Modulators of the Sphingosine 1-phosphate receptor 1

The Sphingosine 1-phosphate receptor (S1P-R) signaling system has proven to be of biological and medical importance in autoimmune settings. S1P₁-R is a validated drug target for multiple sclerosis (MS) for which FTY720 (Fingolimod), a S1P_1,3–5-R pan-agonist, was recently approved as the first orally active drug for the treatment of relapsing-remitting MS. Transient bradycardia and long half-life are the FTY720 critical pitfalls. This review provides the latest advances on next-generation S1P₁-R modulators from 2012 up to date, with an overview of the chemical structures, structure–activity relationships, and relevant biological and clinical properties.     

Fibrillin-1 regulates the bioavailability of TGFbeta1

We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) beta1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFbeta signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44-49 releases endogenous TGFbeta1, thereby stimulating TGFbeta receptor-mediated Smad2 signaling. This altered TGFbeta1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFbeta1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFbeta1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFbeta-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44-49 can contribute to MFS and related diseases.     

Cloning the Tra1 region of RP1

The Tra1 region of RP1 from a derivative with Tn7 inserted into the kanamycin resistance determinant was cloned, using EcoRI, into the multicopy vector plasmid pBR325. For one orientation of the cloned fragment the resultant chimeric plasmid was very frequently lost from the cell, but in the other orientation it was much more stable and also compatible with RP1. Complementation by the stable chimeric plasmid, pED800, of a series of RP1 tra mutants showed that the mutations of all those retaining sensitivity to the P-specific phages PRR1, Pf3, and PR4, or only to PR4, mapped in the Tra1 region, while only 2 out of 20 amber mutations leading to full P-specific phage-resistance did so.     

Translocation of the interleukin-1 receptor-associated kinase-1 (IRAK-1) into the nucleus

Interleukin-1 (IL-1) signal transduction involves the recruitment of the IL-1 receptor-associated kinase-1 (IRAK-1). Subsequent signaling finally leads to nuclear translocation of NFkappaB. We here show that the association and autophosphorylation of IRAK-1 was already detectable 30 s after IL-1 stimulation of ECV 304 cells. Significant levels of IRAK-1 accumulated in the nucleus 30 min after IL-1 stimulation shown by Western blot analysis and confocal laser scanning microscopy. Nuclear transfer of IRAK-1 upon IL-1 stimulation was confirmed in the murine T cell line EL-4. This characterizes nuclear localization of IRAK-1 as a possibly essential event in the IL-1 signaling cascade.     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

Structure-Function Relationship of the Bik1-Bim1 Complex

1. Download high-res image (157KB)
2. Download full-size image

     

Analysis of the cytochrome P450 1A1 (CYP1A1) gene polymorphism in the ethnic groups of the republic of Bashkortostan

In three ethnic groups from the Republic of Bashkortostan, Russians (N = 451), Tatars (N= 333), and Bashkirs (N = 171), allele, genotype, and haplotype frequency distribution patterns of the CYP1A1 gene single nucleotide polymorphisms, A2455G and T33801C, were investigated. Substantial interethnic differences in the allele frequency distribution patterns of the CYP1A1 polymorphisms A2455G and T3801C (χ ² = 15.61, d.f. = 2, P = 0.0001; and χ ² = 22.10, d.f. = 2, P = 0.0001, respectively) were observed. Pairwise comparison showed that ethnic groups of Tatars and Russians were similar in the A2455G allele frequencies (χ ² = 1.10, d.f. = 1, P = 0.30). However, in case of the T3801C marker, statistically significant differences were revealed (χ ² = 4.56, d.f. = 1, P = 0.032). At the same time, Bashkir ethnic group was found to be statistically significantly different from Russians and Tatars in the CYP1A1 polymorphic allele frequency distribution patterns (χ ² = 15.74, d.f. = 2, P = 0.0001; and χ ² = 7.47, d.f. = 1, P = 0.024, for A2455G, and χ ² = 6.46, d.f. = 1, P = 0.011; and χ ² = 21.36, d.f. = 1, P = 0.0001, for T3801C). Analysis of the CYP1A1 haplotype diversity showed that in terms of the CYP1A1 haplotype frequency distribution patterns, Bashkir ethnic group was statistically significantly different from both Russians (χ ² = 30.07, d.f. = 3, P = 0.0001) and Tatars (χ ² = 11.28, d.f. = 3, P = 0.013). The differences observed were caused by the high frequency of haplotype CYP1A1*2B, which was represented by a combination of rare alleles of the CYP1A1 polymorphisms A2455G and T3801C in Bashkirs (5.81%). On the other hand, the ethnic groups of Russians and Tatars residing in the Republic of Bashkortostan were characterized by similar frequencies of the CYP1A1 haplotypes (χ ² = 6.322, d.f. = 3, P = 0.127). The data obtained could be used in further investigations of the genetic bases of ecology dependant diseases and in the risk groups in the Republic of Bashkortostan.     

Topography of the 1:1 alpha-cyclodextrin-nitromethane inclusion complex

Dissolution of alpha-cyclodextrin (alpha-CD) in 9:1 water-nitromethane smoothly generates the title compound, which crystallizes as the pentahydrate in the orthorhombic space group P2(1)2(1)2(1) with a = 9.452(4), b = 14.299(3), c = 37.380(10) A, and Z = 4. Its crystal structure analysis revealed the alpha-CD macrocycle in an unstrained conformation stabilized through a ring of O-2...O-3' hydrogen bonds between five of the six adjacent glucose residues. The nitromethane is located in the alpha-CD cavity in an orientation parallel to the plane of the macrocycle, and assumes two sites of equal population with the nitro group in excessive thermal motion; the guest is held by van der Waals contacts and C-H...O-type hydrogen bonds to the pyranose H-3 and H-5 protons. The packing of the macrocycles in the crystal lattice is of cage herringbone-type with an extensive intra- and intermolecular hydrogen bonding network. The ready formation of a nitromethane inclusion complex in aqueous nitromethane, and the subtleties of its molecular structure amply demonstrate the ease with which water is expelled from the alpha-CD cavity by a more hydrophobic co-solvent.     

Evolution of the CDKN1C-KCNQ1 imprinted domain

Background  

Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting.     

Organization of the human interleukin-1 receptor antagonist gene IL1HY1

 The interleukin (IL)-1 family of proteins plays an important role in inflammatory and defense mechanisms. The recently characterized IL1HY1 cDNA encodes a new member of the IL-1 receptor antagonist family (IL-1ra). In this report, we describe the complete nucleotide sequence of the human IL1HY1 gene. We sequenced approximately 7600 nucleotides and found four coding exons ranging in size from 55 to 2288 nucleotides. The 5′ untranslated region is formed by one of two alternatively used exons and one invariably present exon which also contains the region encoding the first nine amino acids of the protein. IL1HY1 and IL-1ra intron positions are well conserved within the protein-coding region, providing evidence that these genes arose from a duplication of a primordial IL-1 receptor antagonist gene. Received: 15 October 1999 / Revised: 30 December 1999     

On the role of IS1 in the formation of hybrids between the bacteriophage P1 and the R plasmid NR1

Summary The genomes of bacteriophage P1 derivatives carrying drug resistance genes derived from an R plasmid NR1 were analysed by restriction cleavage and be DNA-DNA hybridization. Two representatives of a class of oversized P1CmSmSu phages were identified as P1 carrying the entire r-determinant of NR1 together with its two flanking, directly repeated IS1. In one case the r-determinant insertion is carried at the site of the residential IS1 of P1, in the other case it is transposed into another region of the P1 genome. Models postulate that the first type resulted from reciprocal recombination within IS1 elements and that the formation of the second type of P1-R hybrid depended both on IS1 mediated transposition and reciprocal recombination. Plaque forming P1Cm or P1CmSm phages are explained as IS1 mediated deletion derivatives of P1CmSmSu, although an alternative model postulates that sometimes P1Cm phages might result from two consecutive transposition events of only one IS1 without involving reciprocal recombination. Secondary P1 derivatives carrying only one IS1 at the site of the original r-determinant or of Cm insertions into P1 must have been produced by reciprocal recombination between the two IS1 flanking the insertions. An implication from this study, that any genetic material carried adjacent to an IS1 element may undergo passive transposition, is discussed.     

Crystal structure of the PB1 domain of NBR1

The scaffold protein NBR1 is involved in signal transmission downstream of the serine/protein kinase from the giant muscle protein titin. Its N-terminal Phox and Bem1p (PB1) domain plays a critical role in mediating protein-protein interactions with both titin kinase and with another scaffold protein, p62. We have determined the crystal structure of the PB1 domain of NBR1 at 1.55A resolution. It reveals a type-A PB1 domain with two negatively charged residue clusters. We provide a structural perspective on the involvement of NBR1 in the titin kinase signalling pathway.     

Dissecting the free energy of formation of the 1:1 actomyosin complex

The behaviour of solutions of pure myosin, of pure F-actin and of the equimolar mixture of myosin and of F-actin is studied. It is found that the chemical potential of the two proteins, in separate solutions, increases monotonically with the increase of protein osmotic pressure. A method is presented to determine the chemical potential of the 1:1 actin-myosin complex formed from equimolar solutions of myosin and of F-actin (as monomer).This is the first evaluation of the chemical potential of actomyosin under conditions similar to those of skeletal muscle. It is found that the filament suspensions of myosin and of the 1:1 actin-myosin complex display a high non-ideal behavior as well as distinctly different energy profiles as a function of protein osmotic pressure. This supports the hypothesis that, in muscle: (a) detached cross-bridge change significantly their free energy when sarcomere is shifting from the relaxed to the active or to the rigor state; and (b) the cross-bridge attachment-detachment process is accompanied by changes of muscle protein osmotic pressure.     

Interaction between RNA1 and the primer precursor in the regulation of Co1E1 replication

The analysis of a large number of independent mutants in the target of one of the inhibitors of pMB1 replication suggests that RNA1 regulates primer formation by base-pairing with the complementary sequence in the primer precursor. We conclude that the number of bases that are involved in the hydrogen bonding responsible for the specificity of the mechanism that controls plasmid replication and incompatibility properties is not much larger than seven. Five of these bases are located in the central loop and two in loop I of the RNA primer cloverleaf structure. Twenty-two single, double or triple mutants, with different nucleotide sequences in these seven bases, maintain an active mechanism of control, though with altered specificity. The efficiency of the inhibition mechanism correlates with the delta G value of the hydrogen bonds between the nucleotides of the two heptamers postulated to be involved in the interaction. The implications of these findings are discussed, and a molecular model of the interaction between RNA1 and the primer precursor is presented.