Interactions of the Lysosomotropic Detergent O-Methyl-Serine Dodecylamide Hydrochloride (MSDH) with Lipid Bilayer Membranes-Implications for Cell Toxicity.

O-methyl-serine dodecylamine hydrochloride (MSDH) is a detergent that accumulates selectively in lysosomes, a so-called lysosomotropic detergent, with unexpected chemical properties. At physiological pH, it spontaneously forms vesicles, which disassemble into small aggregates (probably micelles) below pH 6.4. In this study, we characterize the interaction between MSDH and liposomes at different pH and correlate the findings to toxicity in human fibroblasts. We find that the effect of MSDH on lipid membranes is highly pH-dependent. At neutral pH, the partitioning of MSDH into the liposome membrane is immediate and causes the leakage of small fluorophores, unless the ratio between MSDH and lipids is kept low. At pH 5, the partitioning of MSDH into the membrane is kinetically impeded since MSDH is charged and a high ratio between MSDH and the lipids is required to permeabilize the membrane. When transferred to cell culture conditions, the ratio between MSDH and plasma membrane lipids must therefore be low, at physiological pH, to maintain plasma membrane integrity. Transmission electron microscopy suggests that MSDH vesicles are taken up by endocytosis. As the pH of the endosomal compartment progressively drops, MSDH vesicles disassemble, leading to a high concentration of increasingly charged MSDH in small aggregates inside the lysosomes. At sufficiently high MSDH concentrations, the lysosome is permeabilized, the proteolytic content released to the cytosol and apoptotic cell death is induced.

Lipid vesicles affect the aggregation of 4-hydroxy-2-nonenal-modified α-synuclein oligomers.

Parkinson's disease (PD) and other synucleinopathies are characterized by accumulation of misfolded aggregates of α-synuclein (α-syn). The normal function of α-syn is still under investigation, but it has been generally linked to synaptic plasticity, neurotransmitter release and the maintenance of the synaptic pool. α-Syn localizes at synaptic terminals where it can bind to synaptic vesicles as well as to other cellular membranes. It has become clear that these interactions have an impact on both α-syn functional role and its propensity to aggregate. In this study, we investigated the aggregation process of α-syn covalently modified with 4-hydroxy-2-nonenal (HNE). HNE is a product of lipid peroxidation and has been implicated in the pathogenesis of different neurodegenerative diseases by modifying the kinetics of soluble toxic oligomers. Although HNE-modified α-syn has been reported to assemble into stable oligomers, we found that slightly acidic conditions promoted further protein aggregation. Lipid vesicles delayed the aggregation process in a concentration-dependent manner, an effect that was observed only when they were added at the beginning of the aggregation process. Co-aggregation of lipid vesicles with HNE-modified α-syn also induced cytotoxic effects on differentiated SHSY-5Y cells. Under conditions in which the aggregation process was delayed cell viability was reduced. By exploring the behavior and potential cytotoxic effects of HNE-α-syn under acidic conditions in relation to protein-lipid interactions our study gives a framework to examine a possible pathway leading from a physiological setting to the pathological outcome of PD.

Type IV secretion system core component VirB8 from Brucella binds to the globular domain of VirB5 and to a periplasmic domain of VirB6.

Type IV secretion systems are macromolecular assemblies in the cell envelopes of bacteria that function in macromolecular translocation. Structural biology approaches have provided insights into the interaction of core complex components, but information about proteins that undergo transient interactions with membrane components has not been forthcoming. We have pursued an unbiased approach using peptide arrays and phage display to identify interaction partners and interaction domains of type IV secretion system assembly factor VirB8. These approaches identified the globular domain from the VirB5 protein to interact with VirB8. This interaction was confirmed in cross-linking, pull-down, and fluorescence resonance energy transfer (FRET)-based interaction assays. In addition, using phage display analysis, we identified different regions of VirB6 as potential interaction partners of VirB8. Using a FRET-based interaction assay, we provide the first direct experimental evidence of the interaction of a VirB6 periplasmic domain with VirB8. These results will allow us to conduct directed structural biological work and structure-function analyses aimed at defining the molecular details and biological significance of these interactions with VirB8 in the future.

Plasma membrane calcium pump (PMCA) differential exposure of hydrophobic domains after calmodulin and phosphatidic acid activation.

The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.

The structure of calreticulin C-terminal domain is modulated by physiological variations of calcium concentration.

Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor.

Quantitative analysis of VirB8-VirB9-VirB10 interactions provides a dynamic model of type IV secretion system core complex assembly.

Type IV secretion systems are multiprotein complexes that translocate macromolecules across the bacterial cell envelope. The type IV secretion system in Brucella species encodes 12 VirB proteins that permit this pathogen to translocate effectors into mammalian cells, where they contribute to its survival inside the host. The "core" complex proteins are conserved in all type IV secretion systems, and they are believed to form the channel for substrate translocation. We have investigated the in vitro interactions between the soluble periplasmic domains of three of these VirB components, VirB8, VirB9, and VirB10, using enzyme-linked immunosorbent assays, circular dichroism, and surface plasmon resonance techniques. The in vitro experiments helped in the quantification of the self-association and binary interactions of VirB8, VirB9, and VirB10. Individually, distinct binding properties were revealed that may explain their biological functions, and collectively, we provide direct evidence of the in vitro formation of the VirB8-VirB9-VirB10 ternary complex. To assess the dynamics of these interactions in a simplified in vivo model of complex assembly, we applied the bacterial two-hybrid system in studying interactions between the full-length proteins. This approach demonstrated that VirB9 stimulates the self-association of VirB8 but inhibits VirB10-VirB10 and VirB8-VirB10 interaction. Analysis of a dimerization site variant of VirB8 (VirB8(M102R)) suggested that the interactions with VirB9 and VirB10 are independent of its self-association, which stabilizes VirB8 in this model assay. We propose a dynamic model for secretion system assembly in which VirB8 plays a role as an assembly factor that is not closely associated with the functional core complex comprising VirB9 and VirB10.

Stoichiometry of lipid-protein interaction assessed by hydrophobic photolabeling.

Here we undertook a comparative study of the composition of the lipid annulus of three ATPases pertaining to the P-type family: plasma membrane calcium pump (PMCA), sarcoplasmic reticulum calcium pump (SERCA) and Na,K-ATPase. The photoactivatable phosphatidylcholine analogue [(125)I]TID-PC/16 was incorporated into mixtures of dimyristoyl phosphatidylcholine (DMPC) and each enzyme with the aid of the nonionic detergent C(12)E(10). After photolysis, the extent of the labeling reaction was assessed to determine the lipid:protein stoichiometry: 17 for PMCA, 18 for SERCA, 24 for the Na,K-ATPase (alpha-subunit) and 5.6 mol PC/mol protein for the Na,K-ATPase (beta-subunit).

Phospholipid distribution around the plasma membrane calcium pump: a hydrophobic photolabeling study.

The functions of membrane proteins are highly dependent on their phospholipid environment. In this article, we have used a hydrophobic photolabeling method to study the noncovalent interactions between plasma membrane calcium pump (PMCA) and surrounding phospholipids. With this approach, we determined (1) the number of lipid molecules in close contact with the transmembrane surface, i.e., the lipid-protein stoichiometry, and (2) the distribution of lipid molecules among different regions of the protein. PMCA was photolabeled in mixed micelles containing detergent, the phosphatidylcholine photoactivatable analog 1-palmitoyl-2-[9-[2'-[125I]iodo-4'- (trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine, and different amounts of dimyristoyl phosphatidylcholine (PC). The stoichiometry was estimated after the extent of the labeling reaction had been independently assessed. We determined a maximum number of 17 +/- 1 molecules of PC in close contact with the transmembrane surface per PMCA molecule. In addition, a semiquantitative description of the phospholipid environment around different regions of PMCA was carried out after limited proteolysis of the photolabeled protein. The distribution of labels among the N-terminal (1-322), the central (323-660), and the C-terminal (661-1,205) regions was 26, 36, and 38%, respectively.

VirB6 and VirB10 from the Brucella type IV secretion system interact via the N-terminal periplasmic domain of VirB6.

Type IV secretion systems are multi-protein complexes that transfer macromolecules across the cell envelope of bacteria. Identifying the sites of interaction between the twelve proteins (VirB1-VirB11 and VirD4) that form these complexes is key to understanding their assembly and function. We have here used phage display, bacterial two-hybrid and fluorescence-based interaction assays to identify an N-terminal domain of the inner membrane protein VirB6 as a site of interaction with the envelope-spanning VirB10 protein. Our results are consistent with the notion that VirB6 acts in concert with VirB10 as well as with VirB8 during secretion system assembly and function.

A new conformation in sarcoplasmic reticulum calcium pump and plasma membrane Ca2+ pumps revealed by a photoactivatable phospholipidic probe.

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca(2+) pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2'-[(125)I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([(125)I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [(125)I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca(2+). This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca(2+) and calmodulin decreases by approximately the same amount. However, incubation with Ca(2+) alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [(125)I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E(1) conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca(2+) + C28 for SERCA and Ca(2+) alone for PMCA), and one in which the enzyme is fully active (Ca(2+) for SERCA and Ca(2+)-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.