¹Rajarata University of Sri Lanka

Ayurveda is a system of complementary and alternative medicine which originated more than 3500 years ago and is practiced widely in India and Sri Lanka. In Sri Lanka, CAM medicine is dominated by practitioners who practice both Ayurveda and Deshiya Chikithsa; an indigenous system of medicine unique to Sri Lanka [7]

Contrary to the common belief that its pharmacopeia was developed by trial and error over a period of time, Ayurveda is based on a sound philosophical and logical basis. Although not immediately lucid to those trained in conventional science, knowledge of the fundamentals of Ayurveda is required to understand how the system works in the human body. When clinical trials are planned in disciplines such as Ayurveda it is important for the researcher to understand the incommensurability of theories of Ayurveda and western medicine. Not appreciating this will lead to planning studies in conventional formats which are incompatible with principles of Ayurveda. This has already contributed to a poor evidence base in Ayurveda contrary to widely held perceptions of its efficacy [7]. Following are some reasons why a different approach to clinical research and trials is needed when investigating Ayurvedic medicine compared to conventional medical research.

In Ayurveda, approach to treatment, understanding and determining the prakurthi or temperament (constitution) of the patient is a prerequisite to starting treatment. Ayurveda treatment will vary depending on the patient’s temperament. Prakurthi of a patient is a composite outcome of vata, pitta and kapha (these are the three balancing forces or doshas in the body) states of the body. These balancing forces will be dependent on the patients’ general prakurthi at birth as well as subsequent effects caused by patients’ behaviour and interactions with the external environment [1].

According to Ayurveda it is important to understand whether the disease is in ama or nirama state as the treatment may vary depending on it. For example applying herbal oil could be used as a treatment in osteoarthritis in nirama state but would worsen the disease if applied in ama state. It is difficult to explain what ama and nirama are in conventional medical terms. However ama state could be considered as an acute inflammatory state and nirama as disease in remission [2].

A disease could have sub variants according to their aetiology and association with predominant dosha. In haemorrhoids or arshas there are different forms such as sahaja (congenital) and janmottaraja (arising after birth). Janmottaraja is further subdivided into vathaja, piththaja, kapaja, thridosaja (blood) [3].Treatment varies depending on different subcategories. Identification of a distinct genomic and metabolomic profile of rheumatoid arthritis classified according the Chinese medical system points to the possible validity of these Ayurvedic classifications [4].

In conventional research methodology the participants are selected according to standard diagnostic criteria for RCT. However these participants are not suitable for uniform intervention according to the principles of Ayurveda. As Ayurvedic treatment will widely differ according to prakurthi, amanirama and the doshaja state. Similarly the pharmacogenomic approach also targets patients according to their genotypes and RCTs have struggled to keep pace with this [5].

Ayurveda also deals with diseases in a holistic approach. It will not only prescribe a medicine (aushada) but will advocate on certain behaviors (viharana) and dietary regimes (ahara). These interventions will also be adjusted according to age, mental state (satva, rajas and thamas- three states of mind) and the season (climate and other environmental factors) etc. Usually Ayurvedic approach to treatment can be divided into two broader segments. They are shodhana (purificatory or conditioning) and shamana (restorative) treatment. In RCTs both components should be used in interventions. Otherwise the desired outcome cannot be achieved. Shodhana includes procedures of panchakarma such as nasya (nasal instillation of oils), vamana (emesis), virechana (purgatives), rakthamokshana (blood letting), and vasthi (enema) [1]. Obviously panchakarma is immensely difficult to conceal in RCTs.

Furthermore, the taste and smell of Ayurvedic drugs makes it difficult to produce placebos. According to Ayurveda, taste (rasa) will have a particular effect on the body which will contribute to healing. Hence the same taste (rasa) in the placebo could also bring a similar effect to a certain extent. Hence use of placebos and blinding are incompatible with research that is planned in-keeping with fundamentals of Ayurvedic approach.

The assessment of outcomes also needs to be tailor-made to suit the fundamentals of Ayurveda. Ayurvedic treatment sometimes aims to achieve a balanced state of doshas. Such factors will have to be assessed in the outcome of RCTs in addition to the conventional outcomes.

Ayurvedic treatment changes according to the disease state and factors such as climate among many other things. This dynamic therapeutic approach also needs to be considered when planning the intervention arm of RCTs. Therefore conventional study designs may be inappropriate when doing clinical research in CAM. “In such circumstances, the choice of study design should be discussed on a case-by-case basis” with experts in fundamentals of CAM systems [6]. Methodologies such as single case design, black box design, and open label design, ethnographic and observational studies may be more appropriate for clinical research in Ayurveda [6].

Acknowledgement
Samuel Franzen Nuffield Department of Medicine, University of Oxford.

References

1. Sharma RK, Dash B: Agnivesa’s Caraka Samhita text with English translation and critical exposition based on Cakrapani Datta’s Ayurvedha Dipika. Chowkhamba Sanskrit Series Office Varanasi India 1-5; 1977-1997.
2. Dwarakanatha K: Introduction to kayachikitsa. Chaukhambha Orientalia Varanasi; 1986.
3. Vagbhata’s Astanga Hrdayam Vol 2. Translated by KR Srikantha Murthy Krishnadas academy Varnasi; 1998: Page 66.
4. Van Wietmarschen H, Yuan K, Lu C, Gao P, Wang J, Xiao C, Yan X, Wang M, Schroën J, Lu A, et al: Systems Biology Guided by Chinese Medicine Reveals New Markers for Sub-Typing Rheumatoid Arthritis Patients. J. Clin. Rheumatol 2009,15: 330–337.
5. Liu L, Leung E L-H, Tian X:The clinical trial barriers. The Nature 2011, 480: S100doi:10.1038/480S100a.
6. World Health Organization: General Guidelines for methodologies on research and evaluation of traditional medicine. Geneva, WHO;2000.
7. www.wikipedia.org/wiki/Ayurveda

DOI: 10.1016/j.coph.2013.02.007

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology

¹Inst. for Bioscience and Biotechnology Research
²Dept. of Bioengineering
³Dept. of Chemistry and Biochemistry
University of Maryland

While most globular proteins populate relatively homogeneous conformational ensembles under physiological conditions, significant exceptions continue to emerge. Many biological processes involve extensive re-modeling of protein conformation, including switches from disordered to ordered states. Some natural proteins can even undergo large-scale transitions from one ordered state to another involving major shifts in secondary structure, repacking of the protein core, and exposure of new surfaces. Such “metamorphic” proteins [1] are capable of performing alternative functions triggered by binding interactions that stabilize latent conformational states [2, 3]. The ability of these proteins to completely change their fold topologies has implications in a number of important areas discussed here including computational and structural biology, protein evolution, human disease, and protein design.

The structures of protein sequences at the interface between folds are unpredictable to both ab initio and knowledge-based prediction algorithms, demonstrating that current knowledge of protein folding physics is incomplete [4]. Our previous studies on the G_A/G_B system demonstrated that protein structure can be encoded by a small number of essential residues, and that a very limited subset of intra-protein interactions can tip the balance from one fold (4b+a) to another (3a) [5, 6]. These observations reveal an additional layer of complexity in protein folding. While mutations often appear to have little affect on the overall structure, they can destabilize the original fold and simultaneously increase the propensity for new folds and functions. This may in part explain why alternative folds can sometimes have similar or more favorable energies in structure prediction algorithms than the target fold. Could such examples be indicators of switchable folds in some cases? Improved structure prediction algorithms may come from closer collaboration between computational biophysicists and experimentalists in the fold-switching field. For example, the large body of structure and stability data obtained in determining the mutational paths between the G_A and G_B folds may be useful in the calibration and development of more fine-grained protein structure prediction algorithms.

Understanding the capacity of a protein to acquire a completely different structure as a result of minor mutagenic perturbation is central to understanding how new protein structures and functions evolve. In the case of the G_A/G_B system, there are multiple short paths between the 3a and 4b+a folds and new function can become apparent even before the corresponding new fold is significantly populated [6] (Figure 1). This is because thermodynamic linkage between folding and binding allows the interaction energy of binding to stabilize conformations otherwise likely to be overwhelmed by the “standard” conformation. A plausible mechanism for evolutionary fold migration, involving either conformational switching of sub-domains in globular proteins or switching of duplicated regions in multi-domain systems, may illustrate a commonplace in vivo process. Recent examples support these ideas [7].

            Natural and engineered global fold switches share common characteristics that make switching more probable between some folds than others. For example, core residues in one fold that are largely solvent exposed in another fold allow significant folding information for both topologies to coexist in a single polypeptide and may make these 3D structures more prone to switching. Because the extent of hydrophobic core overlap between two arbitrary folds of similar size varies widely, compatible fold switch pairs may be readily identifiable. Some topologies (fold hubs) could be compatible with multiple switches such that folds might be interconverted by mutations to sequences encoding a common topology. These mutational connectivities in fold space create networks of probable fold migrations. Comparing experimentally determined networks to computationally derived networks [8] will improve prediction of switchable folds. Predicting potential fold switches may lead to new approaches for interpreting genetic polymorphisms, alternative splicing, trinucleotide-repeat disorders, the consequences of proteolysis, and other disease-related events.

Global fold switching may be related to observations of physicists and mathematicians studying the phenomenon of self-organizing critical states [9]. Large dynamical systems tend to organize themselves into critical states in which events that would ordinarily be uncoupled become correlated. Criticality seeks to explain why complex systems tend to move from one highly organized state to another through catastrophic events resulting from the accretion of small perturbations.

Finally, there may also be other practical benefits to furthering our understanding of fold switches. Until recently, the design of protein switches with potential therapeutic applications typically involved one of three types of conformational transitions – rigid-body, limited change, or unfolding-folding [10]. A fourth way, however, is the implementation of the global fold switch described here. While fold switching can occur through relatively short mutational paths, it is also clear that some sequences are bi-functional and that changes in global fold topology can be effected environmentally. This opens the door to the development of ligand-induced switches. From an applied standpoint, designing global protein switches that can flip between folds and functions may provide new ways to develop more specific biologic drugs where, for example, a function is masked until the target (such as a receptor) is reached. Other applications in the design of tunable nano-scale devices may also be possible. Overall, the ability of proteins to undergo large amplitude conformational change may have widespread impact in biology.

References

1. Murzin, A. G. (2008) Biochemistry. Metamorphic proteins. Science 320, 1725-1726.
2. Tuinstra, R. L., Peterson, F. C., Kutlesa, S., Elgin, E. S., Kron, M. A., and Volkman, B. F. (2008) Interconversion between two unrelated protein folds in the lymphotactin native state. Proc. Natl. Acad. Sci. USA 105, 5057-5062.
3. Luo, X., Tang, Z., Xia, G., Wassmann, K., Matsumoto, T., Rizo, J., and Yu, H. (2004) The Mad2 spindle checkpoint protein has two distinct natively folded states. Nat. Struct. Mol. Biol. 11, 338-345.
4. Allison, J. R., Bergeler, M., Hansen, N., and van Gunsteren, W. F. (2011) Current computer modeling cannot explain why two highly similar sequences fold into different structures. Biochemistry 50, 10965-10973.
5. Bryan, P. N., and Orban, J. (2010) Proteins that switch folds. Curr. Opin. Struct. Biol. 20, 482-488.
6. He, Y., Chen, Y., Alexander, P. A., Bryan, P. N., and Orban, J. (2012) Mutational tipping points for switching protein folds and functions. Structure 20, 283-291.
7. Roessler, C.G., Hall, B.M., Anderson, W.J., Ingram, W.M., Roberts, S.A., Montfort, W.R. and Cordes, M.H. (2008). Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds. Proc. Natl. Acad. Sci. USA 105, 2343-2348.
8. Cao, B., and Elber, R. (2010) Computational exploration of the network of sequence flow between protein structures. Proteins 78, 985-1003.
9. Bak, P., and Paczuski, M. (1995) Complexity, contingency, and criticality. Proc. Natl. Acad. Sci. USA 92,6689-6696.
10. Ha, J-H., and Loh, S. N. (2012) Protein conformational switches: From nature to design. Chem. Eur. J. 18, 7984-7999.

DOI: 10.1016/j.sbi.2013.03.001

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology

Matteo Fassan

¹University of Verona, Verona – Italy
²Sanofi, Cambridge (MA) – USA

The genesis of the current non-coding RNAs’ scientific paradigm can easily be traced to the seminal discovery of a microRNA (miRNA or miR) gene in 1993 [1]. At that time, no one could have predicted that the discovery of these endogenous small non-coding molecules in Caenorhabditis elegans would have such a profound effect on future research on cancer biology and therapy. Following the original observation, miRNAs were completely overlooked for nearly a decade, becoming culturally relevant only in the first years of the 21^st century, when a link between miRNAs’ dysregulation and human cancer (i.e., miR-15a and miR-16-1 genes) was reported for the first time to be involved in the pathogenesis of chronic lymphocytic leukemia [2]. Concurrently, the revision and experimental validation of the results obtained by the human genome project further pinpointed that many transcripts are actually non-coding transcripts, and that miRNAs represent the most important class of non-coding RNA molecules.

Very quickly, miRNAs became the most promising class of diagnostic, prognostic and predictive biomarkers in human cancer. From a therapeutic perspective, advances in the understanding of the molecular role of miRNAs in the pathological processes has significantly contributed to the identification of alternate molecular pathways which will would undoubtedly influence the selection of new therapeutic modalities [3]. In this exciting scenario, the translation into clinical practice of the discovery by Fire and Mello in 1998 of the process of RNA interference in vivo [4] poses an intriguing dilemma: can miRNAs be used as therapeutics per se?

The intrinsic characteristics that confers stability to miRNAs in vitro, allow a longer molecular/structural resistance and activity in vivo [5]. Preclinical models have consistently underlined the feasibility and efficacy of miRNA-based therapies, either alone or in combination with current targeted therapies. The appealing strength of such therapeutic option dwells in miRNAs ability to concurrently target multiple genes, frequently in the context of a specific network/pathway, making miRNA-based therapy extremely efficient in regulating distinct biological processes relevant to normal and pathological cell homeostasis [6]. There are two main therapeutic strategies to target miRNA expression: miRNA reduction and miRNA replacement (Figure 1) [7].

The use of oligonucleotides or virus-based constructs can either block the expression of an oncogenic miRNA or reintroduce the loss of expression of a tumour suppressor miRNA. A different approach is the use of drugs to modulate miRNA expression by targeting their transcription and their processing [6].

There are some fundamental issues, which have impeded development of miRNA-based treatments. First, we need to clearly demonstrate a tissue-specific delivery and develop a more efficient cellular uptake of synthetic oligonucleotides to achieve sustained target inhibition. This should result in significantly-enhanced patient benefits and reduced drug toxicity. In fact, the second and even more challenging problem to overcome is the biological instability of miRNAs in bodily fluids or tissues, as unmodified oligonucleotides are rapidly degraded by cellular and serum nucleases, requiring huge doses of drugs. As a result, various chemical modifications in oligonucleotides have been investigated, such as morpholinos, peptide nucleic acids, cholesterol conjugation and phosphorothioate backbone modifications. Among others, the locked nucleic acid (LNA) constructs provide the most promising results. LNA nucleosides are a class of nucleic acid analogues in which the ribose ring is “locked” by a methylene bridge connecting the 2′-O atom to the 4′-C atom. This feature confers to LNA oligonucleotides great advantages including: (i) High hybridization affinity towards complementary single-stranded RNA and complementary single-stranded or double-stranded DNA; (ii) Excellent mismatch discrimination, and (iii) High aqueous solubility. The so-called “LNA anti-miR” constructs have been successfully used in several in vitro and in vivo studies to knock-down the expression of specific miRNAs [8, 9]. This success has culminated in the first two miRNA-based clinical trials for the treatment of hepatitis C virus infection by targeting miR-122 with a LNA-antimiR (miravirsen or SPC3649; Santaris Pharma, Denmark) [8, 9]. The phase IIa clinical trial [9] has shown a dose-dependent, long reduction in HCV RNA that continues to fall after completion of treatment without any recorded serious adverse effects.

The discovery of exosome-specific miRNA circulation among bodily fluids provided the “Trojan horse” for the forthcoming development of miRNA delivery vehicles for systemic gene therapy: exosomes, as natural cell-derived nano-carriers, are immunologically inert and possess an intrinsic ability to cross biological barriers [10]. On the other hand, exosome-released miRNAs represent a novel mechanism of cross-talk and genetic exchange between cells. Interestingly, cancer-released exosomes have been shown to carry oncogenic miRNAs, and the inhibition of cancer-related exosome secretion has been demonstrated to significantly reduce the metastatic potential of lung cancer cell lines [10].

The LNA and exosome data drew attention to the potential of miRNAs for cancer tratment. The development of safe and specific methods of delivery of miRNA-based treatments will allow modulation of miRNAs to become in the next few years a central feature of cancer treatment and management.

We are facing a new “Copernican” revolution: with miRNAs we demonstrated that biomarkers could be at the same time tools and effectors for patient treatment; an innovative exciting piece in medical history.

Figure 1. Potential miRNA-based therapeutic strategies. The function of oncogenic miRNAs could be stopped by small-molecule inhibitors (regulation of miRNAs expression at the transcriptional level), antisense oligonucleotides (binding by complementarity miRNAs and inducing either duplex formation or miRNA degradation), miRNAs masking (molecules complementary to the 3′-UTR of the target miRNA, resulting in competitive inhibition of the downstream target effects) or miRNAs sponges (oligonucleotide constructs with multiple complementary miRNA binding sites to the target miRNA). Tumor suppressor miRNAs function can be restored by introducing systemic miRNAs (miRNA mimics) or inserting genes coding for miRNAs into viral constructs.

Disclosure of conflict of interest:  Sanofi has an interest in pursuing miRNA therapeutics through a collaboration with Regulus.

References

1. Lee RC, Feinbaum RL, Ambros V. (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843-854.
2. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, Aldler H, Rattan S, Keating M, Rai K, Rassenti L, Kipps T, Negrini M, Bullrich F, Croce CM. (2002) Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. U. S. A. 99:15524-15529.
3. Fassan M, Baffa R, Kiss A, Zaninotto G, Rugge M. (2012) MicroRNA Dysregulation in Esophageal Neoplasia: The Biological Rationale for Novel Therapeutic Options. Curr Pharm Des In Press.
4. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
5. Baffa R, Fassan M, Volinia S, O’Hara B, Liu CG, Palazzo JP, Gardiman M, Rugge M, Gomella LG, Croce CM, Rosenberg A. (2009) MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets. J. Pathol. 219:214-221.
6. Garzon R, Marcucci G, Croce CM. (2010) Targeting microRNAs in cancer: rationale, strategies and challenges. Nat. Rev. Drug Discov. 9:775-789.
7. Kong YW, Ferland-McCollough D, Jackson TJ, Bushell M. (2012) microRNAs in cancer management. Lancet Oncol 13:e249-e258.
8. Landford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, Kauppinen S, Ørum H. (2010) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327:198-201.
9. Lindow M, Kauppinen S. (2012) Discovering the first microRNA-targeted drug. J Cell Biol 199:407-412.
10. Fabbri M. (2012) TLRs as miRNA Receptors. Cancer Res 72:6333-6337.

DOI: 10.1016/j.gde.2013.01.002

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology

¹Institute of Plant Biology & Zürich-Basel Plant Science Center, University of Zürich, Switzerland
²Institut de Recherche pour le Développement, UMR232; CNRS, ERL5300; Université de Montpellier II, France

In plants and animals, embryo development becomes ultimately controlled by zygotic genes, but the timing of zygotic genome activation (ZGA) varies greatly between organisms[1,2]. We recently showed that the transcriptome of young Arabidopsis embryos is dominated by maternal transcripts with a progressive ZGA under the maternal control of epigenetic pathways [3]. In contrast, another study reported that both parental genomes contribute equally to the transcriptome of young embryos, suggesting that ZGA occurs immediately after fertilization[4]. How to explain such dramatic differences? We propose that the discrepancies between these two studies likely reflect genuine biological differences between the two experiments, paving the road towards exciting discoveries on ZGA mechanisms in plants.

In animals, early stages of embryo development are associated with extensive epigenetic reprogramming to coordinate zygotic genome activation (ZGA) [2]. ZGA is typically delayed, although to a varying extent depending on the species, with a gradual loss of the maternal dominance and increase of zygotic influence [1,2]. In flowering plants, maternal effects on seed development have been recognized, yet are difficult to investigate because of the intricate relationships between the embryo, the embryo-nourishing endosperm, and the maternal seed coat [5]. To understand the interaction of parental genomes following fertilization, allele-specific assays were used to distinguish paternal and maternal contributions for selected loci or at the genome-wide level in dissected embryos (reviewed in [1]), with surprisingly different results. Yet, the diversity of species (Arabidopsis, maize, tobacco) and developmental stages analyzed made it difficult to draw general conclusions. In fact, the observed differences may reflect yet undiscovered biological factors controlling ZGA in flowering plants.

We have previously shown that the transcriptome of Arabidopsis embryos derived from crosses between the accessions Landsberg erecta (Ler) and Columbia (Col) is largely dominated by maternal reads (88%) at early stages (2-4 cells). Despite this maternal dominance, 66% of the genes have transcripts from both parental alleles, consistent with the fact that many embryo lethal mutations with preglobular developmental phenotypes are zygotically recessive [3]. Transcriptome analyses at the globular stage, in conjunction with expression analyses of seven reporter gene loci, confirmed a gradual increase of paternal transcripts during embryogenesis, reflecting progressive ZGA[3]. We also demonstrated that paternal loci are epigenetically regulated by two antagonistic maternal pathways: a siRNA-based mechanism involving genes of the RNA-dependent DNA methylation (RdDM) pathway restricts expression of paternal alleles, while their activation relies on a nucleosome-remodeling pathway[3]. As a result, kyp/KYP embryos derived from mothers lacking the activity of the histone methyltransferase KRYPTONITE (KYP), show both a higher proportion of paternal reads (34% vs 12% in the wild type) and a gene distribution that is skewed towards higher paternal contributions (based on a statistical best-fit model)[3].In contrast, a recent study using Arabidopsis embryos derived from crosses between the accessions Cape Verde Island (Cvi) and Col, showed a transcriptome with an equal contribution of paternal and maternal transcripts[4]. To explain this discrepancy, the authors suggested that transcripts derived from the maternal seed coat might have contaminated our embryo samples. However, this hypothesis does not explain the following observations: First, our genetic results on the regulation of parental contributions obtained in profiling studiesand by reporter gene analyses[3]; second, other studies analyzing expression of specific loci or reporter genes  (reviewed in [1]); and third, the observation that 1003 embryo-expressed genes, which were not detected in a seed coat transcriptome, are covered by 84% maternal reads (Raissig, Baroux, Lenormand, Wittig, Rosenstiel, Grossniklaus, unpublished). So are there possible biological explanations that have not been explored? We believe that there are several exciting hypotheses worth investigating before closing the debate on ZGA, for the benefit of the scientific endeavor.

In fact, the two experiments do not only differ in the way the embryos were isolated (discussed elsewhere[6]) but in at least two other respects (Figure 1): First, different hybrid combinations, Ler x Col [3] and Cvi x Col [4] were used. Cvi is being known for its singular epigenetic configuration involving atypical DNA methylation and transposon insertion patterns as well as structural heterochromatin phenotypes reminiscent of a dominant-negative effect on RdDM control  [7]. In this respect, the results reported by Nodine and Bartel[4] would be clearly consistent with our former conclusion[3] that embryos maternally deficient in RdDM components show a precocious bi-allelic expression of many genes. Alternatively, the diverging genetic relatedness of Cvi with Col and Ler may influence parental contributions in hybrid embryos, consistent with our proposition that the maternal control of paternal expression is expected to become weaker with increasing genetic distance [3]. Second, while we profiled mRNAs irrespective of their polyadenylation status, the other study specifically analyzed polyadenylated mRNAs[4]. In animals, cytoplasmic poly(A)-elongation is prevalent as a mechanism for the regulation of maternal mRNAs during early development [8]. Although data with respect to polyadenylation of plant mRNAs is scarce, it is possible that different mRNAs subpopulations were studied in the two experiments. Given that alternative polyadenylation during development is highly dynamic in plants, that Arabidopsis has a cytoplasmic polyadenylase, and that maternal mRNAs populations with short poly(A)-tails have been reported in maize and rice [9,10, 11] this seems a plausible scenario. Polyadenylated mRNA might represent a distinctive fraction of the embryonic pool of mRNA possibly under-representing maternally provided transcripts. Given these possible biological differences, future investigations on the mechanisms and natural variation in plant zygotic genome activation promise to shed new light onto this essential phase of the plant life cycle, which has consequences for many basic and applied aspects of plant biology.

Figure 1. Different readouts of parental contributions to the embryo transcriptome may reflect genuine biological differences. The parental contribution to the Arabidopsis embryo transcriptome were determined by RNA-Seq (pie charts; white: maternal sequence reads, grey: paternal sequence reads). Differences in the biological materials used in the two studies may explain the different readouts. First, different maternal genotypes were used, with either wild-type Ler (a) [3] or Cvi (c) [4], the latter showing a well-characterized difference in heterochromatin formation (nuclei insets), largely due to a dominant effect conveyed by the HDA6 locus, affecting chromatin organization and gene silencing [7].Second, different mRNA populations were sampled with respect to their polyadenylation status (black line with head: mRNA with a long poly(A) tail; black line without head: mRNA with no or a short poly(A) tail). Interestingly, the higher paternal contribution in Cvi/Col embryos (c) is partially mimicked in Ler/Col embryos maternally deficient in the histone H3K9-methyltransferase KYP (b),a component of the siRNA pathway regulating parental contributions [3]
Image sources (upper panel): seedlings [12] nuclei insets [7, 13] .

1. Baroux C, Autran D, Gillmor CS, Grimanelli D, Grossniklaus U: The maternal to zygotic transition in animals and plants. Cold Spring Harb Symp Quant Biol 2008, 73:89-100.
2. Tadros W, Lipshitz HD: The maternal-to-zygotic transition: a play in two acts. Development 2009, 136:3033-3042.
3. Autran D, Baroux C, Raissig MT, Lenormand T, Wittig M, Grob S, Steimer A, Barann M, Klostermeier UC, Leblanc O, et al.: Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis. Cell 2011, 145:707-719.
4. Nodine MD, Bartel DP: Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos. Nature 2012.
5. Chaudhury AM, Berger F: Maternal control of seed development. Semin Cell Dev Biol 2001, 12:381-386.
6. Raissig MT, Gagliardini V, Jaenisch J, Grossniklaus U, Baroux C: Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds. Journal of Vizualised Experiments 2012, in press.
7. Tessadori F, van Zanten M, Pavlova P, Clifton R, Pontvianne F, Snoek LB, Millenaar FF, Schulkes RK, van Driel R, Voesenek LA, et al.: Phytochrome B and histone deacetylase 6 control light-induced chromatin compaction in Arabidopsis thaliana. PLoS Genet 2009, 5:e1000638.
8. Lasko P: Translational control during early development. Prog Mol Biol Transl Sci 2009, 90:211-254.
9. Grimanelli D, Perotti E, Ramirez J, Leblanc O: Timing of the maternal-to-zygotic transition during early seed development in maize. Plant Cell 2005, 17:1061-1072.
10. Shen Y, Venu RC, Nobuta K, Wu X, Notibala V, Demirci C, Meyers BC, Wang GL, Ji G, Li QQ: Transcriptome dynamics through alternative polyadenylation in developmental and environmental responses in plants revealed by deep sequencing. Genome Res 2011, 21:1478-1486.
11. Luo, M., Taylor, J. M., Spriggs, A., Zhang, H., Wu, X., Russell, S., Singh, M. and Koltunow, A. (2011) A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm, PLoS Genet 7(6): e1002125.
12. Norman Jaimie M. Van, Benfey Philip N.. Arabidopsis thaliana as a model organism in systems biology. WIREs Syst Biol Med 2009, 1: 372-379.
13. Fischer, A.; Hofmann, I.; Naumann, K.; Reuter, G. (2006) Heterochromatin proteins and the control of heterochromatic gene silencing in Arabidopsis. Journal of Plant Physiology, 163, Issue 3, February 2006, Pages 358-368

DOI: 10.1016/j.gde.2013.01.006

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology

Lawrence P. Wackett

¹University of Minnesota, Twin Cities
² Department of Biochemistry and Molecular Biology and Biophysics
BioTechnology Institute
³Department of Mechanical Engineering

Hydraulic fracturing, or fracking, is a controversial process developed for releasing natural gas or petroleum from tight shale formations. The process has opened up large, previously unavailable sources of hydrocarbons for use as fuels and chemical feedstocks. Hydraulic fracturing has been criticized as postponing a shift from non-renewable fossil fuels to renewable biofuels. However, it is questionable that biomass-derived liquid fuels will be sufficient to replace liquid fuels (1). In that context, we feel that hydraulic fracturing to recover abundant oil and gas from shale resources will help meet society’s fuel needs in the short-term. Another criticism of hydraulic fracturing derives from its use of water resources and the contamination of that water with chemicals from the shale and the fracturing process itself. This has brought a focus onto the environmental aspects of hydraulic fracturing.

We have analyzed flowback and produced waters from shale formations in North Dakota and Pennsylvania using methods similar to those employed in the Deepwater Horizon oil spill of 2010 [2] and we have identified similarities in chemical composition as both derive from thermogenic hydrocarbon formations. The oil in the Gulf diminished over time due to weathering, submersion, and biodegradation [3]. The latter was facilitated by the addition of surfactants to break the surface oil layer into micro-droplets that made the oil components more assessable to the indigenous microorganisms in the Gulf waters that are capable of degrading them [4].

Therefore, we believe that biotechnology can play a role in mitigating against the negative impacts of hydraulic fracturing by removing the contaminants in water that derive from fracturing operations. Water discharge into municipal treatment systems is typically not allowed and this has led to the treatment of water using evaporation, filtration, and ozonation processes. The former concentrate wastes, which still require sequestration, typically in hazardous waste landfills. All of these methods are energy intensive. We believe that bioremediation offers unique advantages in not requiring an external energy source to operate and with the potential to degrade organic contaminants completely to carbon dioxide, therefore eliminating the landfilling requirement and expense. In most instances this would clean the waters suitable for re-use in future hydraulic fracturing processes, significantly reducing overall water demand.

Bioremediation can be employed either by utilizing the intrinsic biodegradation capabilities of the bacteria native to the water, or by using engineered systems that contain bacteria selected to degrade the specific contaminants of concern. Flowback and produced waters obtained from fracking operations have been analyzed for microbial populations and are found to be teaming with bacteria [5]. Further analysis is required to determine if those populations might be efficacious in degrading water contaminants. Biodegradation of petroleum hydrocarbons and additives has been an intensive field of study over the last decades and this suggests that engineered systems using characterized bacteria could be effective. The characteristics and biodegradation pathways for many naturally-occurring biodegradative bacteria are freely available from web databases [6]. While there are many publications describing genetically-engineered, biodegrading bacteria, there are regulatory impediments to their use environmentally [7]; thus, most large-scale treatments use naturallyoccurring bacteria. Pure cultures of naturally-occurring bacteria are often used in engineered systems, for example in the biodegradation of trichloroethylene by Burkholderia vietnamiensis G4 [8].

 The question emerges as to what kind of bioremediation would be most useful for hydraulic fracturing water decontamination and where it might be implemented. There is a pressing need currently to recycle water from a hydraulic fracture within one well to be used for other wells. In such an application, the residence time for the water is relatively short and it is more likely that engineered systems would work quickly enough to provide rapid cleaning. In other situations where the water in ponds or storage tanks have long residence times, intrinsic bioremediation by indigenous bacteria might provide a low-cost treatment option. Indigenous populations of bacteria may be stimulated by carbon, nitrogen, or phosphorus-containing nutrients that selectively enhance the biodegrading bacteria present in the medium to be treated [9]. Metagenomic studies [5] can help point the way toward identification and characterization of the bacteria that thrive in the presence of hydrocarbons, high salt concentrations, and high total dissolved solids. Analytical chemistry can reveal what nutrients might be limiting for biodegradation to occur at a maximal rate.

There are several similarities between issues of water re-use following hydraulic fracturing and water contamination due to the extraction of petroleum hydrocarbons from oil sands. The latter process uses large volumes of water and those become contaminated with a complex mixture of high molecular weight hydrocarbons and heterocycles. Treatment of oil sand wastewater by bioremediation has been a very active area of study [10] and research into either area can inform the other.

 In total, we feel that biotechnology offers the potential for cost effective treatment of hydrocarbon remediation stemming from spills and normal operations in hydraulic fracturing and oil sands extraction. Our knowledge of the fundamental biodegradation biochemistry has become extensive; it is necessary to push the envelope on engineered systems to effectively use the biodegradative metabolism that nature has evolved.

References

1. Michel, H: The nonsense of biofuels.Angew Chem Int Ed 2012, 51:2516-2518.
2. Leco Corporation Technical Article: Analysis of Samples from the Gulf of Mexico Oil Spill by GCxGCTOF- MS. 2010, http://www.leco.com/resources/application_notes/pdf/PEG4D_GULF_OF_MEXICO_OIL_SPILL_203-821- 389.pdf
3. Atlas R, Hazen TC: Oil biodegradation and bioremediation: A tale of the two worst spills in U. S. history. 2011, Environ Sci Technol 45:6709-6715.
4. Hazen TC, Dubinsky EA, DeSantis TZ, Andersen GL, Piceno YM, Singh N, Jansson JK, Probst A, Borglin SE, Fortney JL et al: Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. 2010, Science 330:204-208.
5. Christopher G CG Struchtemeyer and Mostafa S MS Elshahed: Bacterial communities associated with hydraulic fracturing fluids in thermogenic natural gas wells in North Central Texas, USA. 2012, FEMS Microbiol Ecol 81:13-25
6. Gao J, Ellis LBM, Wackett LP: The University of Minnesota Biocatalysis/ Biodegradation Database: improving public access. 2010, Nucleic Acids Res 38: D488-D491.
7. Schmidt M, de Lorenzo V: Synthetic constructs in/for the environment: managing the interplay between natural and engineered biology. 2012, FEBS Lett 586:2199-2206.
8. Kumar A, Vercruyssen A, Dewulf J, Lens P, Van Langenhove H: Removal of gaseous trichloroethylene (TCE) in a composite membrane biofilm reactor. 2012, J Environ Sci Health A Tox Hazard Subst Environ Eng 47:1046-1052.
9. Hosoda A, Takahashi T, Numano K, Nakajou K, Higashimoto A, Toda M, Arai H,Hotta Y, Tamura H: Rapid reductive dechlorination of trichloroethene in contaminated ground water using biostimulation agent, BD-1, formulated from canola oil. 2012, J Oleo Sci 61:155-161.
10. Kannel PR, Gan TY: Naphthenic acids degradation and toxicity mitigation in tailings wastewater systems and aquatic environments: a review. 2012, J Environ Sci Health A Tox Hazard Subst Environ Eng 47:1-21.

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology – Structural Biology

Simona Romano¹ and Maria Fiammetta Romano¹  

The use of statins is essential for the treatment of hyperlipidemia as well as for the primary and secondary prevention of coronary artery disease and strokes. Statins decrease low-density lipoprotein (LDL) cholesterol levels by inhibiting 3-hydroxy-3-methyl-glutaryl-CoA HMG-CoA reductase (HMGCR). HMGCR in turn catalyses the conversion of HMG-CoA into mevalonic acid, an important intermediate metabolite in hepatic cholesterol biosynthesis. Statins exert pleiotropic effects independent of their cholesterol-lowering activity, which are in part mediated by mevalonic acid, a precursor of the isoprenoid intermediates farnesyl and geranyl-geranyl pyrophosphate [1]. These compounds participate in the post-translational modification of intracellular G-proteins, such as Rho, Rac, and Ras. In turn, it is well-known that G-proteins drive signalling pathways that are widely involved in carcinogenesis [2], and their inhibition by statins has been proven to efficiently impair the growth of several tumours [3]. Furthermore, statins inhibit cellular matrix metalloproteinases and nuclear factor-kB (NF-kB) transcription factors that are, additionally, often deregulated in cancer. A large body of studies exists that support the efficacy of statins against several cancer types [3]. For this reason, these compounds has been proposed as adjuvant options in cancer therapies and in cancer prevention, Currently, recommendations and guidelines on statin use in malignancies do not exist; in addition, to date, results from randomised controlled trials are inconclusive regarding the question of whether cancer treatment may benefit from statins.

Very recently, a number of studies, have found that atorvastatin is a powerful stimulus for endoglin production [4-6]. For example, atorvastatin was shown to stimulate expression of endoglin in human umbilical vein endothelial cells (HUVEC), at both transcriptional and protein levels [4]. Endoglin mRNA levels meanwhile, were found to be increased in the endothelium of subjects that were statin recipients, in comparison with subjects that were not statin recipients [4]. In addition, high levels of endoglin were measured in the blood of subjects with familial hypercholesterolemia that were under chronic statin treatment [5]. Lastly, Nachtigal et al. also found that statin treatment significantly induced endoglin expression in the aortic endothelium in atherosclerosis models [6].

Endoglin, or CD105 as it was designated at the Fifth International Workshop on Human Leukocyte Differentiation Antigens, is an accessory receptor of the Transforming Growth Factor (TGF) -b family of proteins, which includes TGF-b, bone morphogenetic proteins (BMPs), and activins. Endoglin forms complexes with type I and type II TGF-b receptors (TbRI and TbRII), and modulates TGF-b signalling. Stimuli that increase endoglin expression promote the BMP signalling pathway, which is active during embryonic development and is reactivated during adult vasculogenesis. CD105 expression in vascular endothelial cells of de novo blood vessels is therefore considered to be potentially useful in the detection of cancer patients with high risks of disease recurrence and metastasis formation [7]. In addition, CD105 expression was found to be of particular significance in the evaluation of neoangiogenesis and prediction of prognosis in brain tumours [8]. Endoglin is predominantly expressed in proliferating endothelial cells that participate in tumour angiogenesis, with weak or no expression in the vascular endothelium of normal tissues; for this reason CD105 is considered as a suitable vascular target for antiangiogenetic cancer therapy.

Angiogenesis is a powerful element that drives tumour progression. Blockage of angiogenesis maintains tumours in a harmless state and constrains the outgrowth of metastases. Inability to initiate angiogenesis in disseminated tumour cells is considered as one of the main causes of tumour dormancy. Selective targeting of proliferating endothelial cells within tumour-associated blood vessels represents one of the most pursued approaches to limit tumour progression, with molecules that are overexpressed in actively proliferating endothelial cells being attractive targets for antiangiogenetic strategies. Among these targets, endoglin is considered to be of prime importance, due to the central role this molecule exerts in tumour angiogenesis.

Currently, there are no studies directly linking statins with endoglin production and tumour progression. In addition, an in-depth characterisation of the mechanism by which statins induce CD105 is still lacking. However, it is worth noting that several studies show conversely that statins can promote cancer [9], suggesting that pro-tumoural mechanisms might be, in addition, activated by these drugs.

The undoubtedly striking and specific inhibitory actions on tumourigenic pathways exerted by statins are consistent with a protective action for these agents in cancer prevention [10]. The efficient pro-apoptotic activities exerted by statins, at doses much higher than those used to lower cholesterol, suggest that these compounds could have an effective role, if used in concert with chemotherapy, in cancer treatment. However, the possibility cannot be excluded that statins may promote progression of pre-existing, asymptomatic and even dormant tumours because of their capacity to stimulate CD105. These observations are consistent with a dual role for statins in cancer prevention and progression. The role of statins in cancer represents an open question that requires urgent addressing and careful investigation, in both experimental and pre-clinical settings. Besides prospective studies, clinical trials may be required to define unambiguously the ultimate biological effects of statins in cancer intervention and thus the patient population that could benefit most from this treatment. Finally, it is worth emphasising that millions of healthy people, including both children and adults, are currently taking statin drugs.

References

1. Liao JK, Laufs U: Pleiotropic effects of statins, Annu Rev Pharmacol Toxicol 2005, 45:89-118.
2. Dorsam RT, Gutkind JS: G-protein-coupled receptors and cancer, Nat Rev Cancer 2007, 7:79-94.
3. Hindler K, Cleeland CS, Rivera E, Collard CD: The role of statins in cancer therapy, Oncologist 2006, 11:306-15.
4. Giordano A, Romano S, Monaco M, Sorrentino A, Corcione N, Di Pace AL, Ferraro P, Nappo G, Polimeno M, Romano MF: Differential effect of atorvastatin and tacrolimus on proliferation of vascular smooth muscle and endothelial cells, Am J Physiol Heart Circ Physiol 2012, 302:H135-42.
5. Blaha M, Cermanova M, Blaha V, Jarolim P, Andrysd] C, Blazek M, Maly J, Smolej L, Zajic J, Masin V, Zimova R, Rehacek V: Elevated serum soluble endoglin (sCD105) decreased during extracorporeal elimination therapy for familial hypercholesterolemia, Atherosclerosis 2008, 197:264-70.
6. Nachtigal P, Pospisilova N, Vecerova L, Micuda S, Brcakova E, Pospechova K, Semecky V: Atorvastatin increases endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS expression in ApoE/LDLR double knockout mice, J Atheroscler Thromb 2009, 16:265-74.
7. Marioni G, Marino F, Blandamura S, D’Alessandro E, Giacomelli L, Guzzardo V, Lionello M, De Filippis C, Staffieri A: Neoangiogenesis in laryngeal carcinoma: angiogenin and CD105 expression is related to carcinoma recurrence rate and disease-free survival, Histopathology 2010, 57:535-43.
8. Yao Y, Kubota T, Takeuchi H, Sato K: Prognostic significance of microvessel density determined by an anti-CD105/endoglin monoclonal antibody in astrocytic tumors: comparison with an anti-CD31 monoclonal antibody, Neuropathology 2005, 25:201-6.
9. Chang CC, Ho SC, Chiu HF, Yang CY: Statins increase the risk of prostate cancer: a population-based case-control study, Prostate 2011, 71:1818-24.
10. Pelton K, Freeman MR, Solomon KR: Cholesterol and prostate cancer, Curr Opin Pharmacol 2012, Jul 21 [Epub ahead of print].

DOI: 10.1016/j.coph.2013.02.004 

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology

Curt Alexander Davey

Chromatin organization underlies genomic function, and DNA sequence contributes to nucleosome stability and positioning.  A new study based on chemical footprinting to give the first genome-wide base-pair-resolution map of nucleosome positions showed that the A|T versus G|C preference associated with minor versus major groove-inward orientation is much more pronounced than was evident before from micrococcal nuclease digestion analysis [1].  Thus the contribution of DNA sequence to chromatin structure has been underestimated, but the overall pattern is nonetheless consistent with a passive form of sequence-selectivity, whereby the histones indirectly readout the DNA sequence through systematic wrapping of a double helix with sequence-dependent structure and flexibility.However, the high resolution map also showed a strong rogue signal near the nucleosome centre that is out of phase with the ~10 bp A|T/G|C periodicity.  Nucleosome crystal structures display a unique and conserved histone arginine-base contact at this location showing direct readout of A|T base pairs.  Since direct readout is a hallmark of sequence-specific DNA-binding proteins, this suggests that the core histones have evolved deliberate DNA sequence bias and questions our understanding of chromatin evolution and function.

Sequence-specific DNA binding proteins recognize their substrates by the chemical signature of bases in the major or minor grooves, which is known as direct, or base, readout [2].  In addition, an indirect form of readout is also frequently employed to achieve further selectivity, wherein the protein recognizes structural/flexibility properties of the DNA that are dictated by the sequence [3].  On the other hand, proteins that are supposed to indiscriminately associate with the double helix regardless of sequence must find a way to minimize the contribution of both direct and indirect readout.  Eliminating direct readout is straightforward, as the protein need only avoid making base contacts, and in fact such contacts can still be formed if they are heterogeneous and therefore sequence-neutral.  Avoiding indirect readout is altogether another matter since in order to bind with appreciable affinity, a protein must make multiple, defined contacts with the double helix, which itself has sequence-specific conformational preferences.  Because of the pronounced sequence-dependence of double helix flexibility [4], eliminating indirect readout becomes even more challenging, if not impossible, for systems that significantly bend/distort their DNA targets from the naked conformation.

The histone octamer (composed of an H3-H4 tetramer and two H2A-H2B dimers) may be regarded as the ultimate non-sequence-specific DNA binding protein, since it can wrap almost any sequence into a nucleosome and this encompasses a massive protein-DNA contact area over ~147 bp [5].  The few sequences that inhibit nucleosome formation include repeats that could adopt alternative DNA structures and poly-A:T tracts, which have unusual rigid character.  This surprisingly modest level of sequence-dependency is achieved by the histone octamer recognizing the double helix with a ‘finger tips grip’, making contacts almost exclusively with the sugar-phosphate backbone.  The sequence discrimination that remains appears to be at first glance an unavoidable consequence of systematically deforming the double helix to such an extent.  Due to sequence-dependent flexibility and groove width characteristics, A|T and G|C sequences preferentially localize to positions where the minor and major groove, respectively, face inward towards the histone octamer binding surface [6]. 

The ~10 bp A|T/G|C periodicity associated with nucleosome positioning in vivo [1] and the characteristics of the Widom consensus sequence for maximal histone octamer affinity in vitro [7] are largely consistent with a default indirect readout of the DNA sequence.  However, variations in the magnitude and character of DNA deformation as well as fluctuations in the relative constraints on double helix structure imposed over the octamer binding surface give rise to distinctions in sequence-dependence between the different histone-DNA binding sites [6,8], which is evident in both genome-wide nucleosome maps and the Widom consensus sequence [1,7].  Nonetheless, one could still argue that the resulting translational nucleosome positioning signals may have evolved not deliberately, but rather as an indirect consequence of other structural or dynamic functions.  For instance, distinctions in double helix conformation and binding strength over the different histone sites could be required for chromatin assembly/disassembly and site-specific recognition of nucleosomes.  In this regard, the chromatin system would have developed to minimize sequence-dependency, and what remains is either exploited or overridden in the regulation of the genome.

In fact, the unprecedented resolution of the recent genome-wide chemical nucleosome mapping reveals the true strength of the indirect readout, but with an unexplained rogue signal situated approximately nine bp from the nucleosome centre [1].  This is a location where the DNA major groove faces inward, so that one would expect an increased preference for G|C dinucleotides in accord with the overall pattern [6], whereas the opposite actually occurs.  An inspection of the 19 highest resolution crystal structures of different native nucleosome core particles— composed of ubiquitous, variant and modified histones of many different species from yeast to human and eight distinct DNA sequences [6,9]— reveals that a well-defined arginine side chain of the H3 N-terminal tail (R40) resides in the minor groove at this location and forms hydrogen bonds with the electronegative groups of A|T base pairs.  This indicates both direct readout at play selecting against the presence of the electropositive group of G|C base pairs and indirect readout favouring a narrowed minor groove preferred by A|T sequences [2,3].

So why would the nucleosome engage in ‘premeditated’ DNA sequence bias?  The discriminating arginine in question is part of an N-terminal histone-fold extension unique to H3, which is responsible for holding on to the DNA termini of the nucleosome core [5].  Since these terminal interactions are the first to break in nucleosome breathing dynamics [10] (and last to form, which may explain why the rogue signal [1] is not evident in the Widom consensus sequence that is heavily biased towards the early events in nucleosome reconstitution [7]), it is interesting to speculate that they are involved in regulating nucleosome unfolding and factor access in a DNA sequence-sensitive fashion.  This suggests that DNA sequence may play a more active role in chromatin structure and function than has been assumed, with fascinating attributes yet to be unravelled.

Acknowledgement:  I am grateful to Gabriela E. Davey for helpful comments and to the National Medical Research Council, Ministry of Health, Singapore (NMRC/1312/2011) for funding. 

References

1. Brogaard, K, Xi, L, Wang, JP, Widom, J: A map of nucleosome positions in yeast at base-pair resolution. Nature 2012, 486:496-501.
2. Rohs, R, Jin, X, West, SM, Joshi, R, Honig, B, Mann, RS: Origins of specificity in protein-DNA recognition. Annu Rev Biochem 2010, 79:233-269.
3. Rohs, R, West, SM, Sosinsky, A, Liu, P, Mann, RS, Honig, B: The role of DNA shape in protein-DNA recognition. Nature 2009, 461:1248-1253.
4. Olson, WK, Gorin, AA, Lu, XJ, Hock, LM, Zhurkin, VB: DNA sequence-dependent deformability deduced from protein-DNA crystal complexes. Proc Natl Acad Sci U S A 1998, 95:11163-11168.
5. Davey, CA, Sargent, DF, Luger, K, Maeder, AW, Richmond, TJ: Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J Mol Biol 2002, 319:1097-1113.
6. Chua, EY, Vasudevan, D, Davey, GE, Wu, B, Davey, CA: The mechanics behind DNA sequence-dependent properties of the nucleosome. Nucleic Acids Res 2012, 40:6338-6352.
7. Thastrom, A, Bingham, LM, Widom, J: Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning. J Mol Biol 2004, 338:695-709.
8. Wu, B, Mohideen, K, Vasudevan, D, Davey, CA: Structural insight into the sequence dependence of nucleosome positioning. Structure 2010, 18:528-536.
9. Tan, S, Davey, CA: Nucleosome structural studies. Curr Opin Struct Biol 2011, 21:128-136.
10. Li, G, Levitus, M, Bustamante, C, Widom, J: Rapid spontaneous accessibility of nucleosomal DNA. Nat Struct Mol Biol 2005, 12:46-53..

 
DOI: 10.1016/j.sbi.2013.01.011

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology

¹Lund University, Lund, Sweden
² Yale University, New Haven, CT, USA
³University of Strasbourg, Strasbourg, France

The Yusupov’s paper on eukaryotic ribosome structure includes a proposal for the way the proteins found in those particles should be named (1). This is a topic on which there is no general agreement today.  We urge the ribosome field to take this opportunity to discuss their proposal, the objective being to arrive at a consensus about how ribosomal proteins should be named.

            When work began on the purification and characterization of the ribosomal proteins from E. coli in the late 1960s, all the groups involved developed their own method for naming those molecules, and everyone had to use tables that related the different names that were being applied to each protein.  This ended in the early 1970s as a result of what amounted to a summit meeting in which all the major players agreed on a single naming system [2]. 

            The time has come for a similar discussion and agreement for the eukaryotic ribosomal proteins. The solution to this problem proposed by the Yusupovs makes sense.  Now that there are three-dimensional structures available for both prokaryotic and eukaryotic ribosomes, it is possible to map the proteins found in eukaryotic ribosomes onto those found in the E. coli ribosome unambiguously.  Since the ribosomal proteins from E. coli were the first to be sequenced, standard priority practices in the sciences dictate that their eukaryotic homologs be assigned their E. coli names.  Those eukaryotic ribosomal proteins that have no eubacterial homolog, of which there are many, should be given the name assigned to them either when the rat version was sequenced by Wool and his colleagues [3], or when they were sequenced by the yeast community [4], depending on which happened first.  By appending the letter “e” after the eukaryotic-only names, one can avoid the problems that would otherwise arise because of accidental overlaps in protein numbering schemes, and one also alerts the reader that the protein in question has no eubacterial homolog. Our proposal is presented as Tables 1 and 2.

Table 1: New nomenclature for proteins from the small ribosomal subunit

Table 2: Nomenclature for proteins from the large ribosomal subunit

            These two tables are not quite the same as those presented by Jenner et al (1). Ribosomal protein L10 in bacteria is somewhat longer in eukaryotes and called P0. We propose that the original name, L10, be used. Furthermore, only bacteria have proteins that correspond to the protein called L7/L12 in E. coli. In addition the acetylated variant, L7, is not found in all bacterial species. Therefore we suggest that this protein be called L12 unless its acetylated form is being discussed in which case it could be called L7. In eukaryotes the corresponding proteins, which are not homologous, are called P1 and P2. Sometimes there is also a variant called P3. We suggest that these names be maintained. The corresponding protein in archaea has been called archaeal L12, but this inappropriate since the protein is closely related to P1 and P2, but not at all to L12. Since there is only one variant we suggest that it be called P1.

            We urge everyone who is interested in this issue to register his or her opinions about this proposal on this blog, and to read (and comment) on what others have to say on the matter (all comments must be signed.)  In six months or so, a specific proposal will be made to the entire community that is based on the opinions recorded on this blog.  We hope that a consensus will have emerged about how ribosomal proteins should be named that will eliminate the confusion we now experience because so many ribosomal proteins have several different names associated with them.

            Mitochondria from higher eukaryotes and kinetoplastids have larger number of ribosomal proteins (5). As for the cytoplasmic ribosomal proteins, well-resolved structures of such ribosomes will be needed before one can safely propose names for them that are consistent with the names given cytoplasmic ribosomes.  

The full article “Crystal structure of the 80S yeast ribosome” by Marat Yusupov et al. on this topic is freely accessible here for a limited time.

References

1. Jenner L, Melnikov S, Garreau de Loubresse N, Ben-Shem A, Iskakova M, Urzhumtsev A, Meskauskas A, Dinman J, Yusupova G and Yusupov M: Crystal structure of the 80S yeast ribosome. Curr Op Struct Biol 2012,
2. Wittmann HG, Stöffler G, Hindennach I, Kurland CG, Randall-Hazelbauer L, Birge EA, Nomura M, Kaltschmidt E, Mizushima S, Traut RR, Bickle TA: Correlation of 30S ribosomal proteins of Escherichia coli isolated in different laboratories. Molec Gen Genetics 1971, 111:327-333.
3. Wool IG, Chan Y-L, Gluck A: Structure and evolution of mammalian ribosomal proteins, Biochem Cell Biol 1995, 73:933-947.
4. Planta RI, Mager WH: The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Yeast 1998, 14:471-477.
5. Desmond E, Brochier-Armanet C, Forterre P, Gribaldo S: On the last common ancestor and early evolution of eukaryotes: reconstructing the history of mitochondrial ribosomes. Res Microbiol 2011, 162:53-70.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology

Marian F. Young

¹National Institutes of Health, Bethesda, MD, USA
²Brown University, Providence RI, USA

Biglycan is an extracellular matrix component of many parts of the skeleton including bone, cartilage, tendon, teeth and muscle. Biglycan is predominantly expressed as a proteoglycan, but a mature form lacking GAG side chains (‘nonglycanated’) has recently been shown to have specific functions in muscle, synapses and Wnt signaling in bone. The biglycan gene is on the X (and not Y) chromosome and is dysregulated in Turner (XO) and Kleinfelter’s Syndromes (supernumery X) diseases, characterized by short and tall stature respectively. Biglycan deficient mice have shorter bones as well as lower bone mass (ostepenia/osteoporosis),1 another notable feature observed in Turners Syndrome. The mechanisms underlying the thinner and weaker bones produced without biglycan have been studied in detail and point to the fact that biglycan modulates multiple pathways critical to skeletal metabolism.

While biglycan is not needed for development of the musculoskeletal system, it is required for the maintenance of its integrity. In adult bone turnover is regulated by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. In the absence of biglycan, there is decreased bone formation due to defects in the maturation of osteogenic precursors that form bone 2. Bone Morphogenic Protein 2/4 (BMP-2/4), a well-known inducer of bone formation, is currently being used therapeutically to aid bone repair. Bone-derived cells depleted of biglycan have less BMP-2/4 binding and subsequently less osteogenic differentiation. It is logical to conclude that biglycan could be a prime candidate to enhance BMP-2/4 function in situations where it is commonly used such as in bone regeneration and repair after fracture or trauma.

Mice lacking biglycan also display pathologies typically associated with skeletal aging. Specifically, by three months of age, hallmark signs of osteoarthritis (OA) are evident in the mutant mice, including fissures, cell clustering and loss of the smooth articular cartilage surface on the joints. The OA is detected in all weight bearing joints as well as in the temporomandibular joint of the jaw. The effects of biglycan loss are exacerbated by depletion of the related small leucine-rich proteoglycan fibromodulin (Bgn-/0; Fmod-/- DKO). Molecular studies point to the abnormal sequestration of the potent growth factor TGF- in the combined absence of biglycan and fibromodulin causing it to be “unleashed” and subsequently overactive. The uncontrolled stimulation of TGF- in this context leads to hyper-proliferation, premature differentiation of cartilage derived cells, MMP induction and, ultimately, loss of the condyle tissue integrity 3.

Biglycan can also control the fate of skeletal stem cells by modulating the extracellular niche. This function was demonstrated in ECM-rich tendon tissue that harbors a cell population with stem cell features including clonogenicity, multipotency and regenerative capabilities 4. The combined removal of biglycan and fibromodulin caused tendon stem/progenitor cells to be hypersensitive to BMP-2: instead of differentiating into tendon, these progenitors form multiple ectopic bones within the tendons that affect the gait of the mice . Biglycan also controls other factors critical to bone in addition to TGF- and BMP-2/4. In humans, a mutation in the extracellular domain of the key Wnt signaling molecule LRP-6 (R611C) causes elevated cholesterol and osteopenia. Notably, exogenous application of non-glycanated biglycan repaired the defective Wnt signaling in cells expressing mutant LRP-6 5. Thus, biglycan could potentially ameliorate pathologies caused by defective Wnt signaling. Taken together these findings underscore the importance of biglycan in modulating several key growth factor-mediated signaling pathways that regulate skeletal tissue architecture and function.

Biglycan also plays a role in organizing membrane architecture and function in muscle and at synapses. Muscle membranes are highly specialized to transmit force, protect the cell from contraction-induced damage and orchestrate signaling pathways required for normal function. The dystrophin- and utrophin- membrane glycoprotein complexes (DGC and UGC, respectively) link the cytoskeleton to the extracellular matrix and serve as a scaffold for signaling molecules in adult (DGC) and immature (UGC) muscle. Biglycan binds to three shared components of these complexes: the extracellular peripheral membrane protein -dystroglycan and the transmembrane proteins - and -sarcoglycan 6, 7. Genetic studies show that biglycan regulates the expression of utrophin, the two sarcoglycans and an intracellular membrane-associated signaling complex comprised of dystrobrevin, syntrophins and nNOS (neuronal nitric oxide synthase) in immature muscle 8. Notably, dosing mice with recombinant non-glycanated biglycan (rNG-BGN) can restore the expression of several of these components to the membrane 8.

The role of biglycan in binding and regulating several components of DGC and UGC, coupled with the ability to deliver rNG-BGN systemically, suggested that biglycan could be a therapeutic for Duchenne Muscular Dystrophy (DMD). DMD is the most common form of muscular dystrophy and results from mutations in dystrophin – a large intracellular protein that links the actin cytoskeleton to the membrane and anchors the DGC. Notably, utrophin upregulation can compensate for dystrophin loss in mouse models of DMD (mdx; Davies). Systemically-delivered rNG-BGN recruits utrophin to the membrane and improves muscle health and function in mdx mice 9. The efficacy of the non-glycanated form (i.e. lacking GAG side chains) in this therapeutic approach is most likely based on two reasons. First, this form can be readily manufactured in a homogeneous form. Second, biglycan proteoglycan (PG) but not non-glycanated (core) is proinflammatory 10. A non-glycanated form of biglycan is currently in preclinical development for DMD.

Biglycan is also important for synapse stabilization 11. In biglycan-deficient mice, neuromuscular junctions form normally but then they become unstable about three weeks after birth. The mechanism of biglycan action at the synapses is likely to involve MuSK, a receptor tyrosine kinase that is the master regulator of synapse differentiation and maintenance. Biglycan binds to MuSK and regulates its expression in vivo. Notably, synaptic loss is one of the earliest abnormalities observed in almost all neurodegenerative diseases, including ALS (amyotrophic lateral sclerosis) and SMA (spinal muscular atrophy). Treatments that promote neuromuscular junction stability could prolong function and potentially survival in these devastating motor neuron diseases.

In summary, biglycan plays important roles in the musculoskeletal system. The fact that non-glycanated forms of biglycan are effective in ameliorating muscle defects and that it can be administered systemically makes it particularly amenable for tissue and cell therapy. Taken together, it is reasonable to conclude that biglycan holds promise as a novel therapeutic for numerous musculoskeletal diseases including low bone mass, osteoarthritis, ectopic bone formation and muscular dystrophy.

References

1. Xu T, Bianco P, Fisher LW, Longenecker G, Smith E, Goldstein S, Bonadio J, Boskey A, Heegaard AM, Sommer B, Satomura K, Dominguez P, Zhao C, Kulkarni AB, Robey PG, Young MF: Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice, Nature genetics 1998, 20:78-82
2. Chen XD, Fisher LW, Robey PG, Young MF: The small leucine-rich proteoglycan biglycan modulates BMP-4-induced osteoblast differentiation, FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2004, 18:948-958
3. Embree MC, Kilts TM, Ono M, Inkson CA, Syed-Picard F, Karsdal MA, Oldberg A, Bi Y, Young MF: Biglycan and fibromodulin have essential roles in regulating chondrogenesis and extracellular matrix turnover in temporomandibular joint osteoarthritis, The American journal of pathology 2010, 176:812-826
4. Bi Y, Ehirchiou D, Kilts TM, Inkson CA, Embree MC, Sonoyama W, Li L, Leet AI, Seo BM, Zhang L, Shi S, Young MF: Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche, Nature medicine 2007, 13:1219-1227
5. Berendsen AD, Fisher LW, Kilts TM, Owens RT, Robey PG, Gutkind JS, Young MF: Modulation of canonical Wnt signaling by the extracellular matrix component biglycan, Proceedings of the National Academy of Sciences of the United States of America 2011, 108:17022-17027
6. Bowe MA, Mendis DB, Fallon JR: The small leucine-rich repeat proteoglycan biglycan binds to alpha-dystroglycan and is upregulated in dystrophic muscle, The Journal of cell biology 2000, 148:801-810
7. Rafii MS, Hagiwara H, Mercado ML, Seo NS, Xu T, Dugan T, Owens RT, Hook M, McQuillan DJ, Young MF, Fallon JR: Biglycan binds to alpha- and gamma-sarcoglycan and regulates their expression during development, Journal of cellular physiology 2006, 209:439-447
8. Mercado ML, Amenta AR, Hagiwara H, Rafii MS, Lechner BE, Owens RT, McQuillan DJ, Froehner SC, Fallon JR: Biglycan regulates the expression and sarcolemmal localization of dystrobrevin, syntrophin, and nNOS, FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006, 20:1724-1726
9. Amenta AR, Yilmaz A, Bogdanovich S, McKechnie BA, Abedi M, Khurana TS, Fallon JR: Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice, Proceedings of the National Academy of Sciences of the United States of America 2011, 108:762-767
10. Moreth K, Brodbeck R, Babelova A, Gretz N, Spieker T, Zeng-Brouwers J, Pfeilschifter J, Young MF, Schaefer RM, Schaefer L: The proteoglycan biglycan regulates expression of the B cell chemoattractant CXCL13 and aggravates murine lupus nephritis, The Journal of clinical investigation 2010, 120:4251-4272
11. Amenta AR, Creely HE, Mercado ML, Hagiwara H, McKechnie BA, Lechner BE, Rossi SG, Wang Q, Owens RT, Marrero E, Mei L, Hoch W, Young MF, McQuillan DJ, Rotundo RL, Fallon JR: Biglycan is an extracellular MuSK binding protein important for synapse stability, The Journal of neuroscience : the official journal of the Society for Neuroscience 2012, 32:2324-2334

Acknowledgments
The experiments described in this commentary were supported in part by the Division of Intramural Research, NIDCR of the Intramural Research Program, NIH, DHHS

DOI: 10.1016/j.gde.2012.07.008

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Posted in Genetics & Development

Home – Cell Biology – Genetics & Development – Pharmacology

Mervyn J. Merrilees¹ and Thomas N. Wight

Versican is a chondroitin sulfate (CS) proteoglycan present in the interstitial space of all tissues and is notably elevated during early development, where it plays a key role in organogenesis, and later in numerous pathologies, often those affecting major organs such as vessels, lung and skin [1]. Accumulating evidence indicates that versican is of critical importance in chronic vascular diseases including atherosclerosis, aneurysms and restenosis [2]. Versican is also elevated in many types of cancers and can directly influence the metastatic potential of tumors [3-6]. Versican increases in the airways and parenchyma of lungs affected by emphysema and chronic obstructive pulmonary disease (COPD), and is elevated in some rare lung diseases such as lymphangioleiomyomatosis (LAM) [7]. Recent evidence indicates it is a central player in inflammation, which broadens its importance to acute events in tissue repair. Versican is thus emerging as a potential target promising wide therapeutic benefits.

Versican is highly interactive with versatility reflected in its name. The versican gene and protein follow a domain template with an amino-terminal globular domain (G1) that binds hyaluronan, a carboxy-terminal globular domain (G3) similar to the selectin family of proteins with a C-type lectin adjacent to two epidermal growth factor domains and a complement region, and a middle region encoded by two large exons specifying CS attachment regions [8].

Versican’s innate complexity is further reflected by alternative splicing of exons 7 and 8 that encode the glycosaminoglycan (GAG) attachment domains, giving rise to V0, V1, V2, and V3 variants. V3 has neither of these exons, so is without GAG chains, but paradoxically has a demonstrated ability to dramatically influence structure and composition of the extracellular matrix (ECM) in potentially beneficial ways [2,9]. In addition to variation in core protein size due to splicing, the CS chains also vary in size (25 to 80kDa) as does composition, depending on tissue source and stimuli, with variation in the ratio of chondroitin-6-sulfate to chondroitin-4-sulfate. Expression of the versican gene is regulated by a promoter harboring a typical TATA box, β-catenin/TCF response elements and potential binding sites for transcription factors such as SP1, p53, AP1, AP2, CCAAT enhancer protein, and cAMP-responsive elements [10,11]. Regulation of core protein synthesis can be at transcriptional and posttranscriptional levels.

Studies on vessels have contributed most to our understanding of the roles of versican in pathogenesis. It is present in early intimal thickenings, and is elevated in human vessels susceptible to atherosclerosis. It contributes to intimal expansion and intimal progression and conversely its degradation is accompanied by lesion regression [12]. It is present around the core of large lesions and there is good evidence that its CS chains interact with and retain LDL in the vessel wall; it is thus central to the response-to-retention hypothesis of atherogenesis. Binding sites for versican have been identified in human apoprotein B of LDL, and expression of mutated apoB with defective binding dramatically ameliorates development of lesions in animals fed a high fat diet [13].

Versican is also elevated in aneurysms and restenosis, two conditions in which there is substantial remodelling of tissues, including marked changes to elastic fibers for which the GAG-containing variants are negative regulators. Assembly of elastic fibers, which involves incorporation of tropoelastin onto the microfibrillar scaffold, a process mediated via cell surface elastin binding protein, is inhibited by CS. On the other hand, over-expression of V3 lacking CS chains decreases the tissue content of the CS-containing versican variants and markedly enhances elastogenesis and the assembly of elastic fibers in the matrix [14]. Alternatively, decreasing CS-containing versican variants by over-expression of V3 or application of versican antisense sequences similarly promotes elastogenesis [15]. V3 also increases deposition of elastin by skin fibroblasts, and has been utilized in vitro to correct the elastin deficiency that characterizes Costello syndrome [16]. This inverse relationship between versican and elastin is also present in lungs affected by LAM and COPD, and in the latter versican levels correlate negatively with lung function measures such as FEV1 [7]. This opens up the possibility for V3 or versican antisense interventions in these respiratory diseases.

Recently, we demonstrated that manipulation of versican levels in the repair tissue (neointima) of balloon-injured arterial wall, through over-expression of V3, dramatically remodels the ECM, resulting in a compact, elastin-enriched and layered neointima that is not only resistant to cholesterol deposition, but also to macrophage ingress [9]. The reduced deposition of cholesterol is consistent with versican’s role in retaining lipoproteins, but the additional advantage of reducing inflammation broadens the benefits of targeting the CS-containing versican variants.

Several lines of evidence point to versican as an important mediator of inflammation, as well as being a prometastatic agent. Versican V1 stimulates proinflammatory cytokines tumor necrosis factor- (TNF-) and interleukin 6 through the Toll 2 receptor [4], and mediates leukocyte aggregation through binding to P-selectin glycoprotein ligand-1 [17]. Further, versican promotes adhesion of monocytes in a hyaluronan-dependent manner, an interaction that that is significantly reduced in matrices in which versican has been reduced by versican antisense or by V3-expression [9], versican shRNA [6] or blocked by an antibody to versican [18]. Such findings highlight a potential therapeutic application for targeting versican as anti-inflammatory therapy in the treatment of diseases such as atherosclerosis and cancer.

The association between versican and metastasis is clearly illustrated by breast and prostate cancers, where elevated levels of peritumoral stromal versican correlate positively with tumor grade and invasiveness [5]. Experimental evidence demonstrating that versican is central to metastasis comes from studies on Lewis lung carcinoma (LLC) cells. Versican from LLC cells directly mediates metastasis through activation of Toll-like receptor 2 on macrophages which then up-regulate TNF-. Elevated expression of versican is seen in myeloid progenitor cells in metastatic lungs in a mouse model of breast cancer and in the metastatic lungs of human patients with breast cancer [3]. Knockdown of versican in the bone marrow progenitor cells significantly impairs lung metastasis in this breast cancer mouse model. In addition, lung metastases that develop in a murine model of bladder cancer could be controlled by targeting tumor expression of versican [6].

In summary, the GAG-containing versican variants are emerging as potential targets for therapeutic intervention across a variety of conditions where tissues are undergoing growth and remodelling associated with expansion, degradation and inflammation. Interestingly and seemingly paradoxically, the variant without GAG chains, V3, acts to counter the changes induced by its parent molecules, dampening growth and inflammation and promoting formation of elastin-rich and differentiated tissues.

Leave a Reply

References

1. Wight TN: Versican: a versatile extracellular matrix proteoglycan in cell biology. Curr Opin Cell Biol 2002, 14:617-623.
2. Wight TN, Merrilees MJ: Proteoglycans in atherosclerosis and restenosis: key roles for versican. Circ Res 2004, 94:1158-1167.
3. Gao D, Joshi N, Choi H, Ryu S, Hahn M, Catena R, Sadik H, Argani P, Wagner P, Vahdat LT, et al.: Myeloid progenitor cells in the premetastatic lung promote metastases by Inducing mesenchymal to epithelial transition. Cancer Res 2012, In press.
4. Kim S, Takahashi H, Lin WW, Descargues P, Grivennikov S, Kim Y, Luo JL, Karin M: Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature 2009, 457:102-106.
5. Ricciardelli C, Sakko AJ, Ween MP, Russell DL, Horsfall DJ: The biological role and regulation of versican levels in cancer. Cancer Metastasis Rev 2009, 28:233-245.
6. Said N, Sanchez-Carbayo M, Smith SC, Theodorescu D: RhoGDI2 suppresses lung metastasis in mice by reducing tumor versican expression and macrophage infiltration. J Clin Invest 2012, In press
7. Merrilees M, Ching P, Beaumont B, Hinek A, Wight T, Black P: Changes in elastin,elastin binding protein and versican in alveoli in chronic obstructive pulmonary disease. Respir Res 2008, 18:41-50.
8. Zimmermann DR, Ruoslahti E: Multiple domains of the large fibroblast proteoglycan, versican. EMBO J 1989, 8:2975-2981.
9. Merrilees MJ, Beaumont BW, Braun KR, Thomas AC, Kang I, Hinek A, Passi A, Wight TN: Neointima formed by arterial smooth muscle cells expressing versican variant v3 is resistant to lipid and macrophage accumulation. Arterioscler Thromb Vasc Biol 2011, 31:1309-1316.
10. Domenzain-Reyna C, Hernandez D, Miquel-Serra L, Docampo MJ, Badenas C, Fabra A, Bassols A: Structure and regulation of the versican promoter: the versican promoter is regulated by AP-1 and TCF transcription factors in invasive human melanoma cells. J Biol Chem 2009, 284:12306-12317.
11. Rahmani M, Wong BW, Ang L, Cheung CC, Carthy JM, Walinski H, McManus BM: Versican: signaling to transcriptional control pathways. Can J Physiol Pharmacol 2006, 84:77-92.
12. Kenagy RD, Plaas AH, Wight TN: Versican degradation and vascular disease. Trends Cardiovasc Med 2006, 16:209-215.
13. Borén J, Olin K, Lee I, Chait A, Wight TN, Innerarity TL: Identification of a principal proteoglycan binding site in LDL: A single point mutation in apo B-100 severely affects proteoglycan interaction without affecting LDL receptor binding. J. Clin. Invest. 1998, 101:2658-2664.
14. Merrilees MJ, Lemire JM, Fischer JW, Kinsella MG, Braun KR, Clowes AW, Wight TN: Retrovirally mediated overexpression of versican v3 by arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in vitro and in neointima after vascular injury. Circ Res 2002, 90:481-487.
15. Huang R, Merrilees MJ, Braun K, Beaumont B, Lemire J, Clowes AW, Hinek A, Wight TN: Inhibition of versican synthesis by antisense alters smooth muscle cell phenotype and induces elastic fiber formation in vitro and in neointima after vessel injury. Circ Res 2006, 98:370-377.
16. Hinek A, Braun KR, Liu K, Wang Y, Wight TN: Retrovirally mediated overexpression of versican v3 reverses impaired elastogenesis and heightened proliferation exhibited by fibroblasts from Costello syndrome and Hurler disease patients. Am J Pathol 2004, 164:119-131.
17. Zheng PS, Vais D, Lapierre D, Liang YY, Lee V, Yang BL, Yang BB: PG-M/versican binds to P-selectin glycoprotein ligand-1 and mediates leukocyte aggregation. J Cell Sci 2004, 117:5887-5895.
18. Potter-Perigo S, Johnson PY, Evanko SP, Chan CK, Braun KR, Wilkinson TS, Altman LC, Wight TN: Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion. Am J Respir Cell Mol Biol 2010, 43:109-120.

doi: 10.1016/j.ceb.2013.01.001

Current Comments contain the personal views of the authors who, as experts, reflect on the direction of future research in their field.

Home – Cell Biology – Genetics & Development – Pharmacology - Structural Biology