High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

Reconstitution of Proteoliposomes for Phospholipid Scrambling and Nonselective Channel Assays.

Phospholipid scramblases catalyze the rapid trans-bilayer movement of lipids down their concentration gradients. This process is essential for numerous cellular signaling functions including cell fusion, blood coagulation, and apoptosis. The importance of scramblases is highlighted by the number of human diseases caused by mutations in these proteins. Because of their indispensable function, it is essential to understand and characterize the molecular function of phospholipid scramblases. Powerful tools to measure lipid transport in cells are available. However, these approaches provide limited mechanistic insights into the molecular bases of scrambling. Here we describe in detail an in vitro phospholipid scramblase assay and the accompanying analysis which allows for determination of the macroscopic rate constants associated with phospholipid scrambling. Notably, members of the TMEM16 family of scramblases also function as nonselective ion channels. To better understand the physiological relevance of this channel function as well as its relationship to the scrambling activity of the TMEM16s we also describe in detail an in vitro flux assay to measure nonselective channel activity. Together, these two assays can be used to investigate the dual activities of the TMEM16 scramblases/nonselective channels.

Membrane disruptive antimicrobial potential of NK-lysin from yellow catfish (Pelteobagrus fulvidraco).

NK-lysins, a type of broad-spectrum antimicrobial peptide (AMP), act as an essential effector of innate defense against microbial attack in higher vertebrates and so in fish. The present study delineates the structural and functional characterization of NK-lysin from yellow catfish (Pelteobagrus fulvidrac) (Pelteobagrus fulvidraco). PfNK-lysin encodes a 153-residue peptide, which displays the hallmark features of other known NK-lysins with the ordered array of six well-conserved cysteine residues and five-exon/four-intron structure. It was found to be ubiquitous in tissues, being detected most abundantly in gill and head kidney. In vivo exposure to stimuli (LPS, PolyI:C, and Edwardsiella ictaluri) induced PfNK-lysin expression in head kidney and spleen. Synthetic PfNK-lysin-derived peptide exhibited in vitro bactericidal potency against both Gram-positive and Gram-negative bacteria, with the highest inhibitory effect on pathogen Edwardsiella ictaluri. Fluorescence microscopy and scanning electron microscopy further confirmed its capacity to cause damage to the bacterial plasma membrane. Taken together, these data suggest that PfNK-lysin might participate in antimicrobial defense of yellow catfish by membrane-disruptive action.

Reconstituted Proteoliposome Fusion Mediated by Yeast SNARE-Family Proteins.

Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular membrane trafficking in all eukaryotic cells, which delivers proteins and lipids to their appropriate subcellular membrane compartments such as organelles and plasma membrane. The molecular basis of SNARE-mediated membrane fusion has been revealed by studying fusion of reconstituted proteoliposomes bearing purified SNARE-family proteins and chemically defined lipid species. This chapter describes the detailed experimental protocols for (1) purification of recombinant SNARE-family and SM (Sec1/Munc18-family) proteins in the yeast Saccharomyces cerevisiae; (2) preparation of reconstituted proteoliposomes bearing purified yeast SNARE proteins; and (3) developing an assay to monitor lipid mixing between reconstituted SNARE-bearing proteoliposomes. Lipid mixing assays for reconstituted SNARE-bearing proteoliposomes are useful for evaluating the intrinsic capacity of SNARE-family proteins to directly catalyze membrane fusion and to determine the specificity of membrane fusion.

Protective capacity of proteoliposomes from Mycobacterium bovis BCG in a mouse model of tuberculosis.

Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.

Effects of Ureaplasma urealyticum lipid-associated membrane proteins on rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: As an infectious agent might play a role in rheumatoid arthritis (RA) development, this study investigated effects of Ureaplasma urealyticum lipid-associated membrane proteins (UuLAMPs) on RA synovial fibroblast (RASF) proliferation, and tumour necrosis factor (TNF)-α and interleukin (IL)-1ß production by THP-1 macrophages. Possible immunogenic proteins in UuLAMPs were identified. METHODS: RASFs were cultured from synovial tissue from donors with RA. Serum samples from donors with/without RA and with/without U. urealyticum infection were used for immunogenicity analyses. THP-1 macrophages served as a model for synovial macrophages. TNF-α and IL-1ß mRNA levels were assessed using reverse transcription-polymerase chain reaction; protein levels were estimated using enzyme-linked immunosorbent assay. UuLAMPs underwent separation and Western blot analyses. RESULTS: UuLAMPs (0.025-0.4 µg/ml) stimulated RASF proliferation in a dose- and time-dependent manner, and increased TNF-α and IL-1ß levels in THP-1 macrophages. Several immunogenic UuLAMPs were identified, but antibodies to a 25 kDa protein were only found in RA patients with U. urealyticum infection. CONCLUSIONS: UuLAMPs might induce RASF proliferation and proinflammatory cytokine secretion in synovium from RA patients. A 25 kDa U. urealyticum protein might act as a cross-reactive antigen.

Microfluidic flow cell for sequential digestion of immobilized proteoliposomes.

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 µg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 µg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.

Very small size proteoliposomes derived from Neisseria meningitidis: an effective adjuvant for antigen-specific cytotoxic T lymphocyte response stimulation under leukopenic conditions.

Leukopenia is a severe condition resulting from both pathological processes and some treatments, like chemotherapy in cancer patients. However, the activation of the patient immune system is required for the success of immunotherapeutic strategies, as cancer vaccines. In this regard, leukopenia constitutes a major hurdle to overcome, mainly due to the impairment of cytotoxic T lymphocyte (CTL) responses. Adjuvants are basic components of vaccine formulations, which might be useful to stimulate immunity under this immunosuppressed condition. To this aim, we tested the capacity of a novel nanoparticulated complex, very small size proteoliposomes (VSSP), to promote CTL even in a leukopenic scenario. Noteworthy, we observed that a VSSP-based OVA vaccine induced a normal antigen-specific CTL response in mice rendered leukopenia by the administration of high doses of the chemotherapeutic agent cyclophosphamide (CY), while under the same conditions the OVA antigen formulated in the TLR-3 agonist polyinosinic-polycytidylic acid (P(I:C)) was ineffective. Moreover, an appropriate combination of VSSP with the P(I:C) vaccine was able to restore the CD8(+) T cell effector function in leukopenic mice. VSSP induced not only a faster repopulation of immune cells in CY-receiving animals, but also enhanced the recovery of memory T lymphocytes and myeloid dendritic cells (DCs) while simultaneously abrogated the immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs). Our results suggest that VSSP could be a particularly suitable immunomodulator to be used in CTL-promoting active immunotherapy strategies operating in severe immune compromised scenarios.

Leishmania pifanoi proteoglycolipid complex P8 induces macrophage cytokine production through Toll-like receptor 4.

The P8 proteoglycolipid complex (P8 PGLC) is a glyconjugate expressed by Leishmania mexicana complex parasites. We previously have shown that vaccination with P8 PGLC provides protection against cutaneous leishmaniasis in susceptible BALB/c mice. However, the biological importance of this complex remains unknown. Here we show that P8 PGLC localizes to the surface of Leishmania pifanoi amastigotes and that upon exposure to macrophages, P8 PGLC binds and induces inflammatory cytokine and chemokine mRNAs such as tumor necrosis factor alpha and RANTES early after stimulation. Our studies indicate that cytokine and chemokine induction is dependent upon Toll-like receptor 4 (TLR4). Interestingly, key inflammatory cytokines and chemokines (such as interleukin-6 [IL-6], macrophage inflammatory protein 1beta, and beta interferon [IFN-beta]) that can be induced through TLR4 activation were not induced or only slightly upregulated by P8 PGLC. Activation by P8 PGLC does not occur in the presence of TLR4 alone and requires both CD14 and myeloid differentiation protein 2 for signaling; this requirement may be responsible for the limited TLR4 response. This is the first characterization of a TLR4 ligand for Leishmania. In vitro experiments indicate that L. pifanoi amastigotes induce lower levels of cytokines in macrophages in the absence of TLR4; however, notably higher IL-10/IFN-gamma ratios were found for TLR4-deficient mice than for BALB/c mice. Further, increased levels of parasites persist in BALB/c mice deficient in TLR4. Taken together, these results suggest that TLR4 recognition of Leishmania pifanoi amastigotes is important for the control of infection and that this is mediated, in part, through the P8 PGLC.

Association of two proteolipids of unknown function with ATP synthase from bovine heart mitochondria.

ATP synthase, or F-ATPase, purified from bovine heart mitochondria in the absence of phospholipids is an assembly of 16 different subunits. In the presence of exogenous phospholipids, two additional hydrophobic proteins, a 6.8kDa proteolipid and diabetes associated protein in insulin sensitive tissue (DAPIT), were associated with the purified complex, with DAPIT at sub-stoichiometric levels. Both proteins are conserved in vertebrates and invertebrates, but not in fungi, and prokaryotic F-ATPases do not contain orthologues of either of them. Therefore, their roles are likely to be peripheral to the synthesis of ATP.

Very small size proteoliposomes derived from Neisseria meningitidis: an effective adjuvant for dendritic cell activation.

Recent findings in the interactions between pathogens and the innate immune system, particularly with dendritic cells (DC), have opened new opportunities for adjuvants designs. We have elaborated a new approach, in which gangliosides are incorporated into the outer membrane complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). VSSP demonstrated a unique ability to render highly tolerated gangliosides immunogenic. These results drove our attention to the immunopotentiating properties of VSSP. Here we examined the VSSP adjuvant effect on dendritic cell activation. Also the role of LPS on this effect was dissected. This study reveals that VSSP is a potent adjuvant for DC activation.

Strategy for determination of an efficient Cochleate particle size.

Cochleate structures obtained from the outer membrane of Neisseria meningitidis serotype B have demonstrated to be high immunogenicity when administrated by intramuscular, oral or intranasal routes, and could be used as adjuvant and meningococcal nasal vaccine candidate. Due to the microparticulate nature of Cochleate it is necessary to control the particle size since it capture by cells of the immune system could be affected by this aspect. We combined optic microscopy and immunisation experiments to select the optimum particle size. Six different processes of producing Cochleate obtaining were evaluated and different mechanical stress conditions were carried out to homogenize and modulate the particles size. The more immunogenic particles were selected on the basis of the levels of specific IgA and IgG antibodies induced after intranasal immunisation in mice. The best treatment parameter for mechanical stress of the Cochleate was prolonged treatment with untrasonic low frequency waves.

Scale up of proteoliposome derived Cochleate production.

Cochleate are highly stable structures with promising immunological features. Cochleate structures are usually obtaining from commercial lipids. Proteoliposome derived Cochleate are derived from an outer membrane vesicles of Neisseria meningitidis B. Previously, we obtained Cochleates using dialysis procedures. In order to increase the production process, we used a crossflow system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The raw material and solutions used in the production process are already approved for human application. This work demonstrates that CFS is very efficient process to obtain Cochleate structures with a yield of more than 80% and the immunogenicity comparable to that obtained by dialysis membrane.

Concanamycin and indolyl pentadieneamide inhibitors of the vacuolar H+-ATPase bind with high affinity to the purified proteolipid subunit of the membrane domain.

The macrolide antibiotic concanamycin is a potent and specific inhibitor of the vacuolar H(+)-ATPase (V-ATPase), binding to the V(0) membrane domain of this eukaryotic acid pump. Although binding is known to involve the 16 kDa proteolipid subunit, contributions from other V(0) subunits are possible that could account for the apparently different inhibitor sensitivities of pump isoforms in vertebrate cells. In this study, we used a fluorescence quenching assay to directly examine the roles of V(0) subunits in inhibitor binding. Pyrene-labeled V(0) domains were affinity purified from Saccharomyces vacuolar membranes, and the 16 kDa proteolipid was subsequently extracted into chloroform and methanol and purified by size exclusion chromatography. Fluorescence from the isolated proteins was strongly quenched by nanomolar concentrations of both concanamycin and an indolyl pentadieneamide compound, indicating high-affinity binding of both natural macrolide and synthetic inhibitors. Competition studies showed that these inhibitors bind to overlapping sites on the proteolipid. Significantly, the 16 kDa proteolipid in isolation was able to bind inhibitors as strongly as V(0) did. In contrast, proteolipids carrying mutations that confer resistance to both inhibitors showed no binding. We conclude that the extracted 16 kDa proteolipid retains sufficient fold to form a high-affinity inhibitor binding site for both natural and synthetic V-ATPase inhibitors and that the proteolipid contains the major proportion of the structural determinants for inhibitor binding. The role of membrane domain subunit a in concanamycin binding and therefore in defining the inhibitor binding properties of tissue-specific V-ATPases is critically re-assessed in light of these data.

Expression of recombinant forms of human 21.5 kDa myelin basic protein and proteolipid protein in CHO cells.

MBP and PLP are major structural protein components of myelin. Both proteins play a functional role in formation of myelin sheath and in maintenance of its compaction. Immune responses to MBP and PLP have been implicated in the pathogenesis of multiple sclerosis (MS), an auto-immune disease of the central nervous system. Recombinant forms of both proteins isolated and purified from bacterial or insect cell systems are commonly used to study the specificity of auto-response in MS. We have prepared recombinant forms of MBP and PLP stably expressed in CHO cells. Several clones with proper cytoplasmic MBP or surface PLP localization were obtained and characterized by flow cytometry and indirect immunostaining. CHO cells expressing the recombinant forms of MBP and PLP can be very useful in studies on the autoimmune mechanism of MS.

Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies.

Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the Ca(2+)-ATPases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport properties. PLB interacts with and regulates the cardiac Ca(2+)-ATPase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. SLN interacts with and regulates the skeletal muscle Ca(2+)-ATPase in a mechanism analogous to that used by PLB. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies, such as electron cryo-microscopy and X-ray crystallography, large quantities of over-expressed and purified protein are required. Herein, we report a modified method for producing large quantities of PLB and SLN in a rapid and efficient manner. Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins. A tobacco etch virus protease site allowed specific cleavage of the fusion protein and release of recombinant PLB or SLN. Selective solubilization with guanidine-hydrochloride followed by reverse-phase HPLC permitted the rapid, large-scale production of highly pure protein. Reconstitution and measurement of ATPase activity confirmed the functional interaction between our recombinant regulatory proteins and Ca(2+)-ATPase. The inhibitory properties of the over-produced proteins were consistent with previous studies, where the inhibition was relieved by elevated calcium concentrations. In addition, we show that our recombinant PLB and SLN are suitable for high-resolution structural studies.

Surface-exposed glycopeptidolipids of Mycobacterium smegmatis specifically inhibit the phagocytosis of mycobacteria by human macrophages. Identification of a novel family of glycopeptidolipids.

Phagocytosis by macrophages represents the early step of the mycobacterial infection. It is governed both by the nature of the host receptors used and the ligands exposed on the bacteria. The outermost molecules of the nonpathogenic Mycobacterium smegmatis were extracted by a mechanical treatment and found to specifically and dose dependently inhibit the phagocytosis of both M. smegmatis and the opportunistic pathogen M. kansasii by human macrophages derived from monocytes. The inhibitory activity was attributed to surface lipids because it is extracted by chloroform and reduced by alkaline hydrolysis but not by protease treatment. Fractionation of surface lipids by adsorption chromatography indicated that the major inhibitory compounds consisted of phospholipids and glycopeptidolipids (GPLs). Mass spectrometry and nuclear magnetic resonance spectroscopy analyses, combined with chemical degradation methods, demonstrated the existence of a novel family of GPLs that consists of a core composed of the long-chain tripeptidyl amino-alcohol with a di-O-acetyl-6-deoxytalosyl unit substituting the allo-threoninyl residue and a 2-succinyl-3,4-di-O-CH3-rhamnosyl unit linked to the alaninol end of the molecules. These compounds, as well as diglycosylated GPLs at the alaninol end and de-O-acylated GPLs, but not the non-serovar-specific di-O-acetylated GPLs, inhibited the phagocytosis of M. smegmatis and M. avium by human macrophages at a few nanomolar concentration without affecting the rate of zymosan internalization. At micromolar concentrations, the native GPLs also inhibit the uptake of both M. tuberculosis and M. kansasii. De-O-acylation experiments established the critical roles of both the succinyl and acetyl substituents. Collectively, these data provide evidence that surface-exposed mycobacterial glycoconjugates are efficient competitors of the interaction between macrophages and mycobacteria and, as such, could represent pharmacological tools for the control of mycobacterial infections.

Amoebapores and NK-lysin, members of a class of structurally distinct antimicrobial and cytolytic peptides from protozoa and mammals: a comparative functional analysis.

Amoebapores, the pore-forming polypeptides of the protozoan parasite Entamoeba histolytica, and NK-lysin, an effector molecule of porcine NK (natural killer) and cytotoxic T cells, belong to the same protein family, the saposin-like proteins. As both types of protein are implicated in the killing of microbes in vivo, it appears that phylogenetically diverse organisms such as amoebae and mammals use similar effector molecules to fulfil a comparable task. However, structural features have led to the assumption that the proteins display their activities according to different modes of action. To address this question, we analysed the antibacterial, cytotoxic and pore-forming activities of these proteins in parallel and in comprehensive detail. Interestingly, the comparison of activities revealed significant differences. Whereas NK-lysin, recombinantly expressed, is efficient at a broad range of pH values, the amoebapores exhibited a pronounced pH dependence of all their activities, with markedly decreased activity at pH values above 6. Moreover, increasing salinity affects amoebapores more drastically than NK-lysin. All of the proteins compared were found to be potently active against Gram-positive bacteria, but only NK-lysin was equally efficient against Gram-negative bacteria. However, the amoebapores displayed five times higher pore-forming activity than NK-lysin, which is in accordance with the more hydrophobic character of the amoebapores compared with the essentially cationic NK-lysin.

Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies.

Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct interaction with the Ca-ATPase in cardiac and skeletal muscle, respectively. The molecular details of these protein-protein interactions are as yet undetermined. Solution and solid-state NMR spectroscopies have proven to be effective tools for deciphering such regulatory mechanisms to a high degree of resolution; however, large quantities of pure recombinant protein are required for these studies. Thus, recombinant PLB and SLN production in Escherichia coli was optimized for use in NMR experiments. Fusions of PLB and SLN to maltose binding protein (MBP) were constructed and optimal conditions for protein expression and purification were screened. This facilitated the large-scale production of highly pure protein. To confirm their functionality, the biological activities of recombinant PLB and SLN were compared to those of their synthetic counterparts. The regulation of Ca-ATPase activity by recombinant PLB and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLB and SLN.