Histone variant macroH2A contains two distinct macrochromatin domains capable of directing macroH2A to the inactive X chromosome

Chromatin on the inactive X chromosome (Xi) of female mammals is enriched for the histone variant macroH2A that can be detected at interphase as a distinct nuclear structure referred to as a macro chromatin body (MCB). Green fluorescent protein-tagged and Myc epitope-tagged macroH2A readily form an MCB in the nuclei of transfected female, but not male, cells. Using targeted disruptions, we have identified two macrochromatin domains within macroH2A that are independently capable of MCB formation and association with the Xi. Complete removal of the non-histone C-terminal tail does not reduce the efficiency of association of the variant histone domain of macroH2A with the Xi, indicating that the histone portion alone can target the Xi. The non-histone domain by itself is incapable of MCB formation. However, when directed to the nucleosome by fusion to core histone H2A or H2B, the non-histone tail forms an MCB that appears identical to that of the endogenous protein. Mutagenesis of the non-histone portion of macroH2A localized the region required for MCB formation and targeting to the Xi to an ~190 amino acid region.     

Deletions in Xq26.3–q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern

High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3–q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother’s white blood cells and 80–85% of the sister’s leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing ∼13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed ∼12 Mb of DNA around the FMR1 gene. Cytogenetic and molecular data revealed that ∼50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3–q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3–q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients. Received: 19 December 1996 / Accepted: 13 March 1997     

ELECTRON MICROSCOPE STUDIES OF CRYSTAL-COLLAGEN RELATIONSHIPS IN BONE : IV. THE OCCURRENCE OF CRYSTALS WITHIN COLLAGEN FIBRILS

Crystals are seen occasionally within the diameter of transversely sectioned collagen fibrils near the calcification front of newly formed bone.     

A 2.6 A structure of a serpin polymer and implications for conformational disease.

The function of the serpins as proteinase inhibitors depends on their ability to insert the cleaved reactive centre loop as the fourth strand in the main A beta-sheet of the molecule upon proteolytic attack at the reactive centre, P1-P1'. This mechanism is vulnerable to mutations which result in inappropriate intra- or intermolecular loop insertion in the absence of cleavage. Intermolecular loop insertion is known as serpin polymerisation and results in a variety of diseases, most notably liver cirrhosis resulting from mutations of the prototypical serpin alpha1-antitrypsin. We present here the 2.6 A structure of a polymer of alpha1-antitrypsin cleaved six residues N-terminal to the reactive centre, P7-P6 (Phe352-Leu353). After self insertion of P14 to P7, intermolecular linkage is affected by insertion of the P6-P3 residues of one molecule into the partially occupied beta-sheet A of another. This results in an infinite, linear polymer which propagates in the crystal along a 2-fold screw axis. These findings provide a framework for understanding the uncleaved alpha1-antitrypsin polymer and fibrillar and amyloid deposition of proteins seen in other conformational diseases, with the ordered array of polymers in the crystal resulting from slow accretion of the cleaved serpin over the period of a year.     

The Epidemiology of Helicobacter pylori and Public Health Implications

This article presents a review of the literature on the epidemiology and public health implications of Helicobacter pylori infection published from April 2008 through to March 2009. The authors used MeSH terms "Helicobacter infections epidemiology,""Helicobacter infections prevention and control" to search multiple databases (PubMed, Embase, Cochrane, Cochrane Library, EBMR, BIOSIS), and independently searched PubMed using the term "Helicobacter" with "Epidemiology,""Transmission,""Prevalence" or "Environment." Articles without topical relevance were excluded. Two additional papers known to the authors were added. The identified literature is summarized by subtopic: reviews; prevalence; incidence; transmission; risk factors; and public health policy.     

New insight into serpin polymerization and aggregation

We recently solved the crystallographic structure of a dimeric form of the serpin antithrombin which has fundamentally changed the way we think about serpin polymerization. Like for other diseases that have protein deposition as a hallmark, the serpinopathies are associated with discrete inter-protomer linkage followed by subsequent association into larger fibrils and aggregates. Polymerization of the serpins is an off-pathway event that occurs during folding in the endoplasmic reticulum. Our structure reveals the nature of the polymerogenic folding intermediate, the reason that the inter-protomer linkage is hyperstable, and suggests a mechanism of lateral association of polymers into soluble fibrils and insoluble aggregates. While the basis of cellular toxicity is still unclear, novel therapeutic approaches targeting the folding intermediate or the lateral association event are now conceivable.Key words: aggregation, conformation, folding, polymerization, serpin, structure, toxicityThe serpins are serine protease inhibitors that utilize a unique and well characterized β-sheet expansion event as a necessary part of their mechanism.^1–3 This conformational/topological change from a five-stranded (N in Fig. 1) to a six-stranded β-sheet A (L in Fig. 1) results in a doubling of the protein''s stability, and is driven by a free-energy term of around −32 kcal/mol.⁴ Thus, the native form of serpins is metastable in defiance of the Anfinsen principle,⁵ requiring a folding pathway that kinetically traps the five-stranded state. This unusual protein folding requirement allows for an off-pathway event known as polymerization, where one protomer completes the A sheet of another.⁶ For secreted serpins, this occurs in the endoplasmic reticulum (ER) where polymers are seen to accumulate as insoluble proteinaceous inclusions. Polymerization has been described for several serpins and is always associated with a loss of functional levels due to accumulation within the cell, and occasionally it is associated with the death of the secretory cells through a poorly understood mechanism.^7–9 The best examples of the latter are provided by the Z variant of α₁-antitrypsin (α₁AT) leading to liver disease and the Syracuse variant of neuroserpin that causes early onset dementia.Open in a separate windowFigure 1Serpin folding and polymerization. The pathway of serpin folding proceeds from the unfolded state (U) to the native state (N) via a stable intermediate (M*). The native conformation is the only active state, and is composed of a five-stranded A sheet (red) and a 20 residue reactive centre loop (RCL, yellow). Serpin inhibitory function requires the native conformation to be a kinetically trapped metastable state. Completion of sheet A by incorporation of the RCL as strand 4, to form the latent (L) state, results in the doubling of the serpin''s thermodynamic stability (the six strands are labelled on L). Folding and unfolding of native serpins is known to proceed via a stable intermediate denoted M*, which also corresponds to the polymerogenic form.^24–26 The key feature of the M* state is that strand 5 is not yet incorporated into sheet A, and can thus insert in an intermolecular fashion to form off-pathway polymers (P, each protomer of the pentamer is in a different colour). The polymers have complete A sheets and are thus hyperstable. As a consequence of polymerization, the linker region (cyan), containing helix I, remains unfolded. We hypothesize that the hydrophobic linker (indicated by the oval) is responsible for the lateral association of polymers into insoluble aggregates.We recently solved a crystal structure of a self-terminating (closed) serpin dimer that revealed a large domain swap including the fourth and fifth strands of β-sheet A.¹⁰ We then modelled an open polymerization competent polymer based on the structure (P in Fig. 1) that explained their facile propagation, the hyperstability and flexibility of the inter-protomer linkage, and also suggested a structure for the polymerogenic folding intermediate (M* in Fig. 1). We proposed that the final step in folding to the native state is the insertion of strand 5 into β-sheet A and the association of the coiled linker domain to the ‘bottom’ of the molecule. This event would leave the fourth strand (the reactive centre loop) accessible to serve as bait for proteolytic attack, necessary for the functioning of the serpin mechanism. While many details are yet to be confirmed, the position of certain polymerogenic mutations on and underlying strand 5A supports the proposal.¹⁰One unexpected implication of our model is the requisite unfolding and exposure of helix I and the following coiled region in linear serpin polymers. Exposure of this ‘linker region’ was verified in linear polymers of serpins antithrombin and α₁AT through limited proteolysis and fluorescence studies, and explains the observation that polymers are hydrophobic and exhibit an increased propensity towards aggregation. Unglycosylated serpins typically aggregate when polymerized in vitro (with heat or low concentrations of chaotrophes), even at vanishingly low concentrations, whereas high concentrations are required to observe aggregation of glycosylated serpin polymers. We hypothesized that aggregation/precipitation occurs via lateral association of linear polymers, either through specific β-strand linkage or non-specific hydrophobic interactions involving the linker region. Sequence analysis of helix I suggest that it is a ‘frustrated’ β-strand¹¹ for several serpins including α₁AT, supporting the idea that aggregates of serpin polymers form through an extended β-sheet mechanism akin to other ‘conformational diseases.’¹²While there are clear parallels between the ‘serpinopathies’ and conformational diseases such as Alzheimer, Huntington and the prion encephalopathies (e.g., ordered intermolecular linkage, β-sheet expansion, cell death, dementia, accumulation of insoluble aggregates, domain-swapping),^12,13 the detailed molecular mechanism revealed by our crystal structure is unique to the serpins. Domain swaps in other proteins are generally characterized by normal activity and stability, and may not play a role in the secondary association event that leads to the toxic species.^14,15 For serpins the domain swap leads to hyperstability and the exposure of hydrophobic regions not seen in the monomeric state. Another key difference is the manner of cellular toxicity and the nature of the toxic species. It is becoming clear that for Alzheimer, Huntington and other conformational diseases the toxic fragments are likely to be the soluble (proto)-fibrils, not the insoluble aggregates (inclusions).^16,17 Serpin polymerization generally leads to disease through loss of secretion of the active species, and only in two special cases is it through gain-of-function cellular toxicity, and although the toxic mechanisms are incompletely resolved, they appear to involve the insoluble aggregates.The most common cause of cirrhosis among children is the homozygous Z mutation in α₁AT.¹⁸ Antitrypsin is expressed at high levels by hepatocytes (1.3–3.5 g/l in blood plasma) and its expression can increase in response to infection and other stimuli.¹⁹ However, only about one-third of the homozygous carriers ever manifest liver disease¹⁸ and it never occurs in carriers of a single Z allele, indicating that hepatocytes are generally well equipped to deal with the mutant protein. Soluble Z α₁AT in the ER binds to chaperones and is subsequently targeted to the ERAD pathway for clearance by the proteosome, and insoluble polymers and aggregates are thought to activate autophagy for degradation in the lysosomes.^20,21 In such a model, accumulation of polymers and cellular toxicity only occur when the proteosomal and autophagic pathways have been saturated by the high level of expression of mutant α₁AT in the liver.²¹ In contrast, neuroserpin is expressed at low levels in neurons and mutation leads to polymerization and dementia in an autosomal dominant fashion.^22,23 However, cell death and disease are still associated with accumulation of inclusion bodies within the ER, and the toxic mechanisms are likely to be similar.⁹In summary, we have elucidated a novel mechanism of serpin polymerization that involves a hyperstable domain swap of a folding intermediate. Formation of linear polymers exposes hydrophobic regions that mediate lateral polymer association and eventually leads to intra ER accretion and cellular toxicity. Our proposal suggests new avenues for the rational design of compounds to combat the diseases caused by serpin polymerization, either through targeting the folding intermediate or the lateral association of soluble polymers.     

Crystal structure of monomeric native antithrombin reveals a novel reactive center loop conformation

The poor inhibitory activity of circulating antithrombin (AT) is critical to the formation of blood clots at sites of vascular damage. AT becomes an efficient inhibitor of the coagulation proteases only after binding to a specific heparin pentasaccharide, which alters the conformation of the reactive center loop (RCL). The molecular basis of this activation event lies at the heart of the regulation of hemostasis and accounts for the anticoagulant properties of the low molecular weight heparins. Although several structures of AT have been solved, the conformation of the RCL in native AT remains unknown because of the obligate crystal contact between the RCL of native AT and its latent counterpart. Here we report the crystallographic structure of a variant of AT in its monomeric native state. The RCL shifted approximately 20 A, and a salt bridge was observed between the P1 residue (Arg-393) and Glu-237. This contact explains the effect of mutations at the P1 position on the affinity of AT for heparin and also the properties of AT-Truro (E237K). The relevance of the observed conformation was verified through mutagenesis studies and by solving structures of the same variant in different crystal forms. We conclude that the poor inhibitory activity of the circulating form of AT is partially conferred by intramolecular contacts that restrain the RCL, orient the P1 residue away from attacking proteases, and additionally block the exosite utilized in protease recognition.