Nuclear envelope disease and chromatin organization

The fifth U.K. meeting on nuclear envelope disease and chromatin brought together international experts from across the field of nuclear envelope biology to discuss the advancements in a class of tissue-specific degenerative diseases called the laminopathies. Clinically, these range from relatively mild fat-wasting disorders to the severe premature aging condition known as Hutchinson-Gilford progeria syndrome. Since the first association of the nuclear envelope with human inherited disease in 1994, there has been an exponential increase in an unexpected variety of functions associated with nuclear envelope proteins, ranging from mechanical support and nucleocytoskeletal connections to regulation of chromatin organization and gene expression. This Biochemical Society Focused Meeting reinforced the functional complexity of nuclear-associated diseases, revealed new avenues to be investigated and highlighted the signalling pathways suitable as therapeutic targets.     

Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies

Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.     

Nuclear envelope proteins: Identification of lamin B subtypes

The lamins are a group of nuclear envelope proteins thought to form a structural layer at the nuclear periphery. Lamins A, B and C occur in many cell types although lamin C, a subtype of lamin A, is relatively decreased in chicken cells. A subtype of lamin B has been found in chicken cells. This subtype, called lamin B1, is slightly larger and more acidic than the quantitatively major subtype now called lamin B2. The lamin B subtypes have very similar primary sequences and share a distinctive topology. Two lamin B subtypes have not been observed in mammalian tissues but have been found in three avian tissues, erythrocytes of mature chickens and liver and erythrocytes of 11- to 13-day-old embryos. As these tissues differ widely in metabolic activity, both subtypes appear to be constitutive nuclear proteins.     

The arrangement of proteins on the deoxyribonucleic acid in chromatin

The arrangement of the protein component on the DNA of the chromatin complex was studied by comparing the rate of release of oligonucleotides and of protein after addition of deoxyribonuclease I and deoxyribonuclease II to rat thymus chromatin. Also the action of deoxyribonuclease I on normal chromatin and on chromatin depleted of non-histone protein was compared, to elucidate the role of the latter protein in chromatin structure. As a preliminary to the above, the rate of action of deoxyribonuclease I on DNA and on chromatin at the same DNA concentration, and the dependence of the action of this enzyme on the Mg(2+) concentration, were studied. It was found that: (1) little if any DNA in chromatin is present in extensive, truly ;free' zones, i.e. completely uncovered by protein; (2) at relatively low concentrations of added Mg(2+), deoxyribonuclease I degrades chromatin more rapidly than DNA; (3) the non-histone protein is not attached directly to the DNA in chromatin.     

Nuclear envelope proteins from Spisula solidissima germinal vesicles

Biochemical characterization of the nuclear pore complex requires quantities of highly enriched nuclear pore complex material which could not be obtained with available procedures. We have developed a technique for the mass isolation of nuclear envelopes from germinal vesicles of Spisula solidissima oocytes. The nuclear pore complex is intact after isolation as judged ultrastructurally. The nuclear envelope and the pore complex fibrous lamina fraction are highly purified with respect to nuclear and cytoplasmic protein contaminants. The fibrous lamina pore complex (FLPC) as presently isolated consists of about eight major proteins, three of which are phosphorylated. Comparison of the FLPC of clams with that of rat reveals three proteins of similar molecular weights, which may be pore complex-specific proteins. The clam nuclear envelope has only one protein (67000) in the molecular weight range which is comparable to the three lamina of rat nuclei. The solubility, intermolecular cross-linking and in vitro phosphorylation of this protein resemble that of one of the lamina of rat nuclei. The other lamina of the rat nuclear envelope are not essential proteins of the pore complex because they are not present in the clam FLPC preparation. They also seem non-essential for the maintenance of the fibrous lamina.     

New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.     

New arrangement of proteins and lipids in the stratum corneum cornified envelope

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer-Glx bonds and FFA-Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.     

Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos

Chicken sperm chromatin initiated an assembly of interphase-like nuclei in a cell-free cytoplasmic preparation from 1-6 h old Drosophila melanogaster embryos. The formation of these interphase-like nuclei from the condensed sperm chromatin happened in a series of distinct steps. Anti-Drosophila lamin monoclonal antibody stained the assembled nuclei in a pattern indistinguishable from normal Drosophila nuclei. This assembly process required an ATP regenerating system and could be blocked by the addition of novobiocin into the cell-free extract.     

Spontaneous heteromerization of gammaretrovirus envelope proteins: a possible novel mechanism of retrovirus restriction

The env gene of gammaretroviruses encodes a glycoprotein conserved among diverse retroviruses, except for the domains involved in receptor binding. Here we show that pairs of gammaretrovirus envelope proteins (from Friend virus and GALV or xenotropic viruses) assemble into heteromers when coexpressed. This assembly results in a strong inhibition of infectivity. An unrelated envelope protein does not assemble in heteromers with the gammaretrovirus glycoproteins tested and does not affect their infectivity, demonstrating the specificity of the mechanism we describe. We propose that the numerous copies of endogenous retroviral env genes conserved within mammalian genomes act as restriction factors against infectious retroviruses.     

Protamine-like proteins: evidence for a novel chromatin structure.

Protamine-like (PL) proteins are DNA-condensing proteins that replace somatic-type histones during spermatogenesis. Their composition suggests a function intermediate to that of histones and protamines. Although these proteins have been well characterized at the chemical level in a large number of species, particularly in marine invertebrates, little is known about the specific structures arising from their interaction with DNA. Speculation concerning chromatin structure is complicated by the high degree of heterogeneity in both the number and size of these proteins, which can vary considerably even between closely related species. After careful examination and comparison of the protein sequences available to date for the PL proteins, we propose a model for a novel chromatin structure in the sperm of these organisms that is mediated by somatic-type histones, which are frequently found associated with these proteins. This structure supports the concept that the PL proteins may represent various evolutionary steps between a sperm-specific histone H1 precursor and true protamines. Potential post-translational modifications and the control of PL protein expression and deposition are also discussed.     

Nuclear envelope: nuclear pore complexity

A new study shows that the filamentous fungus, Aspergillus nidulans, which has a closed mitosis, does not maintain a continuous permeability barrier during mitosis. This work challenges current views of the differences between closed and open mitosis and has implications for understanding mitotic specific changes in the nuclear pore complex and Ran GTPase system in lower eukaryotes.     

Topoisomerase II: a fitted mechanism for the chromatin landscape

The mechanism by which type-2A topoisomerases transport one DNA duplex through a transient double-strand break produced in another exhibits fascinating traits. One of them is the fine coupling between inter-domainal movements and ATP usage; another is their preference to transport DNA in particular directions. These capabilities have been inferred from in vitro studies but we ignore their significance inside the cell, where DNA configurations markedly differ from those of DNA in free solution. The eukaryotic type-2A enzyme, topoisomerase II, is the second most abundant chromatin protein after histones and its biological roles include the decatenation of newly replicated DNA and the relaxation of polymerase-driven supercoils. Yet, topoisomerase II is also implicated in other cellular processes such as chromatin folding and gene expression, in which the topological transformations catalysed by the enzyme are uncertain. Here, some capabilities of topoisomerase II that might be relevant to infer the enzyme performance in the context of chromatin architecture are discussed. Some aspects addressed are the importance of the DNA rejoining step to ensure genome stability, the regulation of the enzyme activity and of its putative structural role, and the selectively of DNA transport in the chromatin milieu.