The role of antigen presenting cells in multiple sclerosis

Multiple sclerosis (MS) is a debilitating T cell mediated autoimmune disease of the central nervous system (CNS). Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) have given light to cellular mechanisms involved in the initiation and progression of this organ-specific autoimmune disease. Within the CNS, antigen presenting cells (APC) such as microglia and astrocytes participate as first line defenders against infections or inflammation. However, during chronic inflammation they can participate in perpetuating the self-destructive environment by secretion of inflammatory factors and/or presentation of myelin epitopes to autoreactive T cells. Dendritic cells (DC) are also participants in the presentation of antigen to T cells, even within the CNS. While the APCs alone are not solely responsible for mediating the destruction to the myelin sheath, they are critical players in perpetuating the inflammatory milieu. This review will highlight relevant studies which have provided insight to the roles played by microglia, DCs and astrocytes in the context of CNS autoimmunity.     

6-Carboxyfluorescein and structurally similar molecules inhibit DNA binding and repair by O⁶-alkylguanine DNA alkyltransferase

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. These activities protect normal cells and tumor cells against drugs that alkylate DNA; drugs that inactivate AGT are under test as chemotherapeutic enhancers. In studies using 6-carboxyfluorescein (FAM)-labeled DNAs, AGT reduced the fluorescence intensity by ∼40% at binding saturation, whether the FAM was located at the 5′ or the 3′ end of the DNA. AGT protected residual fluorescence from quenching, indicating a solute-inaccessible binding site for FAM. Sedimentation equilibrium analyses showed that saturating AGT-stoichiometries were higher with FAM-labeled DNAs than with unlabeled DNAs, suggesting that the FAM provides a protein binding site that is not present in unlabeled DNAs. Additional fluorescence and sedimentation measurements showed that AGT forms a 1:1 complex with free FAM. Active site benzylation experiments and docking calculations support models in which the primary binding site is located in or near the active site of the enzyme. Electrophoretic analyses show that FAM inhibits DNA binding (IC₅₀ ∼ 76 μM) and repair of DNA containing an O⁶-methylguanine residue (IC₅₀ ∼ 63 μM). Similar results were obtained with other polycyclic aromatic compounds. These observations demonstrate the existence of a new class of non-covalent AGT-inhibitors. After optimization for binding-affinity, members of this class might be useful in cancer chemotherapy.     

Identification of the allosteric regulatory site of insulysin

Background

Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer''s disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP.

Principal Findings

The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits.

Conclusions/Significance

Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.     

Nanostructured transducer surfaces for electrochemical biosensor construction—Interfacing the sensing component with the electrode

For fabrication of effective electrochemical biosensors, interfacing the biomolecular receptor with the underlying transducer represents a critical step. The actual approach taken depends on the tethering layer covering the transducer, which is typically either a conducting polymeric matrix, or a thin film, such as an alkanethiol monolayer. Non-specific immobilisation methods can be either covalent, or non-covalent affinity attachment, with multipoint electrostatic attachment of the sensing biomolecule to either a polyanionic or polycationic layer representing the most common approach. Many specific affinity immobilisation strategies exist, but the majority make use of one of two binding systems. The first relies on the specific and strong affinity between biotin and proteins of the avidin family, with both bioreceptor and transducer bearing pendant biotins and avidin used as the crosslinker. The second approach employs a metal chelating group on the transducer to which can be bound a polyhistidine tag present on the N- or C-terminus of the receptor protein and which can be introduced genetically, when the expression sequence for a recombinant proteins is designed.     

Building a tree of knowledge: analysis of bitter molecules

A phylogenetic-like tree of structural fragments has been constructed to extract useful insights from a structural database of bitter molecules. The tree of structural fragments summarizes the substructural groups present in the molecules from the bitter database. These structural fragments are compared with a large number of random molecules to highlight substructures specific to bitter molecules. This organization of the structures enabled the detection of structure-activity relationships for the bitter molecules through the construction of R-tables. Key structural groups, able to distinguish between bitter and random molecules, were identified through an analysis of the tree. This information can be used to further understand which structural components are involved in producing a bitter taste.     

Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish

The myostatin (MSTN)-null phenotype in mammals is characterized by extreme gains in skeletal muscle mass or "double muscling" as the cytokine negatively regulates skeletal muscle growth. Recent attempts, however, to reproduce a comparable phenotype in zebrafish have failed. Several aspects of MSTN biology in the fishes differ significantly from those in mammals and at least two distinct paralogs have been identified in some species, which possibly suggests functional divergence between the different vertebrate classes or between fish paralogs. We therefore conducted a phylogenetic analysis of the entire MSTN gene sub-family. Maximum likelihood, Bayesian inference, and bootstrap analyses indicated a monophyletic distribution of all MSTN genes with two distinct fish clades: MSTN-1 and -2. These analyses further indicated that all Salmonid genes described are actually MSTN-1 orthologs and that additional MSTN-2 paralogs may be present in most, if not all, teleosts. An additional zebrafish homolog was identified by BLAST searches of the zebrafish Hierarchical Tets Generation System database and was subsequently cloned. Comparative sequence analysis of both genes (zebrafish MSTN (zfMSTN)-1 and -2) revealed many differences, primarily within the latency-associated peptide regions, but also within the bioactive domains. The 2-kb promoter region of zfMSTN-2 contained many putative cis regulatory elements that are active during myogenesis, but are lacking in the zfMSTN-1 promoter. In fact, zfMSTN-2 expression was limited to the early stages of somitogenesis, whereas zfMSTN-1 was expressed throughout embryogenesis. These data suggest that zfMSTN-2 may be more closely associated with skeletal muscle growth and development. They also resolve the previous ambiguity in classification of fish MSTN genes.     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

A possible role for the covalent heme-protein linkage in cytochrome c revealed via comparison of N-acetylmicroperoxidase-8 and a synthetic, monohistidine-coordinated heme peptide

N-Acetylmicroperoxidase-8 (1) contains heme and residues 14-21 of horse mitochondrial cytochrome c (cyt c). The two thioether bonds linking protein to heme in cyt c are present in 1, and the native axial ligand His-18 remains coordinated to iron. As an approach to probing structural or functional roles played by the double covalent heme-protein linkage in cyt c, we have initiated a study in which the properties of 1 are compared with those of a synthetic mono-His coordinated heme peptide containing a single covalent linkage (2). One consequence of the greater conformational restriction imposed on peptide conformation in 1 is that His-Fe(III) coordination is approximately 1.4 kcal/mol more favorable in 1 than in 2. This highlights a clear advantage conferred to cyt c by having two covalent heme-protein linkages rather than one: greater thermodynamic stability of the protein fold. EPR (11 K) and resonance Raman (298 K) studies reveal that 1 and 2 exhibit a thermal high-spin/low-spin ferric equilibrium but that low-spin character is considerably more pronounced in 1. In addition, the thioether 2-(methylthio)ethanol (MTE) coordinates 0.5 kcal/mol more strongly to 1 than to 2 in 60:40 H(2)O/CH(3)OH and only triggers the expected conversion of iron to the low-spin state characteristic of ferric cyt c in the case of 1. This demonstrates that the axial ligand field provided by an imidazole and a thioether is too weak to induce a high-spin to low-spin conversion in a ferric porphyrin. Our results suggest that a conformationally constrained double covalent heme-protein linkage, as exists in 1 and its parent protein cyt c, is an effective solution that nature has evolved to circumvent this limitation. We propose that the stronger His-Fe(III) coordination enabled by such a linkage serves to markedly enhance the effective ligand field strength of His-18. Our studies with 1 and 2 suggest that a double covalent linkage in cyt c may also enable energetically more favorable trans ligation of Met-80 than would be possible if only a single linkage were present. This would serve to further increase the stability of the protein fold and perhaps to increase the effective ligand field strength of Met-80 as well.     

Certainly can't live without this: SIRT6

Cellular metabolic rates might regulate aging by impinging on genomic stability through the DNA repair pathways. A new study published in Cell (Mostoslavsky et al., 2006) reports that deficiency in one of the mammalian Sir2 homologs, SIRT6, results in genome instability through the DNA base excision repair pathway and leads to aging-associated degenerative phenotypes.