SUMOylation and PARylation cooperate to recruit and stabilize SLX4 at DNA damage sites

SUMOylation plays important roles in the DNA damage response. However, whether it is important for interstrand crosslink repair remains unknown. We report that the SLX4 nuclease scaffold protein is regulated by SUMOylation. We have identified three SUMO interaction motifs (SIMs) in SLX4, mutating all of which abrogated the binding of SLX4 to SUMO-2 and covalent SLX4 SUMOylation. An SLX4 mutant lacking functional SIMs is not recruited to PML nuclear bodies nor stabilized at laser-induced DNA damage sites. Additionally, we elucidated a novel role for PARylation in the recruitment of SLX4 to sites of DNA damage. Combined, our results uncover how SLX4 is regulated by post-translational modifications.     

Role of interleukin-7 in degenerative and inflammatory joint diseases

IL-7 is known foremost for its immunostimulatory capacities, including potent T cell-dependent catabolic effects on bone. In joint diseases like rheumatoid arthritis and osteoarthritis, IL-7, via immune activation, can induce joint destruction. Now it has been demonstrated that increased IL-7 levels are produced by human articular chondrocytes of older individuals and osteoarthritis patients. IL-7 stimulates production of proteases by IL-7 receptor-expressing chondrocytes and enhances cartilage matrix degradation. This indicates that IL-7, indirectly via immune activation, but also by a direct action on cartilage, contributes to joint destruction in rheumatic diseases.     

Structural studies of the carbohydrate moieties of lectins from potato (Solanum tuberosum) tubers and thorn-apple (Datura stramonium) seeds

1. Methylation analysis of potato (Solanum tuberosum) lectin and thorn-apple (Datura stramonium) lectin confirmed previous conclusions that both glycoproteins contained high proportions of l-arabinofuranosides and lesser amounts of d-galactopyranosides. The arabinofuranosides are present in both lectins as short unbranched chains containing 1→2- and 1→3-linkages, which are known to be linked to hydroxyproline. Galactopyranosides are present as monosaccharides, which are known to be attached to serine, in potato lectin and as both the monosaccharide and the 1→3-linked disaccharide in Datura lectin. 2. Alkaline digestion of potato lectin and subsequent separation of the components by gel filtration led to the isolation of four fractions corresponding to the mono-, di-, tri- and tetra-arabinosides of hydroxyproline. The latter two fractions accounted for over 70% of the total hydroxyproline. 3. Methylation analysis was used to show that the triarabinoside contained only 1→2-linkages between sugars, but that the tetra-arabinoside contained both 1→2- and 1→3-linkages. Direct-insertion mass spectrometry of these compounds using electron impact and chemical ionization, in a comparison with other known structural patterns, was used to determine the sequences of the sugars, which were Araƒ1→2Araƒ1→2Araƒ1→Hyp and Araƒ1→3Araƒ1→2Araƒ1→2Araƒ 1→Hyp. 4. On the basis of optical rotation it had previously been suggested [Allen, Desai, Neuberger & Creeth (1978) Biochem. J. 171, 665–674] that all the arabinose of potato lectin was present as the β-l-furanoside. However, measurement of the optical rotations of the hydroxyprolyl arabinosides showed that whereas the diarabinoside had a molar rotation ([m]) value close to that predicted, the triarabinoside was more dextrorotatory and the tetra-arabinoside was less dextrorotatory than expected. Possible explanations for these findings are that, although the di- and tri-arabinosides contain exclusively β-arabinofuranosides, in the tri-arabinoside, interactions between pentose units lead to an enhanced positive rotation. The tetra-arabinoside, however, is proposed to contain a single α-arabinofuranoside residue, which is responsible for the lower than expected positive rotation. The observed rotation of the tetra-arabinoside was found to be close to the theoretical value predicted on that basis. Furthermore, the action of a specific α-arabinofuranosidase on the tetrasaccharide was to remove a single arabinose residue, presumably the terminal non-reducing sugar, and to produce a product that was indistinguishable on electrophoresis from the triarabinoside. Changes in rotation were compatible with this assumption. 5. It is concluded that the structures of the hydroxyprolyl tri- and tetra-arabinosides of potato lectin are: βAraƒ1→2βAraƒ1→2βAraƒ1→Hyp and αAraƒ1→3βAraƒ1→2βAraƒ 1→2βAraƒ1→Hyp. These are identical with compounds that have been isolated from the insoluble hydroxyproline-rich glycoproteins of plant cell walls.     

Activating and inhibitory Fcγ receptors in rheumatoid arthritis: from treatment to targeted therapies

Fcγ receptors (FcγRs) bind the constant Fc region of IgG molecules. IgG/antigen-containing immune complexes elicit a variety of effector functions in cells that express activating FcγRs. Because activating FcγRs are present on cells from the innate immune system, such as dendritic cells, monocytes/macrophages and granulocytes, these IgG receptors form a crucial link between the innate and the acquired immune systems. Recently, the ability to detect the inhibitory FcγRIIb on cells has indicated an imbalance between activating and inhibitory FcγRs in rheumatoid arthritis. This progress offers an opportunity to study modulation of FcγR balance and could stimulate development of FcγR-directed immunotherapy.     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Length variation and secondary structure of introns in the Mlc1 gene in six species of Drosophila

A nearly universal feature of intron sequences is that even closely related species exhibit a large number of insertion/deletion differences. The goal of the analysis described here is to test whether the observed pattern of insertion/deletion events in the genealogy of the myosin alkali light chain (Mlc1) gene is consistent with neutrality, and if not, to determine the underlying forces of evolutionary change. Mlc1 pre-mRNA is alternatively spliced, and one constraint is that signals necessary for tissue-specificity of directed splicing must be conserved. If the total length of an intron is functionally constrained, then the distribution of indels on branches of the gene genealogy should reflect a departure from randomness. Here we perform a phylogenetic analysis, inferring ancestral states wherever possible on a phylogeny of 29 alleles of Mlc1 from six species of Drosophila. Observed patterns of indels on the genealogy were compared to those from simulated data, with the result that we cannot reject the null hypothesis of neutrality. A clear departure from a neutral prediction was seen in the excess folding free energy predicted for the introns flanking the alternatively spliced exon. Relative rate tests also suggest a retardation in the rate of Mlc1 sequence evolution in the simulans clade.      

Some properties of the lectin from Datura stramonium (thorn-apple) and the nature of its glycoprotein linkages

The lectin from Datura stramonium (thorn-apple; Solanaceae) has been purified by affinity chromatography and shown to be a glycoprotein containing about 40% (w/w) of carbohydrate. The most abundant amino acids are hydroxyproline, cystine, glycine and serine. Results obtained by gel filtration in 6m-guanidinium chloride on Sepharose 4B suggest that it has a subunit mol.wt. of about 30000 and that it probably associates into dimers. The lectin is inhibited specifically by chitin oligosaccharides and bacterial-cell-wall oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean (Glycine max) lectin and fetuin are also strong inhibitors of Datura lectin, indicating that it interacts with internal N-acetylglucosamine residues. Its specificity is similar to, but not identical with, that of potato (Solanum tuberosum) lectin. After prolonged proteolytic digestion of reduced and S-carboxymethylated or S-aminoethylated derivatives of the lectin, glycopeptides of mol.wt. of about 18000 were isolated. The glycopeptides contained all the carbohydrate and hydroxyproline of the original glycoprotein, and lesser amounts of serine, S-carboxymethylcysteine and other amino acids. The arabinose residues of the glycoprotein are present as β-l-arabinofuranosides linked to the polypeptide chain through the hydroxyproline residues, and can be removed by mild acid treatment; the ratio of arabinose to hydroxyproline is 3.4:1. Some of the serine residues of the polypeptide chain are substituted with one or two α-galactopyranoside residues, most of which can be removed by the action of α-galactosidase. The galactose residues are more easily removed from the acid-treated glycopeptide (from which arabinose has been removed) than from the complete glycopeptide, indicating a steric hindrance of the galactosidase action by the adjacent chains of arabinosides. There is a slow release of galactose residues by a process of β-elimination in 0.5m-NaOH (pH13.7) from the complete glycopeptide, and a fairly rapid release of galactose by this process from the acid-treated glycopeptide, which lacks arabinose. This is probably due to the inhibitory effect of the negative charge on the adjacent arabinofuranoside residues. The similarities and differences between the lectins from Datura and potato are discussed, as are their structural resemblance to glycopeptides that have been isolated from plant cell walls.     

Regulation of the nitric oxide system in human adipose tissue

Nitric oxide (NO) is involved in adipose tissue biology by influencing adipogenesis, insulin-stimulated glucose uptake, and lipolysis. The enzymes responsible for NO formation in adipose cells are endothelial NO synthase (eNOS) and inducible NO synthase (iNOS), whereas neuronal NO synthase (bNOS) is not expressed in adipocytes. We characterized the expression pattern and the influence of adipogenesis, obesity, and weight loss on genes belonging to the NO system in human subcutaneous adipose cells by combining in vivo and in vitro studies. Expression of most of the genes known to belong to the NO system (eNOS, iNOS, subunits of the soluble guanylate cyclase, and both genes encoding cGMP-dependent protein kinases) in human adipose tissue and isolated human adipocytes was detected. In vitro adipogenic differentiation increased the expression level of iNOS significantly, whereas eNOS expression levels were not influenced. The genes encoding eNOS, iNOS, and cGMP-dependent protein kinase 1 were expressed at higher levels in obese women. Expression of these genes, however, was not influenced by 5% weight loss. Insulin and angiotensin II (Ang II) increased NO production by human preadipocytes in vitro. Increased eNOS and iNOS expression in adipocytes and local effects of insulin and Ang II may increase adipose tissue production of NO in obesity.     

Subunit exchange of lens α-crystallin: a fluorescence energy transfer study with the fluorescent labeled αA-crystallin mutant W9F as a probe

A Trp-free αA-crystallin mutant (W9F) was prepared by site-directed mutation. This mutant appears to be identical to the wild-type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl-specific fluorescent probe, 2-(4′-maleimidylanilino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange between αA- and αA-crystallins as well as between αA- and αB-crystallins, studied by measurement of fluorescence resonance energy transfer. Energy transfer was observed between Trp (donor, with emission maximum at 336 nm) of wild-type αA- or αB-crystallin and MIANS (acceptor, with absorption maximum at 313 nm) of labeled W9F when subunit exchange occurred. Time-dependent decrease of Trp and increase of MIANS fluorescence were recorded. The exchange was faster at 37°C than at 25°C. The energy transfer efficiency was greater between homogeneous subunits (αA-αA) than between heterogeneous subunits (αA-αB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between αA and αB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between αA-αA and αA-αB indicated that αA- and αB-crystallins were not necessarily structurally equivalent.