Loss of major nutrient sensing and signaling pathways suppresses starvation lethality in electron transport chain mutants

The electron transport chain (ETC) is a well-studied and highly conserved metabolic pathway that produces ATP through generation of a proton gradient across the inner mitochondrial membrane coupled to oxidative phosphorylation. ETC mutations are associated with a wide array of human disease conditions and to aging-related phenotypes in a number of different organisms. In this study, we sought to better understand the role of the ETC in aging using a yeast model. A panel of ETC mutant strains that fail to survive starvation was used to isolate suppressor mutants that survive. These suppressors tend to fall into major nutrient sensing and signaling pathways, suggesting that the ETC is involved in proper starvation signaling to these pathways in yeast. These suppressors also partially restore ETC-associated gene expression and pH homeostasis defects, though it remains unclear whether these phenotypes directly cause the suppression or are simply effects. This work further highlights the complex cellular network connections between metabolic pathways and signaling events in the cell and their potential roles in aging and age-related diseases.     

Parvalbumin,calbindin, and calretinin mark distinct pathways during development of monkey dorsal lateral geniculate nucleus

Immunocyochemical labeling was applied to follow the developmental changes in the calcium-binding proteins parvalbumin (PV), calbindin D28k (CaB), and calretinin (CaR) during fetal and infant development of Macaca monkey dorsal lateral geniculate nucleus (LGN). For all three proteins, LGN cell body and retinal ganglion cell (RGC) axon labeling patterns changed temporally and spatially over development, and many of these were LGN laminar specific. CaR+ and CaB+ cells were present at the youngest age studied, fetal day 55 (F55). After lamination of the LGN occurred between F90 and F115, CaR+ and CaB+ neurons were specific markers for the S, intercalated, and interlaminar layers. Double label immunocytochemistry showed that all CaR+ cells contained CaB, and none contained GABA. CaR+ cell bodies decreased in number soon after birth so that adult LGN contained only a very small number of CaR+ cells. These patterns and cell counts indicated that a downregulation of CaR had occurred in the CaB+ population. Although CaB+ cell density in S and interlaminar zones declined in the adult, cell counts indicated that this is due to dilution of a stable population into a much larger nucleus during development. PV+ cells appeared at F85 only within the putative magnocellular (M) and parvocellular (P) layers, and PV remained a marker for these layers throughout development. Fetal PV cells also contained GABA, indicating that they were LGN interneurons. After birth, GABA−/PV+ cell numbers increased dramatically throughout the whole nucleus so that by the end of the first year, P and M layers were filled with PV+ cells. Their number and size indicated that these were the LGN projection neurons. Beginning at F66, bundles of PV+ axons occupied the anterior-middle LGN and filled the optic tract. Up to F101, PV+ synaptic terminals were restricted to P layers, but after F132 labeling in M layers was heavier than in P layers. Axonal labeling for CaR began at F125. Prenatally CaR+ terminals were present mainly in P layers, whereas by postnatal 9 weeks labeling in M layers much exceeded P layers. Axonal labeling for CaB was present at F132, but CaB+ terminals were observed only after birth with labeling always heavier in M than P layers. By postnatal 9 weeks, PV, CaR, and CaB were colocalized in the same axons and terminals. These experiments indicated that during development and in the adult LGN, both CaR and CaB were markers for the LGN neurons in the S and intercalated pathway. CaR was present transiently while CaB persisted into adulthood. PV was a M and P layer marker first for interneurons and later for projection cells. The complex temporal developmental patterns found in this study suggested that viewing PV, CaB, and CaR simply as calcium-buffering proteins severely underestimates their functional roles during visual system maturation. © 1996 John Wiley & Sons, Inc.     

Ligand sensitivity in dimeric associations of the serotonin 5HT2c receptor

G-protein-coupled receptors (GPCRs) respond to external stimuli by activating heterotrimeric G proteins inside the cell. There is increasing evidence that many GPCRs exist as dimers or higher oligomers, but the biochemical nature of such dimers and what roles they have, if any, in signal transduction remains unclear. We conducted a comprehensive study of dimerization of the 5HT2c serotonin receptor using disulphide-trapping experiments. We found a dimer interface between transmembrane (TM) helices IV and V that is markedly sensitive to the state of receptor activation. This dimer seems to be quasisymmetrical in interfacial geometry and asymmetrical in its association with its cognate G alpha protein. We also found a second interface at TM I helices, which is insensitive to the state of activation.     

Differential mass spectrometry of rat plasma reveals proteins that are responsive to 17beta-estradiol and a selective estrogen receptor modulator PPT

Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) alpha and beta. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17beta-estradiol (E2) or an ERalpha-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT-responsive proteins, such as serine protease inhibitor family members.     

Association of Y chromosome haplogroup I with HIV progression, and HAART outcome

The host genetic basis of differential outcomes in HIV infection, progression, viral load set point and highly active retroviral therapy (HAART) responses was examined for the common Y haplogroups in European Americans and African Americans. Accelerated progression to acquired immune deficiency syndrome (AIDS) and related death in European Americans among Y chromosome haplogroup I (Y-I) subjects was discovered. Additionally, Y-I haplogroup subjects on HAART took a longer time to HIV-1 viral suppression and were more likely to fail HAART. Both the accelerated progression and longer time to viral suppression results observed in haplogroup Y-I were significant after false-discovery-rate corrections. A higher frequency of AIDS-defining illnesses was also observed in haplogroup Y-I. These effects were independent of the previously identified autosomal AIDS restriction genes. When the Y-I haplogroup subjects were further subdivided into six I subhaplogroups, no one subhaplogroup accounted for the effects on HIV progression, viral load or HAART response. Adjustment of the analyses for population stratification found significant and concordant haplogroup Y-I results. The Y chromosome haplogroup analyses of HIV infection and progression in African Americans were not significant. Our results suggest that one or more loci on the Y chromosome found on haplogroup Y-I have an effect on AIDS progression and treatment responses in European Americans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural genomics target selection for the New York consortium on membrane protein structure

The New York Consortium on Membrane Protein Structure (NYCOMPS), a part of the Protein Structure Initiative (PSI) in the USA, has as its mission to establish a high-throughput pipeline for determination of novel integral membrane protein structures. Here we describe our current target selection protocol, which applies structural genomics approaches informed by the collective experience of our team of investigators. We first extract all annotated proteins from our reagent genomes, i.e. the 96 fully sequenced prokaryotic genomes from which we clone DNA. We filter this initial pool of sequences and obtain a list of valid targets. NYCOMPS defines valid targets as those that, among other features, have at least two predicted transmembrane helices, no predicted long disordered regions and, except for community nominated targets, no significant sequence similarity in the predicted transmembrane region to any known protein structure. Proteins that feed our experimental pipeline are selected by defining a protein seed and searching the set of all valid targets for proteins that are likely to have a transmembrane region structurally similar to that of the seed. We require sequence similarity aligning at least half of the predicted transmembrane region of seed and target. Seeds are selected according to their feasibility and/or biological interest, and they include both centrally selected targets and community nominated targets. As of December 2008, over 6,000 targets have been selected and are currently being processed by the experimental pipeline. We discuss how our target list may impact structural coverage of the membrane protein space.     

Hydra: software for tailored processing of H/D exchange data from MS or tandem MS analyses

Background  

Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) experiments implemented to characterize protein interaction and protein folding generate large quantities of data. Organizing, processing and visualizing data requires an automated solution, particularly when accommodating new tandem mass spectrometry modes for H/DX measurement. We sought to develop software that offers flexibility in defining workflows so as to support exploratory treatments of H/DX-MS data, with a particular focus on the analysis of very large protein systems and the mining of tandem mass spectrometry data.     

The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.