Understanding intramembrane proteolysis: from protein dynamics to reaction kinetics.

Intramembrane proteolysis - cleavage of proteins within the plane of a membrane - is a widespread phenomenon that can contribute to the functional activation of substrates and is involved in several diseases. Although different families of intramembrane proteases have been discovered and characterized, we currently do not know how these enzymes discriminate between substrates and non-substrates, how site-specific cleavage is achieved, or which factors determine the rate of proteolysis. Focusing on γ-secretase and rhomboid proteases, we argue that answers to these questions may emerge from connecting experimental readouts, such as reaction kinetics and the determination of cleavage sites, to the structures and the conformational dynamics of substrates and enzymes.

Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane.

The quaternary structure and functional properties of synaptophysin, a major integral membrane protein of small presynaptic vesicles, were investigated. Cross-linking and sedimentation studies indicate that synaptophysin is a hexameric homo-oligomer, which in electron micrographs exhibits structural features common to channel-forming proteins. On reconstitution into planar lipid bilayers, purified synaptophysin displays voltage-sensitive channel activity with an average conductance of about 150 picosiemens. Because specific channels and fusion pores have been implicated in vesicular uptake and release of secretory compounds, synaptophysin may have a role in these processes.

Identification of a gephyrin binding motif on the glycine receptor beta subunit.

The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GlyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GlyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the beta subunit. GlyR alpha subunits and GABAA receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GlyR beta subunit into the GABAA receptor beta 1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that beta subunit expression is essential for the formation of a postsynaptic GlyR matrix.

Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein.

A 93 kd polypeptide associated with the mammalian inhibitory glycine receptor (GlyR) is localized at central synapses and binds with high affinity to polymerized tubulin. This protein, named gephyrin (from the Greek gamma epsilon phi upsilon rho alpha, bridge), is thought to anchor the GlyR to subsynaptic microtubules. Here we report its primary structure deduced from cDNA and show that corresponding transcripts are found in all rat tissues examined. In brain, at least five different gephyrin mRNAs are generated by alternative splicing. Expression of gephyrin cDNAs in 293 kidney cells yields polypeptides reactive with a gephyrin-specific antibody, which coprecipitate with polymerized tubulin. Thus, gephyrin may define a novel type of microtubule-associated protein involved in membrane protein-cytoskeleton interactions.

Self assembly of the transmembrane domain promotes signal transduction through the erythropoietin receptor.

Hematopoietic cytokine receptors, such as the erythropoietin receptor (EpoR), are single membrane-spanning proteins. Signal transduction through EpoR is crucial for the formation of mature erythrocytes. Structural evidence shows that in the unliganded form EpoR exists as a preformed homodimer in an open scissor-like conformation precluding the activation of signaling. In contrast to the extracellular domain of the growth hormone receptor (GHR), the structure of the agonist-bound EpoR extracellular region shows only minimal contacts between the membrane-proximal regions. This evidence suggests that the domains facilitating receptor dimerization may differ between cytokine receptors. We show that the EpoR transmembrane domain (TM) has a strong potential to self interact in a bacterial reporter system. Abolishing self assembly of the EpoR TM by a double point mutation (Leu 240-Leu 241 mutated to Gly-Pro) impairs signal transduction by EpoR in hematopoietic cells and the formation of erythroid colonies upon reconstitution in erythroid progenitor cells from EpoR(-/-) mice. Interestingly, inhibiting TM self assembly in the constitutively active mutant EpoR R129C abrogates formation of disulfide-linked receptor homodimers and consequently results in the loss of ligand-independent signal transduction. Thus, efficient signal transduction through EpoR and possibly other preformed receptor oligomers may be determined by the dynamics of TM self assembly.

Ion channel formation by synthetic transmembrane segments of the inhibitory glycine receptor--a model study.

The inhibitory glycine receptor (GlyR) of rat spinal cord contains an intrinsic transmembrane channel mediating agonist-gated anion flux. Here, synthetic peptides modelled after the predicted transmembrane domains M2 and M4 of its ligand-binding subunit were incorporated into lipid vesicle membranes and black lipid bilayers to analyze their channel forming capabilities. Both types of peptides prohibited the establishment of, or dissipated, preexisting transmembrane potentials in the vesicle system. Incorporation of peptide M2 into the black lipid bilayer elicited randomly gated single channel events with various conductance states and life-times. Peptide M4 increased the conductance of the bilayer without producing single channels. Exchange of the terminal arginine residues of peptide M2 by glutamate resulted in a significant shift towards cation selectivity of the respective channels as compared to peptide M2. In conclusion, the peptide channels observed differed significantly from native GlyR in both conductivity and ion-selectivity indicating that individual synthetic transmembrane segments are not sufficient to mimic a channel protein composed of subunits with multiple transmembrane segments.

Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator.

Specific interactions between membrane spanning polypeptide segments are important for folding and oligomerisation of integral membrane proteins. Previously the dimerisation of glycophorin A has been shown to depend on interactions between its transmembrane segment by studying chimeric proteins in detergent solution. Here, we examined dimerisation of the glycophorin A transmembrane segment in a natural membrane employing the ToxR transcription activator from Vibrio cholerae. The ToxR protein is integral to the bacterial inner membrane and its activity requires a dimeric state. Therefore, the ToxR protein is suited to monitor quantitative homophilic interactions. We replaced the ToxR transmembrane segment with parts of the glycophorin A transmembrane segment containing the amino acid motif LIxxGVxxGVxxT previously shown to be sufficient for dimerisation in detergent solution. Expression of these chimeric proteins in an indicator strain resulted in strong transcription activation. This is indicative of efficient dimerisation mediated by the glycophorin transmembrane segment inserted into the inner membrane. Analysis of individual point mutants revealed that at least four residues out of this motif are critical for dimer formation in membranes. However, dimerisation of the glycophorin A transmembrane segment appears to be less sensitive to mutations when localised within a natural lipid bilayer compared to measurements in detergent solution. This may be related to a slightly altered structure of the dimer and/or to a higher local concentration and preorientation of the interacting molecules in a membrane. This makes the ToxR system well suited for probing low-affinity interactions between the transmembrane segments of other proteins.

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity.

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.

The dimerization motif of the glycophorin A transmembrane segment in membranes: importance of glycine residues.

The glycophorin A transmembrane segment homo-dimerizes to a right-handed pair of alpha-helices. Here, we identified the amino acid motif mediating this interaction within a natural membrane environment. Critical residues were grafted onto two different hydrophobic host sequences in a stepwise manner and self-assembly of the hybrid sequences was determined with the ToxR transcription activator system. Our results show that the motif LIxxGxxxGxxxT elicits a level of self-association equivalent to that of the original glycophorin A transmembrane segment. This motif is very similar to the one previously established in detergent solution. Interestingly, the central GxxxG motif by itself already induced strong self-assembly of host sequences and the three-residue spacing between both glycines proved to be optimal for the interaction. The GxxxG element thus appears to be the most crucial part of the interaction motif.

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase.

The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide-dependent kinases, calcium/calmodulin-dependent kinases, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.

Baculovirus-driven expression and purification of glycine receptor alpha 1 homo-oligomers.

The glycine receptor is a ligand-gated anion channel protein of postsynaptic membranes. We expressed a homo-oligomeric receptor composed of human alpha 1 subunits in Spodoptera frugiperda cells by infection with a recombinant Autographa californica nuclear polyhedrosis virus. A substantial fraction of the recombinant receptor was incorporated as a functional channel protein into the cell's plasma membrane at expression levels 4- to 30-fold higher than in other eukaryotic heterologous expression systems or native rat spinal cord membranes, respectively. Upon detergent solubilization, the alpha 1 receptor was found to exist in a predominantly monodisperse state and could be affinity-purified to near homogeneity. This preparation is a potential starting point for future crystallisation studies.

Importance of Arg-219 for correct biogenesis of alpha 1 homooligomeric glycine receptors.

The inhibitory glycine receptor is characterized by a pentameric arrangement of subunits with four predicted transmembrane segments (M1-M4) each. Here, we have mutagenized arginine residues located at both termini of the alpha 1 subunit segment, M2, which lines the receptor's anion channel. No glycine-gated channel formation could be detected in the plasma membrane of expressing cells for any of the mutants. In addition, mutating the arginine at the cytoplasmic terminus of M2 (R219) generated proteins which were only core-glycosylated, retained within intracellular compartments, and aggregated to high molecular weight complexes. Thus, residue R219, which corresponds to an arginine/lysine conserved in other ligand-gated ion channel polypeptides, is essential for correct biogenesis of the receptor.

Hyperekplexia mutations of the glycine receptor unmask the inhibitory subsite for beta-amino-acids.

beta-Alanine and taurine are agonists of the glycine receptor (GlyR) which, at low concentrations, antagonize the action of the principal agonist glycine. We analysed the potency of these ligands on alpha 1 subunits mutated at residue R271. GlyRs formed from alpha 1R271K subunits showed a reduction of beta-alanine and taurine affinities and maximal inducible currents; the mutants alpha 1R271Q and alpha 1R271L associated with human hyperekplexia gave no responses to these ligands. Inhibition of glycine-evoked currents by beta-alanine and taurine, however, was similar for all mutant GlyRs. These data are consistent with the existence of two subdomains within the ligand binding region of the GlyR, an agonistic one, which depends on arginine 271, and an antagonistic subsite, which is not connected to this residue.

Structure, diversity and synaptic localization of inhibitory glycine receptors.

The inhibitory glycine receptor (GlyR) mediates postsynaptic inhibition in spinal cord, brain stem and other regions of the vertebrate central nervous system. Biochemical and molecular approaches have identified different developmentally and regionally regulated GlyR isoforms that result from the differential expression of at least four genes coding for different variants of the ligand-binding alpha subunit. Molecular studies have allowed identification of GlyR subunit domains implicated in ligand binding, channel formation and receptor assembly. At the postsynaptic membrane, the GlyR colocalizes with a 93-kDa tubulin-binding peripheral membrane protein, gephyrin. Antisense inhibition of gephyrin expression prevents GlyR accumulation at postsynaptic membrane specialization. Thus, gephyrin is essential for postsynaptic receptor topology.

A preventive intervention program for bereaved children: problems of implementation.

A brief, standardized intervention program to facilitate children's adjustment to the terminal illness and death of a parent posed the following implementation problems: estimation of patient life expectancy; engagement of the family in crisis; adhesion to the parental guidance model; and termination of formal clinical intervention. Resolution of these issues is described, and adaptation of such programs to other high-risk populations is considered.

Impact of parental terminal cancer on latency-age children.

The underlying psychological and emotional components of distress were examined in 87 children with a parent in the terminal phase of cancer. Common fears, concerns, misperceptions, and behavioral consequences are analyzed, as are more severe psychological and behavioral reactions. Implications for psychoeducational parent-guidance intervention are discussed.

Secondary structure and distribution of fusogenic LV-peptides in lipid membranes.

LV-peptides were designed as membrane-spanning low-complexity model structures that mimic fusion protein transmembrane domains. These peptides harbor a hydrophobic core sequence that consists of helix-promoting and helix-destabilizing residues at different ratios. Previously, the fusogenicity of these peptides has been shown to increase with the conformational flexibility of their hydrophobic cores as determined in isotropic solution. Here, we examined the secondary structure, orientation, and distribution of LV-peptides in membranes. Our results reveal that the peptides are homogeneously distributed within the membranes of giant unilamellar liposomes and capable of fusing them. Increasing the valine content of the core up to the level of the beta-branched residue content of SNARE TMDs (approximately 50%) enhances fusogenicity while maintaining a largely alpha-helical structure in liposomal membranes. A further increase in valine content or introduction of a glycine/proline pair favors beta-sheet formation. In planar bilayers, the alpha-helices adopt oblique angles relative to the bilayer normal and the ratio of alpha-helix to beta-sheet responds more sensitively to valine content. We propose that the fusogenic conformation of LV-peptides is likely to correspond to a membrane-spanning alpha-helix. Beta-sheet formation in membranes may be considered a side-reaction whose extent reflects conformational flexibility of the core.