Identification of follicle-stimulating hormone-selective beta-strands in the N-terminal hormone-binding exodomain of human gonadotropin receptors

Glycoprotein hormone receptors contain large N-terminal extracellular domains (ECDs) that distinguish these receptors from most other G protein-coupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N- and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which beta-strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R) counterparts. Introduction of hFSH-R beta-strand 1 into hLH-R conferred responsiveness to hFSH, whereas hLH-R mutants harboring one of the other hFSH-R beta-strands displayed none or very limited sensitivity to hFSH. However, combined substitution of hFSH-R beta-strand 1 and some of the other hFSH-R beta-strands further increased the sensitivity of the mutant hLH-R to hFSH. The apparent contribution of multiple hFSH-R beta-strands in providing a selective hormone binding interface corresponds well with their position in relation to hFSH as recently determined in the crystal structure of hFSH in complex with part of the hFSH-R ECD.     

Parallel network simulations with NEURON

The NEURON simulation environment has been extended to support parallel network simulations. Each processor integrates the equations for its subnet over an interval equal to the minimum (interprocessor) presynaptic spike generation to postsynaptic spike delivery connection delay. The performance of three published network models with very different spike patterns exhibits superlinear speedup on Beowulf clusters and demonstrates that spike communication overhead is often less than the benefit of an increased fraction of the entire problem fitting into high speed cache. On the EPFL IBM Blue Gene, almost linear speedup was obtained up to 100 processors. Increasing one model from 500 to 40,000 realistic cells exhibited almost linear speedup on 2000 processors, with an integration time of 9.8 seconds and communication time of 1.3 seconds. The potential for speed-ups of several orders of magnitude makes practical the running of large network simulations that could otherwise not be explored. Action Editor: Alain Destexhe     

Crystal structure of the C2 domain of class II phosphatidylinositide 3-kinase C2alpha

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3'-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel beta-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.     

Critical role of the C-terminal domains of factor H in regulating complement activation at cell surfaces

The plasma protein factor H primarily controls the activation of the alternative pathway of complement. The C-terminal of factor H is known to be involved in protection of host cells from complement attack. In the present study, we show that domains 19-20 alone are capable of discriminating between host-like and complement-activating cells. Furthermore, although factor H possesses three binding sites for C3b, binding to cell-bound C3b can be almost completely inhibited by the single site located in domains 19-20. All of the regulatory activities of factor H are expressed by the N-terminal four domains, but these activities toward cell-bound C3b are inhibited by isolated recombinant domains 19-20 (rH 19-20). Direct competition with the N-terminal site is unlikely to explain this because regulation of fluid phase C3b is unaffected by domains 19-20. Finally, we show that addition of isolated rH 19-20 to normal human serum leads to aggressive complement-mediated lysis of normally nonactivating sheep erythrocytes and moderate lysis of human erythrocytes, which possess membrane-bound regulators of complement. Taken together, the results highlight the importance of the cell surface protective functions exhibited by factor H compared with other complement regulatory proteins. The results may also explain why atypical hemolytic uremic syndrome patients with mutations affecting domains 19-20 can maintain complement homeostasis in plasma while their complement system attacks erythrocytes, platelets, endothelial cells, and kidney tissue.     

In vitro activation of CD8 interphotoreceptor retinoid-binding protein-specific T cells requires not only antigenic stimulation but also exogenous growth factors

In a previous study, we demonstrated that immunization with the uveitogenic peptide interphotoreceptor retinoid-binding protein (IRBP) 1-20 induces both CD4 and CD8 uveitogenic T cells in the B6 mouse. In the current study, we determined the role of the CD8 IRBP-specific T cells in the pathogenesis of experimental autoimmune uveitis. We also determined the conditions that facilitated the activation of CD8 autoreactive T cells. Our results showed that the beta2-microglobulin(-/-) mouse had a greatly decreased susceptibility to induction of experimental autoimmune uveitis by adoptive transfer of IRBP-specific T cells from B6 mice. We also showed that unlike CD4 autoreactive T cells, activated CD8 autoreactive T cells produced only a limited number and amounts of growth factors. As a result, in the absence of exogenously supplied growth factor(s), CD8 T cell activation and expansion were aborted. However, the growth and expansion of triggered CD8 autoreactive T cells could be supported by various cytokines. In addition to factors produced by activated CD4 autoreactive T cells, factors produced by nonlymphoid cells, such as IL-7 and IL-15, and unidentified factors in the culture supernatants of astrocytes and retinal pigment epithelial cells support the CD8 autoreactive T cells as well. Finally, we showed that, although several cytokines augmented the CD8 T cell response in vitro, different cytokines appeared to act on different CD8 subsets or on different activation/differentiation phases of CD8 autoreactive T cells. As a result, cytokines, such as IL-7, supported the proliferation and survival of CD8 IRBP-specific T cells, while others had only a growth-promoting effect.     

Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses

Immunodominance is variably used to describe either the most frequently detectable response among tested individuals or the strongest response within a single individual, yet factors determining either inter- or intraindividual immunodominance are still poorly understood. More than 90 individuals were tested against 184 HIV- and 92 EBV-derived, previously defined CTL epitopes. The data show that HLA-B-restricted epitopes were significantly more frequently recognized than HLA-A- or HLA-C-restricted epitopes. HLA-B-restricted epitopes also induced responses of higher magnitude than did either HLA-A- or HLA-C-restricted epitopes, although this comparison only reached statistical significance for EBV epitopes. For both viruses, the magnitude and frequency of recognition were correlated with each other, but not with the epitope binding affinity to the restricting HLA allele. The presence or absence of HIV coinfection did not impact EBV epitope immunodominance patterns significantly. Peptide titration studies showed that the magnitude of responses was associated with high functional avidity, requiring low concentration of cognate peptide to respond in in vitro assays. The data support the important role of HLA-B alleles in antiviral immunity and afford a better understanding of the factors contributing to inter- and intraindividual immunodominance.