Crystal and solution structures of fragments of the human leucocyte common antigen-related protein.

Leucocyte common antigen-related protein (LAR) is a post-synaptic type I transmembrane receptor protein that is important for neuronal functionality and is genetically coupled to neuronal disorders such as attention deficit hyperactivity disorder (ADHD). To understand the molecular function of LAR, structural and biochemical studies of protein fragments derived from the ectodomain of human LAR have been performed. The crystal structure of a fragment encompassing the first four FNIII domains (LARFN1-4) showed a characteristic L shape. SAXS data suggested limited flexibility within LARFN1-4, while rigid-body refinement of the SAXS data using the X-ray-derived atomic model showed a smaller angle between the domains defining the L shape compared with the crystal structure. The capabilities of the individual LAR fragments to interact with heparin was examined using microscale thermophoresis and heparin-affinity chromatography. The results showed that the three N-terminal immunoglobulin domains (LARIg1-3) and the four C-terminal FNIII domains (LARFN5-8) both bound heparin, while LARFN1-4 did not. The low-molecular-weight heparin drug Innohep induced a shift in hydrodynamic volume as assessed by size-exclusion chromatography of LARIg1-3 and LARFN5-8, while the chemically defined pentameric heparin drug Arixtra did not. Together, the presented results suggest the presence of an additional heparin-binding site in human LAR.

Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor-ligand complex.

Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin-AF40431 complex were obtained by co-crystallization and the structure of the complex was solved to 2.7 Šresolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine.

The identification of AF38469: an orally bioavailable inhibitor of the VPS10P family sorting receptor Sortilin.

The identification of the novel, selective, orally bioavailable Sortilin inhibitor AF38469 is described. Structure-activity relationships and syntheses are reported, along with an X-ray crystal structure of the sortilin-AF38469 protein-inhibitor complex.

Expression of the RAI gene is conducive to apoptosis: studies of induction and interference.

The RAI gene is also known as iASPP and PPP1R13L. Recent investigations have shown that the region encompassing RAI is important for the development of cancer in young and middle-aged persons. It has been speculated that the RAI product induces apoptosis by blocking NF-kappaB or inhibits apoptosis by blocking p53. Either way the gene could influence the survival of precancerous lesions. Here we report that the expression of RAI mRNA was increased in non-transformed lymphocytes and fibroblasts induced to undergo apoptosis by various means, such as treatment with etoposide, calcium ions, or interleukin-2 and/or serum deprivation. Treatment with etoposide increased the content of RAI protein, too, and caused it to translocate to the nucleus. Inhibition of RAI expression in lymphocytes and fibroblasts with siRNA reduced apoptosis, but treatment with the NF-kappaB-inhibiting substance sulfasalazine relieved this dependence. In the transformed cell line HEK-293 the association between RAI induction and apoptosis seemed broken. Thus, we hypothesize that RAI induction is necessary but not sufficient for apoptosis induction in non-transformed cells. Our results could be explained by a NF-kappaB mediated mechanism.

Interaction between the 2'-5' oligoadenylate synthetase-like protein p59 OASL and the transcriptional repressor methyl CpG-binding protein 1.

The human 2'-5' oligoadenylate synthetases (OAS) form a conserved family of interferon-induced proteins consisting of four genes: OAS1, OAS2, OAS3 and the 2'-5' oligoadenylate synthetase-like gene (OASL). When activated by double-stranded RNA, OAS1-3 polymerize ATP into 2'-5'-linked oligoadenylates; 2'-5'-linked oligoadenylates, in turn, activate a latent endoribonuclease that degrades viral and cellular RNAs. In contrast, while the p59 OASL protein is highly homologous to the OAS family (45% identity), its 350 amino acid N-terminal domain lacks 2'-5' oligoadenylate synthetase activity. A C-terminal 164 amino acid domain, which is 30% homologous to a tandem repeat of ubiquitin, further distinguishes the p59 OASL protein and suggests that it serves a biological role which is distinct from other OAS family members. To dissect the function of p59 OASL, we utilized the yeast two-hybrid system to identify interacting proteins. Methyl CpG-binding protein 1 (MBD1), which functions as a transcriptional repressor, was identified as a strong p59 OASL interactor. Interestingly, like p59 OASL, transcription of the MBD1 gene was induced by interferon, indicating that these genes are co-ordinately regulated. The interaction was confirmed in vitro and in vivo and was mapped to the ubiquitin-like domain of p59 OASL. The p59 OASL-MBD1 interaction was specific, because p59 OASL did not interact with any of the other MBD family members and MBD1 did not interact with OAS1. These findings link the p59 OASL with MBD1 transcriptional control in the context of an interferon-stimulated cell, and provide the basis for future studies to examine the functional role of this interaction.