Analyzing protein-protein interactions in cell membranes

Interactions among membrane proteins regulate numerous cellular processes, including cell growth, cell differentiation and apoptosis. We need to understand which proteins interact, where they interact and to which extent they interact. This article describes a set of novel approaches to measure, on the surface of living cells, the number of clusters of proteins, the number of proteins per cluster, the number of clusters or membrane domains that contain pairs of interacting proteins and the fraction of one protein species that interacts with another protein within these domains. These data can then be interpreted in terms of the function of the protein-protein interactions.     

Long-term follow-up of spontaneous development in a boy with familial male precocious puberty

BACKGROUND/AIMS: Limited data are available about spontaneous growth, pubertal growth spurt and the long-term outcome of patients suffering from familial male precocious puberty (FMPP). We report on a boy with FMPP whose growth pattern and pubertal development was studied longitudinally without treatment. METHODS: Long-term prospective follow-up without treatment of a 6.2-year-old boy with FMPP having inherited a mutation of the LH receptor gene (A568V) from his father. RESULTS: The pubertal growth spurt was of unusual maximal amplitude (growth rate 12.4 cm/year at the age of 5-6 years) and of extraordinary duration lasting for 5.2 years from age 3.8 to 9.0 years. No deterioration of height potential was observed. Height (174 cm) was within target height range (171.5-188.5 cm) at age 13 years. No central precocious puberty occurred. CONCLUSION: FMPP is an experiment of nature demonstrating that the amplitude and duration of the pubertal growth spurt are much more variable than previously described. Furthermore, this case emphasizes that the indication for treatment is highly dependent on intrafamilial and individual factors.     

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.     

Biochemical association of poly(ADP-ribose) polymerase-1 and its apoptotic peptide fragments with DNA polymerase beta

We have characterized the biochemical association of two DNA damage-dependent enzymes, poly(ADP-ribose) polymerase-1 (PARP-1) [EC 2.4.2.30] and DNA polymerase beta (pol beta) [2.7.7.7]. We reproducibly observed that pol beta is an efficient covalent target for ADP-ribose polymers under standard conditions of enzymatically catalyzed ADP-ribosylation of betaNAD+ as a substrate. The efficiency of poly(ADP-ribosyl)ation increased as a function of the pol beta and betaNAD+ concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP-1 to peptide-specific enzymatic degradation with either caspase-3 or caspase-7 in vitro. This proteolytic treatment, commonly referred to as 'a hallmark of apoptosis', generated the two physiologically relevant peptide fragments of PARP-1, e.g., a 24-kDa amino-terminus and an 89-kDa carboxy-terminal domain. Interestingly, co-incubation of the two peptide fragments of PARP-1 with full-length pol beta resulted in their domain-specific molecular association as determined by co-immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP-1 is proteolyzed by either caspase-3 or caspase-7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol beta, may serve a regulatory molecular role in the execution phase of apoptosis.     

Expression, purification, and aggregation studies of His-tagged thermoalkalophilic lipase from Bacillus thermocatenulatus

The His-tagged lipase BTL2 from Bacillus thermocatenulatus was expressed in Escherichia coli and purified to homogeneity by a simple, one-step purification protocol using immobilized metal affinity chromatography. The success of protein separation and purification was pH-dependent and increased with decreasing pH. The purified BTL2 lipase showed a strong tendency to aggregate upon concentration, which prevented a reproducible crystallization. Aggregation studies using dynamic light-scattering (DLS) analysis were performed to improve the purification and concentration of BTL2 lipase. Different chemical classes of additives were tested to manipulate the aggregation behaviour of BTL2 lipase with the aim of obtaining a monodisperse sample to use for crystallization. For the process of concentration of BTL2 lipase in monomeric form, the alcohol 2-propanol and the ionic detergent dodecyl dimethylamine-N-oxide (LDAO) were found to be necessary. For the concentrated lipase, the availability of 5% 2-propanol was sufficient to hold the lipase in monomeric form and no additional detergent was needed.     

Bird orientation: compensation for wind drift in migrating raptors is age dependent

Despite the potentially strong effect of wind on bird orientation, our understanding of how wind drift affects migrating birds is still very limited. Using data from satellite-based radio telemetry, we analysed the effect of changing winds on the variation of the track direction of individual birds. We studied adults and juveniles of two raptor species, osprey Pandion haliaetus and honey buzzard Pernis apivorus, on autumn migration between North Europe and Africa, and demonstrate an important difference between the age categories of both species in the extent of wind drift. For juveniles, side- and following-wind components affected the rates of movement perpendicular to and along the mean direction, respectively, to a similar degree, suggesting full wind drift. By contrast, for adults the rate of crosswind displacement was significantly smaller than the effect of wind on forward movement, showing much reduced wind drift (29%). This indicates that adults have acquired a more sophisticated orientation system, permitting detection of and compensation for wind drift, than juveniles. These drift effects are likely to reduce the ability of juveniles to locate species-specific wintering areas in case of rapid climatic wind change.     

Strong differential regulation of serum and mucosal IgA responses as revealed in CD28-deficient mice using cholera toxin adjuvant

In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved.     

Vectorial acylation in Saccharomyces cerevisiae. Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex

In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions between these proteins. Multicopy extragenic suppressors were selected in strains carrying deletions in FAA1 and FAA4 or FAA1 and FAT1. Each strain is unable to grow under synthetic lethal conditions when exogenous long-chain fatty acids are required, and neither strain accumulates the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) indicating a fatty acid transport defect. By using these phenotypes as selective screens, plasmids were identified encoding FAA1, FAT1, and FAA4 in the faa1Delta faa4Delta strain and encoding FAA1 and FAT1 in the faa1Delta fat1Delta strain. Multicopy FAA4 could not suppress the growth defect in the faa1Delta fat1Delta strain indicating some essential functions of Fat1p cannot be performed by Faa4p. Chromosomally encoded FAA1 and FAT1 are not able to suppress the growth deficiencies of the fat1Delta faa1Delta and faa1Delta faa4Delta strains, respectively, indicating Faa1p and Fat1p play distinct roles in the fatty acid import process. When expressed from a 2-mu plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import. Evidence of a physical interaction between Fat1p and FACS was provided using three independent biochemical approaches. First, a C-terminal peptide of Fat1p deficient in fatty acid transport exerted a dominant negative effect against long-chain acyl-CoA synthetase activity. Second, protein fusions employing Faa1p as bait and portions of Fat1p as trap were active when tested using the yeast two-hybrid system. Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which consists of Fat1p and Faa1p or Faa4p.