Changes in ubiquitin immunoreactivity in developing rat brain: a putative role for ubiquitin and ubiquitin conjugates in dendrite outgrowth and differentiation.

The role of ubiquitin in development of the mammalian brain has been studied using a monoclonal antibody, RHUb1, specific for ubiquitin. Immunodevelopment of western blots of homogenate samples of the cerebral cortex, hippocampus and cerebellum prepared from animals of known postnatal age show marked developmental changes in conjugate level. Striking decreases in the level of a prominent conjugate of molecular weight 22,000, which is identified as ubiquitinated histone, are observed during the first postnatal week in the cerebral cortex and hippocampus, but not the cerebellum. A marked overall developmental decrease in the level of high-molecular-weight (> 40,000) ubiquitin conjugates which occurs predominantly during the third, but also the fourth, postnatal week is observed in all three regions. Immunocytochemical data obtained with the RHUb1 antibody show intense staining of neuronal perikarya, nuclei and dendrites in early postnatal cerebral cortex and hippocampus. Staining of pyramidal cell perikarya and dendrites is particularly prominent. The intensity of dendritic staining, particularly for the cerebral cortex, shows a striking decrease after postnatal day 14 and only faint dendritic staining is observed in the adult. In early postnatal cerebellum, immunoreactivity is predominantly nuclear, though some staining of the proximal regions of Purkinje cell dendrites is observed between postnatal days 4 and 19. As with the cerebral cortex and hippocampus, most of the ubiquitin reactivity is lost in adult animals. The loss of dendritic staining, particularly in the cerebral cortex, correlates with the decrease in the level of high-molecular-weight ubiquitin conjugates observed on the western blots. Immunodevelopment of western blots of a range of subcellular fractions prepared from developing rat forebrain shows that the developmental decrease in the level of high-molecular-weight ubiquitin conjugates is not uniform for all fractions. The decrease in conjugate level is most marked for the cell-soluble, mitochondrial and detergent-insoluble cytoskeletal fractions. Taken overall, the data suggest a role for ubiquitin in dendrite outgrowth and arborization, loss of dendritic ubiquitin immunoreactivity correlating with completion of these processes.

Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis.

Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237+/-13 versus 279+/-3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.

SwissParam: a fast force field generation tool for small organic molecules.

The drug discovery process has been deeply transformed recently by the use of computational ligand-based or structure-based methods, helping the lead compounds identification and optimization, and finally the delivery of new drug candidates more quickly and at lower cost. Structure-based computational methods for drug discovery mainly involve ligand-protein docking and rapid binding free energy estimation, both of which require force field parameterization for many drug candidates. Here, we present a fast force field generation tool, called SwissParam, able to generate, for arbitrary small organic molecule, topologies, and parameters based on the Merck molecular force field, but in a functional form that is compatible with the CHARMM force field. Output files can be used with CHARMM or GROMACS. The topologies and parameters generated by SwissParam are used by the docking software EADock2 and EADock DSS to describe the small molecules to be docked, whereas the protein is described by the CHARMM force field, and allow them to reach success rates ranging from 56 to 78%. We have also developed a rapid binding free energy estimation approach, using SwissParam for ligands and CHARMM22/27 for proteins, which requires only a short minimization to reproduce the experimental binding free energy of 214 ligand-protein complexes involving 62 different proteins, with a standard error of 2.0 kcal mol(-1), and a correlation coefficient of 0.74. Together, these results demonstrate the relevance of using SwissParam topologies and parameters to describe small organic molecules in computer-aided drug design applications, together with a CHARMM22/27 description of the target protein. SwissParam is available free of charge for academic users at www.swissparam.ch.

Expanding molecular modeling and design tools to non-natural sidechains.

Protein-protein interactions encode the wiring diagram of cellular signaling pathways and their deregulations underlie a variety of diseases, such as cancer. Inhibiting protein-protein interactions with peptide derivatives is a promising way to develop new biological and therapeutic tools. Here, we develop a general framework to computationally handle hundreds of non-natural amino acid sidechains and predict the effect of inserting them into peptides or proteins. We first generate all structural files (pdb and mol2), as well as parameters and topologies for standard molecular mechanics software (CHARMM and Gromacs). Accurate predictions of rotamer probabilities are provided using a novel combined knowledge and physics based strategy. Non-natural sidechains are useful to increase peptide ligand binding affinity. Our results obtained on non-natural mutants of a BCL9 peptide targeting beta-catenin show very good correlation between predicted and experimental binding free-energies, indicating that such predictions can be used to design new inhibitors. Data generated in this work, as well as PyMOL and UCSF Chimera plug-ins for user-friendly visualization of non-natural sidechains, are all available at http://www.swisssidechain.ch. Our results enable researchers to rapidly and efficiently work with hundreds of non-natural sidechains.

Cis-trans interactions of cell surface receptors: biological roles and structural basis.

Cell surface receptors bind ligands expressed on other cells (in trans) in order to communicate with neighboring cells. However, an increasing number of cell surface receptors are found to also interact with ligands expressed on the same cell (in cis). These observations raise questions regarding the biological role of such cis interactions. Specifically, it is important to know whether cis and trans binding have distinct functional effects and, if so, how a single cell discriminates between interactions in cis versus trans. Further, what are the structural features that allow certain cell surface receptors to engage ligand both on the same as well as on an apposed cell membrane? Here, we summarize known examples of receptors that display cis-trans binding and discuss the emerging diversity of biological roles played by these unconventional two-way interactions, along with their structural basis.

New insights into the molecular basis of desmoplakin- and desmin-related cardiomyopathies.

Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

Isolated integrin beta3 subunit cytoplasmic domains require membrane anchorage and the NPXY motif to recruit to adhesion complexes but do not discriminate between beta1- and beta3-positive complexes.

Integrin adhesion receptors consist of non-covalently linked alpha and beta subunits each of which contains a large extracellular domain, a single transmembrane domain and a short cytoplasmic tail. Engaged integrins recruit to focal structures globally termed adhesion complexes. The cytoplasmic domain of the beta subunit is essential for this clustering. beta1 and beta3 integrins can recruit at distinct cellular locations (i.e. fibrillar adhesions vs focal adhesions, respectively) but it is not clear whether individual beta subunit cytoplasmic and transmembrane domains are by themselves sufficient to drive orthotopic targeting to the cognate adhesion complex. To address this question, we expressed full-length beta3 transmembrane anchored cytoplasmic domains and truncated beta3 cytoplasmic domains as GFP-fusion constructs and monitored their localization in endothelial cells. Membrane-anchored full-length beta3 cytoplasmic domain and a beta3 mutant lacking the NXXY motif recruited to adhesion complexes, while beta3 mutants lacking the NPXY and NXXY motifs or the transmembrane domain did not. Replacing the natural beta subunit transmembrane domain with an unrelated (i.e. HLA-A2 alpha chain) transmembrane domain significantly reduced recruitment to adhesion complexes. Transmembrane anchored beta3 and cytoplasmic domain constructs, however, recruited without discrimination to beta1- and beta3-rich adhesions complexes. These findings demonstrate that membrane anchorage and the NPXY (but not the NXXY) motif are necessary for beta3 cytoplasmic domain recruitment to adhesion complexes and that the natural transmembrane domain actively contributes to this recruitment. The beta3 transmembrane and cytoplasmic domains alone are insufficient for orthotopic recruitment to cognate adhesion complexes.

International Union of Pharmacology. XXXV. The glucagon receptor family.

Peptide hormones within the secretin-glucagon family are expressed in endocrine cells of the pancreas and gastrointestinal epithelium and in specialized neurons in the brain, and subserve multiple biological functions, including regulation of growth, nutrient intake, and transit within the gut, and digestion, energy absorption, and energy assimilation. Glucagon, glucagon-like peptide-1, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, growth hormone-releasing hormone and secretin are structurally related peptides that exert their actions through unique members of a structurally related G protein-coupled receptor class 2 family. This review discusses advances in our understanding of how these peptides exert their biological activities, with a focus on the biological actions and structural features of the cognate receptors. The receptors have been named after their parent and only physiologically relevant ligand, in line with the recommendations of the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR).

Regulation of PIDD auto-proteolysis and activity by the molecular chaperone Hsp90.

In response to DNA damage, p53-induced protein with a death domain (PIDD) forms a complex called the PIDDosome, which either consists of PIDD, RIP-associated protein with a death domain and caspase-2, forming a platform for the activation of caspase-2, or contains PIDD, RIP1 and NEMO, important for NF-κB activation. PIDDosome activation is dependent on auto-processing of PIDD at two different sites, generating the fragments PIDD-C and PIDD-CC. Despite constitutive cleavage, endogenous PIDD remains inactive. In this study, we screened for novel PIDD regulators and identified heat shock protein 90 (Hsp90) as a major effector in both PIDD protein maturation and activation. Hsp90, together with p23, binds PIDD and inhibition of Hsp90 activity with geldanamycin efficiently disrupts this association and impairs PIDD auto-processing. Consequently, both PIDD-mediated NF-κB and caspase-2 activation are abrogated. Interestingly, PIDDosome formation itself is associated with Hsp90 release. Characterisation of cytoplasmic and nuclear pools of PIDD showed that active PIDD accumulates in the nucleus and that only cytoplasmic PIDD is bound to Hsp90. Finally, heat shock induces Hsp90 release from PIDD and PIDD nuclear translocation. Thus, Hsp90 has a major role in controlling PIDD functional activity.

How do thyroid hormone receptors bind to structurally diverse response elements?

Three classes of thyroid hormone response elements have been described. They are composed of two half-sites arranged either as a palindromic, a direct repeat or as an inverted palindromic array. Receptor homodimers as well as heterodimers can bind to all three types of response element. While the ligand binding domain of the receptors provides the major dimerization surface, asymmetric contacts between the DNA binding domains are necessary for binding to a direct repeat. Moreover, some recent findings suggest that in TR, compared to RXR, the ligand binding domain has a 180 degrees rotation with respect to the DNA binding domain. This feature could explain the preferential binding of the RXR-TR heterodimer to the direct repeat response element, in which RXR exclusively binds the 5' half-site, and of the TR homodimer to the inverted palindrome response element.

Pharmacologic profile of UR-7247, an orally active angiotensin II AT1 receptor antagonist, in healthy volunteers. p6.

This study was conducted to assess the pharmacologic properties of the new orally active angiotensin II subtype I (AT1) antagonist UR-7247, a product with a half-life >100 h in humans. The experiment was designed as an open-label, single-dose administration study with four parallel groups of four healthy men receiving increasing single oral doses (2.5, 5, and 10 mg) of UR-7247 or losartan, 100 mg. Angiotensin II receptor blockade was investigated < or =96 h after drug intake, with three independent methods [i.e., the inhibition of blood pressure (BP) response to exogenous Ang II, an in vitro Ang II-receptor assay (RRA), and the reactive increase in plasma angiotensin II. Plasma drug levels also were measured. The degree of blockade observed in vivo was statistically significant < or = 96 h with all UR-7247 doses for diastolic BP (p < 0.05) and < or =48 h for systolic BP. The maximal inhibition achieved with 10 mg UR-7247 was measured 6-24 h after drug intake and reached 54 +/- 17% and 48 +/- 20% for diastolic and systolic responses, respectively. Losartan, 100 mg, induced a greater short-term AT1-receptor blockade than 2.5- and 5.0-mg doses of UR-7247 (p < 0.001 for diastolic BP), but the UR-7247 effect was longer lasting. In vivo, no significant difference was observed between 10 mg UR-7247 and 100 mg losartan 4 h after drug intake, but in vitro, the blockade achieved with 100 mg losartan was higher than that seen with UR-7247. Finally, the results confirm that UR-7247 has a very long plasma elimination half-life, which may be due to a high but also tight binding to protein binding sites. In conclusion, UR-7247 is a long-lasting, well-tolerated AT1 receptor in healthy subjects.

Ceftriaxone--bilirubin-albumin interactions in the neonate: an in vivo study.

The in vivo bilirubin-albumin binding interaction of ceftriaxone (CRO) was investigated in 14 non-jaundiced newborns, aged 33-42 weeks of gestation, during the first few days of life after they had reached stable clinical condition. CRO (50 mg/kg) was infused intravenously over 30 min. The competitive binding effect of CRO on the bilirubin-albumin complex was estimated by determining the reserve albumin concentration (RAC) at baseline, at the end of CRO infusion, and at 15 and 60 min thereafter. Immediately after the end of drug administration, RAC decreased from 91.9 (+/- 25.1) mumol/l to 38.6 (+/- 10.1) mumol/l (P = 0.0001). At the same time the plasma bilirubin toxicity index (PBTI) increased from 0.64 (+/- 0.40) before drug infusion to 0.96 (+/- 0.44) thereafter (P = 0.0001). The highest displacement factor (DF) was calculated to be 2.8 (+/- 0.6) at the end of drug infusion. Average total serum bilirubin concentrations decreased from a baseline value of 59.6 (+/- 27.0) mumol/l to 55.2 (+/- 27.1) mumol/l (P = 0.026). Sixty minutes after the end of CRO infusion, RAC was 58.3 (+/- 21.7) mumol/l, PBTI regained baseline, but DF was still 1.9 (+/- 0.2). No adverse events were recorded. Our results demonstrate significant competitive interaction of CRO with bilirubin-albumin binding in vivo. Thus, ceftriaxone should not be given to the neonate at risk of developing bilirubin encephalopathy.

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion.

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.

TCR-alpha CDR3 loop audition regulates positive selection.

How positive selection molds the T cell repertoire has been difficult to examine. In this study, we use TCR-beta-transgenic mice in which MHC shapes TCR-alpha use. Differential AV segment use is directly related to the constraints placed on the composition of the CDR3 loops. Where these constraints are low, efficient selection of alphabeta pairs follows. This mode of selection preferentially uses favored AV-AJ rearrangements and promotes diversity. Increased constraint on the alpha CDR3 loops leads to inefficient selection associated with uncommon recombination events and limited diversity. Further, the two modes of selection favor alternate sets of AJ segments. We discuss the relevance of these findings to the imprint of self-MHC restriction and peripheral T cell activation.

Role of CD8 in aberrant function of cytotoxic T lymphocytes.

Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR-ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR-ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.