhD, DSci, Program on Reproductive and Adult Endocrinology, NICHD, Bethesda, MD
Charlotte L. Phillips, PhD, University of Missouri, Columbia, MO
Antonio Rossi, PhD, Università degli Studi di Pavia, Pavia, Italy
Dan L. Sackett, PhD, Program on Physical Biology, NICHD, Bethesda, MD
Constantine A. Stratakis, MD, DSci, Program on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD

Contact

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

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