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As an antimicrobial peptide, NK-lysin ( NKL ) plays an important role in the innate immune system of organisms. In this study, 300 piglets (68 Landrace pigs, 158 Large White pigs and 74 Songliao Black pigs) were used to further explore the function of NLK gene in porcine immune system. The quantitative real-time PCR analysis detected the NKL gene's expression, and the result demonstrated that NKL mRNA was expressed in lung, spleen, stomach, kidney, liver and heart, and the expression level decreased sequentially. A single-nucleotide polymorphism (SNP, g.59070355 G > A) in intron 3 of the NKL gene was detected by PCR amplification and sequencing. The results of the Chi-square (χ2) test showed that the genotype of the SNP was consistent with the Hardy-Weinberg equilibrium. What's more, association analysis results showed the SNP in NKL gene was significantly associated with T lymphocyte subpopulations. Different genotypes had significant effects on the proportion of CD4 - CD8 - , CD4 - CD8 + , CD4 + CD8 + , CD8 + , CD4 + /CD8 + in peripheral blood ( p < 0.05). These results further suggested that NKL could be recognized as a promising immune gene for swine disease resistance breeding.

Proteolipids are proteins with unusual lipid-like properties. It has long been established that PLP and plasmolipin, which are two unrelated membrane-tetra-spanning myelin proteolipids, can be converted in vitro into a water-soluble form with a distinct conformation, raising the question of whether these, or other similar proteolipids, can adopt two different conformations in the cell to adapt their structure to distinct environments. Here, we show that MALL, another proteolipid with a membrane-tetra-spanning structure, distributes in membranes outside the nucleus and, within the nucleus, in membrane-less, liquid-like PML body biomolecular condensates. Detection of MALL in one or other environment was strictly dependent on the method of cell fixation used, suggesting that MALL adopts different conformations depending on its physical environment -lipidic or aqueous- in the cell. The acquisition of the condensate-compatible conformation requires PML expression. Excess MALL perturbed the distribution of the inner nuclear membrane proteins emerin and LAP2β, and that of the DNA-binding protein BAF, leading to the formation of aberrant nuclei. This effect, which is consistent with studies identifying overexpressed MALL as an unfavorable prognostic factor in cancer, could contribute to cell malignancy. Our study establishes a link between proteolipids, membranes and biomolecular condensates, with potential biomedical implications., (© 2022. The Author(s).)

The Na + /H + exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na + /Li + exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na + /H + exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity., (© 2022. The Author(s).)

Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in Escherichia coli and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, E. coli K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to E. coli K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon E. coli K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the E. coli K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Spectral editing is crucial to simplify the crowded solid-state NMR spectra of proteins. New techniques are introduced to edit 13 C- 13 C correlations of uniformly labeled proteins under moderate magic-angle spinning (MAS), based on our recent frequency-selective homonuclear recoupling sequences [Zhang et al., J. Phys. Chem. Lett. 2020, 11, 8077-8083]. The signals of alanine, serine, or threonine residues are selected out by selective 13 Cα- 13 Cβ double-quantum filtering (DQF). The 13 Cα- 13 Cβ correlations of alanine residues are selectively established with efficiency up to ~ 1.8 times that by dipolar-assisted rotational resonance (DARR). The techniques are shown in 2D/3D NCCX experiments and applied to the uniformly 13 C, 15 N labeled Aquaporin Z (AqpZ) membrane protein, demonstrating their potential to simplify spectral analyses in biological solid-state NMR.

Photosynthesis is tightly regulated in order to withstand dynamic light environments. Under high light intensities, a mechanism known as non-photochemical quenching (NPQ) dissipates excess excitation energy, protecting the photosynthetic machinery from damage. An obstacle that lies in the way of understanding the molecular mechanism of NPQ is the large gap between in vitro and in vivo studies. On the one hand, the complexity of the photosynthetic membrane makes it challenging to obtain molecular information from in vivo experiments. On the other hand, a suitable in vitro system for the study of quenching is not available. Here we have developed a minimal NPQ system using proteoliposomes. With this, we demonstrate that the combination of low pH and PsbS is both necessary and sufficient to induce quenching in LHCII, the main antenna complex of plants. This proteoliposome system can be further exploited to gain more insight into how PsbS and other factors (e.g. zeaxanthin) influence the quenching mechanism observed in LHCII.

De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

This study aimed to determine the transcription profile of NK-lysin gene in native chickens. Moreover, it was targeted toward determining the primary, three-dimensional, and molecular dynamic structures of NK-lysin and granulysin peptides to understand their mode of action and intracellular transduction pathways using in silico analysis. The results revealed that NK-lysin gene in native chickens and Gallus gallus were closely related to those of other avian species. However, there was a low sequence homology when aligned with the mammalian peptides. The coding region of NK-lysin peptide in native chickens encoded 140 amino acids as found in G. gallus with a homology of 98% that declined to 20%, particularly in mammalian species. The results revealed that the NK-lysin in native chickens was closely related to that in avian species at a range of 71-76%. However, it was different from that of other mammalians in terms of nucleotide and amino acid identities. The mRNA transcripts of NK-lysin had high and moderate expression levels in the testis and pancreas, respectively. Nonetheless, the small intestine, kidney, spleen, and liver had a low expression level. The NK-lysin peptides contained more than 50% of the total AA with a nonpolar feature, whereas polar AA constituted up to 30% of AA. The results also indicated that the hydrophilic regions and positively charged amino acids were predominant on the surface of the investigated peptides. The NK-lysin was folded in 4-5 helical units and 3-4 loop structures in their saposin domain. The third helical peptide was long in both avian and bovine species (104-123 residues). However, the fourth helical peptide was short in humans, pigs, and chimpanzees (101-123, 104-123, and 102-124 residues, respectively), with the helical unit residues of 95-97, 96-99, and 96-98, respectively. The obtained results can be helpful in developing novel approaches that could be used as alternatives or adjuncts to the existing means of control., (Copyright © 2020. Published by Elsevier Inc.)

Aggregation of human α-synuclein (αSyn) is linked to Parkinson's disease (PD) pathology. The central region of the αSyn sequence contains the non-amyloid β-component (NAC) crucial for aggregation. However, how NAC flanking regions modulate αSyn aggregation remains unclear. Using bioinformatics, mutation and NMR, we identify a 7-residue sequence, named P1 (residues 36-42), that controls αSyn aggregation. Deletion or substitution of this 'master controller' prevents aggregation at pH 7.5 in vitro. At lower pH, P1 synergises with a sequence containing the preNAC region (P2, residues 45-57) to prevent aggregation. Deleting P1 (ΔP1) or both P1 and P2 (ΔΔ) also prevents age-dependent αSyn aggregation and toxicity in C. elegans models and prevents αSyn-mediated vesicle fusion by altering the conformational properties of the protein when lipid bound. The results highlight the importance of a master-controller sequence motif that controls both αSyn aggregation and function-a region that could be targeted to prevent aggregation in disease.

Integral membrane proteins are exposed to a complex and dynamic lipid environment modulated by nonbilayer lipids that can influence protein functions by lipid-protein interactions. The nonbilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in plant photosynthetic thylakoid membranes, but its impact on the functionality of energy-converting membrane protein complexes is unknown. Here, we optimized a detergent-based reconstitution protocol to develop a proteoliposome technique that incorporates the major light-harvesting complex II (LHCII) into compositionally well-defined large unilamellar lipid bilayer vesicles to study the impact of MGDG on light harvesting by LHCII. Using steady-state fluorescence spectroscopy, CD spectroscopy, and time-correlated single-photon counting, we found that both chlorophyll fluorescence quantum yields and fluorescence lifetimes clearly indicate that the presence of MGDG in lipid bilayers switches LHCII from a light-harvesting to a more energy-quenching mode that dissipates harvested light into heat. It is hypothesized that in the in vitro system developed here, MGDG controls light harvesting of LHCII by modulating the hydrostatic lateral membrane pressure profile in the lipid bilayer sensed by LHCII-bound peripheral pigments.

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [ 14 C]-tetraethylamonium or [ 3 H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli . Removal of cholesterol from the native transporter by MβCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MβCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [ 14 C]-tetraethylamonium or [ 3 H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

Myelin and lymphocyte protein (MAL) is a tetraspan integral membrane protein that resides in detergent-insoluble membrane fractions enriched in condensed membranes. MAL is expressed in oligodendrocytes, in Schwann cells, where it is essential for the stability of myelin, and at the apical membrane of epithelial cells, where it has a critical role in transport. In T lymphocytes, MAL is found at the immunological synapse and plays a crucial role in exosome secretion. However, no involvement of MAL in viral infections has been reported so far. Here, we show that herpes simplex virus 1 (HSV-1) virions travel in association with MAL-positive structures to reach the end of cellular processes, which contact uninfected oligodendrocytes. Importantly, the depletion of MAL led to a significant decrease in infection, with a drastic reduction in the number of lytic plaques in MAL-silenced cells. These results suggest a significant role for MAL in viral spread at cell contacts. The participation of MAL in the cell-to-cell spread of HSV-1 may shed light on the involvement of proteolipids in this process. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establish latent infections in neurons. HSV-1 may spread from infected to uninfected cells by two main routes: by cell-free virus or by cell-to-cell spread. In the first case, virions exit into the extracellular space and then infect another cell from the outside. In the second case, viral transmission occurs through cell-to-cell contacts via a mechanism that is still poorly understood. A third mode of spread, using extracellular vesicles, also exists. In this study, we demonstrate the important role for a myelin protein, myelin and lymphocyte protein (MAL), in the process of cell-to-cell viral spread in oligodendrocytes. We show that MAL is involved in trafficking of virions along cell processes and that MAL depletion produces a significant alteration in the viral cycle, which reduces cell-to cell spread of HSV-1., (Copyright © 2020 López-Guerrero et al.)

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn 4 -Ala and Thr 5 -Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na + /H + antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na + /H + antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na + /H + antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is a powerful technique to determine the topology of membrane proteins in oriented lipid bilayers. Separated local field (SLF) experiments are central to this technique as they provide first-order orientational restraints, i.e., dipolar couplings and anisotropic chemical shifts. Despite the use of low-E (or E-free) probes, the heat generated during the execution of 2D and 3D SLF pulse sequences causes sizeable line-shape distortions. Here, we propose a new heat-compensated SE-SAMPI4 (hcSE-SAMPI4) pulse sequence that holds the temperature constant for the duration of the experiment. This modification of the SE-SAMPI4 results in sharper and more intense resonances without line-shape distortions. The spectral improvements are even more apparent when paramagnetic relaxation agents are used to speed up data collection. We tested the hcSE-SAMPI4 pulse sequence on a single-span membrane protein, sarcolipin (SLN), reconstituted in magnetically aligned lipid bicelles. In addition to eliminating peak distortions, the hcSE-SAMPI4 experiment increased the average signal-to-noise ratio by 20% with respect to the original SE-SAMPI4.

Erythrocytes or red blood cells (RBCs) represent a promising cell-mediated drug delivery platform due to their inherent biocompatibility. Here, we developed an antigen delivery system based on the nanoerythrosomes derived from RBCs, inspired by the splenic antigen-presenting cell targeting capacity of senescent RBCs. Tumor antigens were loaded onto the nanoerythrosomes by fusing tumor cell membrane-associated antigens with nanoerythrosomes. This tumor antigen-loaded nanoerythrosomes (nano-Ag@erythrosome) elicited antigen responses in vivo and, in combination with the anti-programmed death ligand 1 (PD-L1) blockade, inhibited the tumor growth in B16F10 and 4T1 tumor models. We also generated a tumor model showing that "personalized nano-Ag@erythrosomes" could be achieved by fusing RBCs and surgically removed tumors, which effectively reduced tumor recurrence and metastasis after surgery., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA 2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF 3 . Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.

2D homonuclear NMR spectroscopy is an essential technique to characterize small and large molecules, such as organic compounds, metabolites, and biomacromolecules at atomic resolution. However, for complex samples 2D homonuclear spectra display poor resolution, making spectral assignment very cumbersome. Here, we propose a new method that exploits the differential T 2 * relaxation times of individual resonances and resolves the 2D NMR peaks into pseudo-3D spectra, where time is the 3 rd dimension. T 2 * weIghted DEconvolution or TIDE analyzes individual free induction decays (FIDs) and dissects them into sub-FIDs that are transformed into pseudo-3D spectra combining Fourier transformation and covariance NMR. TIDE achieves higher resolution and sensitivity for NMR spectra than classical covariance NMR reducing offset-dependent artifacts. We demonstrate the performance of TIDE for magic angle spinning (MAS) [ 13 C, 13 C]-DARR NMR spectra of single- and multi-span membrane proteins embedded in lipid bilayers. Since TIDE is applicable to all type of homonuclear correlation experiments for liquid and solid samples, we anticipate that it will be a general method for processing NMR data of biomacromolecules, complex mixtures of metabolites as well as material samples.

PglK is a lipid-linked oligosaccharide (LLO) flippase essential for asparagine-linked protein glycosylation in Campylobacter jejuni. Previously we have proposed a non-alternating-access LLO translocation mechanism, where postulated outward-facing states play a primary role. To investigate this unusual mechanistic proposal, we have determined a high-resolution structure of PglK that displays an outward semi-occluded state with the two nucleotide binding domains forming an asymmetric closed dimer with two bound ATPγS molecules. Based on this structure, we performed extensive molecular dynamics simulations to investigate LLO recognition and flipping. Our results suggest that PglK may employ a "substrate-hunting" mechanism to locally increase the LLO concentration and facilitate its jump into the translocation pathway, for which sugars from the LLO head group are essential. We further conclude that the release of LLO to the outside occurs before ATP hydrolysis and is followed by the closing of the periplasmic cavity of PglK., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot ( Scophthalmus maximus ) NK-lysine short peptide (Nkl 71⁻100 ) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this work several biophysical methods, such as vesicle aggregation, leakage and fluorescence anisotropy, are employed to investigate the interaction of Nkl 71⁻100 with different glycerophospholipid vesicles. At acidic pH, Nkl 71⁻100 preferably interacts with phosphatidylserine (PS), disrupts PS membranes, and allows the content leakage from vesicles. Furthermore, Nkl 71⁻100 exerts strong antiviral activity against spring viremia of carp virus (SVCV) by inhibiting not only the binding of viral particles to host cells, but also the fusion of virus and cell membranes, which requires a low pH context. Such antiviral activity seems to be related to the important role that PS plays in these steps of the replication cycle of SVCV, a feature that is shared by other families of virus-comprising members with health and veterinary relevance. Consequently, Nkl 71⁻100 is shown as a promising broad-spectrum antiviral candidate.

Although orphan G protein-coupled receptors (GPCRs) have been used as targets to discover unidentified natural ligands, increasing numbers of non-GPCRs have been found to mediate important biological functions. Bioinformatics of genome and cDNA resources predict putative bioactive peptides, demanding an alternative approach to efficiently unravel cell surface targets. In silico analysis of a full-length cDNA library previously allowed us to identify salusin-β, a parasympathomimetic/pro-atherosclerotic peptide with unique physicochemical properties. Here, we show that the β-chain of ATP synthase is a cell surface receptor for salusin-β by utilizing artificial liposomes embedded with endogenous membrane proteins directly transferred from animal tissues while retaining the ligand-binding capability. Conventional techniques using detergents identified a β-actin-profilin complex as membrane-associated salusin-β-binding proteins, but failed to identify the cell surface receptor. Since the α-chain of ATP synthase is a principal cell surface target for angiostatin, a potent endogenous angiogenesis inhibitor, we investigated whether salusin-β modulates angiogenesis. Salusin-β inhibited cell surface ATP synthase activity and prevented sarcoma cell-induced angiogenesis in an in vivo mouse air sac model. Therefore, salusin-β binds to membrane-bound ATP synthase and acts as an angiogenesis inhibitor. The current methodology allows the identification of novel cell surface targets, irrespective of the receptor structure.

Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H + /organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney and liver. To investigate the mechanism of transport, we have established an in vitro assay procedure that includes its expression in insect cells, solubilization with octyl glucoside, purification, and reconstitution into liposomes. The resultant proteoliposomes containing hMATE1 as the sole protein component took up radiolabeled tetraethylammonium (TEA) in a ∆pH-dependent and electroneutral fashion. Furthermore, lipid-detergent micelle containing hMATE1 showed ∆pH-dependent TEA binding similar to transport. Mutated hMATE1 with replacement E273Q completely lacked these TEA binding and transport. In the case of divalent substrates, transport was electrogenic. These observations indicate that the stoichiometry of OC/H + exchange is independent of substrate charge. Purification and reconstitution of hMATE1 is considered to be suitable for understanding the detailed molecular mechanisms of hMATE1. The results suggest that Glu273 of hMATE1 plays essential roles in substrate binding and transport., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Acquisition of the trace element copper (Cu) is critical to drive essential eukaryotic processes such as oxidative phosphorylation, iron mobilization, peptide hormone biogenesis, and connective tissue maturation. The Ctr1/Ctr3 family of Cu importers, first discovered in fungi and conserved in mammals, are critical for Cu + movement across the plasma membrane or mobilization from endosomal compartments. Whereas ablation of Ctr1 in mammals is embryonic lethal, and Ctr1 is critical for dietary Cu absorption, cardiac function, and systemic iron distribution, little is known about the intrinsic contribution of Ctr1 for Cu + permeation through membranes or its mechanism of action. Here, we identify three members of a Cu + importer family from the thermophilic fungus Chaetomium thermophilum : Ctr3a and Ctr3b, which function on the plasma membrane, and Ctr2, which likely functions in endosomal Cu mobilization. All three proteins drive Cu and isoelectronic silver (Ag) uptake in cells devoid of Cu + importers. Transport activity depends on signature amino acid motifs that are conserved and essential for all Ctr1/3 transporters. Ctr3a is stable and amenable to purification and was incorporated into liposomes to reconstitute an in vitro Ag + transport assay characterized by stopped-flow spectroscopy. Ctr3a has intrinsic high-affinity metal ion transport activity that closely reflects values determined in vivo , with slow turnover kinetics. Given structural models for mammalian Ctr1, Ctr3a likely functions as a low-efficiency Cu + ion channel. The Ctr1/Ctr3 family may be tuned to import essential yet potentially toxic Cu + ions at a slow rate to meet cellular needs, while minimizing labile intracellular Cu + pools., (© 2018 Logeman and Thiele.)

Aquaporins (AQPs) are widely applied in biomimetic membranes for water recycling and desalination. In this study, a novel aquaporin was isolated from Photobacterium profundum SS9 (AQP SS9), which showed high water permeability and potential for practical water purification applications. To improve the stability of the AQP SS9 embedded biomimetic membranes, a modified AQP SS9 was obtained by incorporation of an unnatural amino acid ( p -propargyloxyphenylalanine, p Ppa) (P-AQP SS9) in vitro using a mutated Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and the cell-free expression system. The modified AQP SS9 can covalently link with phospholipids and hence significantly improve the stability of biomimetic membranes. The concentration of Mg 2+ and fusion expression with signal peptides were evaluated to enhance the expression level of P-AQP SS9, resulting in a highest yield of 49 mg/L. The modified AQP SS9 was then reconstituted into DOPC liposomes and analyzed by a stopped-flow spectrophotometer. The obtained water permeability coefficient (P f ) of 7.46×10 -4 m/s was 5.7 times higher than that of proteoliposomes with the wild-type AQP SS9 (P f =1.31×10 -4 m/s) and 12.1 times higher than that of the DOPC liposomes (P f =6.15×10 -5 m/s). This study demonstrates the development of a cell-free system for the expression of membrane proteins with much higher stability and the potential application of the modified aquaporins for water filtration.

Despite the many advantages of small interfering RNA (siRNA) inhalation therapy and a growing prevalence of respiratory pathologies, its clinical translation is severely hampered by inefficient intracellular delivery. To this end, we previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). Interestingly, the PS shell was shown to markedly improve particle stability as well as intracellular siRNA delivery in vitro and in vivo. The major aim of this work was to identify the key molecular components of PS responsible for the enhanced siRNA delivery and evaluate how the complexity of the PS coat could be reduced. We identified surfactant protein B (SP-B) as a potent siRNA delivery enhancer when reconstituted in proteolipid coated hydrogel nanocomposites. Improved cytosolic siRNA delivery was achieved by inserting SP-B into a simplified phospholipid mixture prior to nanogel coating. This effect was observed both in vitro (lung epithelial cell line) and in vivo (murine acute lung injury model), albeit that distinct phospholipids were required to achieve these results. Importantly, the developed nanocomposites have a low in vivo toxicity and are efficiently taken up by resident alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Our results demonstrate the potential of the endogenous protein SP-B as an intracellular siRNA delivery enhancer, paving the way for future design of nanoformulations for siRNA inhalation therapy., Statement of Significance: Despite the therapeutic potential of small interfering RNA (siRNA) and a growing prevalence of lung diseases for which innovative therapies are needed, a safe and effective siRNA inhalation therapy remains non-existing due to a lack of suitable formulations. We identified surfactant protein B (SP-B) as a potent enhancer of siRNA delivery by proteolipid coated nanogel formulations in vitro in a lung epithelial cell line. The developed nanocomposites have a low in vivo toxicity and show a high uptake by alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Importantly, in vivo SP-B is also critical for the developed formulation to obtain a significant silencing of TNFα in a murine LPS-induced acute lung injury model., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Ion channels are transmembrane proteins that mediate ion transport across biological membranes. Ion channel function is traditionally characterized by electrical parameters acquired with techniques such as patch-clamping and reconstitution in lipid bilayer membranes (BLM) that provide relevant information such as ionic conductance, selectivity, and gating properties. High resolution structural information of ion channels however, requires independent technologies, of which atomic force microscopy (AFM) is the only one that provides topological features of single functional channel proteins in their native environments. To date practically no data exist on direct correlations between electrical features and topological parameters from functional single channel complexes. Here, we report the design and construction of a BLM reconstitution microchamber that supports the simultaneous recording of electrical currents and AFM imaging from single channel complexes. As proof-of-principle, we tested the technique on polycystin-2 (PC2, TRPP2), a TRP channel family member from which we had previously elucidated its tetrameric topology by AFM imaging, and single channel currents by the BLM technique. The experimental setup provided direct structural-functional correlates from PC2 single channel complexes that disclosed novel topological changes between the closed and open sub-conductance states of the functional channel, namely, an inverse correlation between conductance and height of the channel. Unexpectedly, we also disclosed intrinsic PC2 mechanosensitivity in response to external forces. The platform provides a suitable means of accessing topological information to correlate with ion channel electrical parameters essential to understand the physiology of these transmembrane proteins., Competing Interests: The authors have declared that no competing interests exist.

Cell-free (CF) protein synthesis has emerged as a powerful technique platform for efficient protein production in vitro. Liposomes have been widely studied as therapeutic carriers due to their biocompatibility, biodegradability, low toxicity, flexible surface manipulation, easy preparation, and higher cargo encapsulation capability. However, rapid immune clearance, insufficient targeting capacity, and poor cytoplasmic delivery efficiency substantially restrict their clinical application. The incorporation of functional membrane proteins (MPs) or peptides allows the transfer of biological properties to liposomes and imparts them with improved circulation, increased targeting, and efficient intracellular delivery. Liposome-chaperoned CF synthesis enables production of proteoliposomes in one-step reaction, which not only substantially simplifies the production procedure but also keeps protein functionality intact. Building off these observations, proteoliposomes with integrated MPs represent an excellent candidate for therapeutic delivery. In this review, we describe recent advances in CF synthesis with emphasis on detailing key factors for improving CF expression efficiency. Furthermore, we provide insights into strategies for rational design of proteoliposomal nanodelivery systems via CF synthesis., Statement of Significance: Liposome-chaperoned CF synthesis has emerged as a powerful approach for the design of recombinant proteoliposomes in one-step reaction. The incorporation of bioactive MPs or peptides into liposomes via CF synthesis can facilitate the development of proteoliposomal nanodelivery systems with improved circulation, increased targeting, and enhanced cellular delivery capacity. Moreover, by adapting lessons learned from natural delivery vehicles, novel bio-inspired proteoliposomes with enhanced delivery properties could be produced in CF systems. In this review, we first give an overview of CF synthesis with focus on enhancing protein expression in liposome-chaperoned CF systems. Furthermore, we intend to provide insight into harnessing CF-synthesized proteoliposomes for efficient therapeutic delivery., (Copyright © 2018. Published by Elsevier Ltd.)

Antimicrobial peptides (AMPs) are a class of natural peptides with varying numbers of amino acids. They are principal components of innate immunity in vertebrates, encoding natural antibiotics and providing a protective response against a broad range of microbes including those responsible for tuberculosis, an important disease in bison. NK-lysins are AMPs that have been described in various organisms and are coded by a single gene in several mammalian species, including human. Recently, we described a family of 4 NK-lysin genes in cattle. Here, we examined NK-lysin genes in bison and identified 4 bison paralogs (NK1, NK2A, NK2B, and NK2C), although the current bison genome assembly annotates only 2 (NK1 and NK2). Sequence and phylogenetic analysis support the triplication of NK2 prior to the most recent common ancestor of bison and cattle. Comparative mapping of bison and cattle paralogs indicates that the NK-lysin family is located on bison chromosome 11 with well-conserved synteny of flanking genes relative to cattle. The 3 bison NK-lysin2 genes share high sequence similarity with each other. RNA-seq analysis demonstrates that NK2A, NK2B, and NK2C are expressed primarily in the lung, whereas NK1 is expressed at low levels in all tissues studied. This tissue expression pattern differs from that previously reported for cattle, suggesting some divergence in function since the evolutionary separation of the 2 species.

This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0-2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 μM, 94.6 ± 47.6 μM, and 0.21 ± 0.07 μM, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10-7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 μM for verapamil IC50, 61.8 ± 34.8 μM for colchicine IC50, and 0.23 ± 0.09 μM for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates., Competing Interests: The authors have declared that no competing interests exist.

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality., Competing Interests: The authors have declared that no competing interests exist.

The field of membrane protein structural biology has been revolutionized over the last few years with a number of high profile structures being solved using cryo-EM including Piezo, Ryanodine receptor, TRPV1 and the Glutamate receptor. Further developments in the EM field hold the promise of even greater progress in terms of greater resolution, which for membrane proteins is still typically within the 4-7Å range. One advantage of a cryo-EM approach is the ability to study membrane proteins in more "native" like environments for example proteoliposomes, amphipols and nanodiscs. Recently, styrene maleic acid co-polymers (SMA) have been used to extract membrane proteins surrounded by native lipids (SMALPs) maintaining a more natural environment. We report here the structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution, with the resulting map being consistent with high resolution crystal structures and other EM derived maps. However, both the C-terminal helix (TM12) and TM7 are poorly defined in the map. These helices are at the exterior of the helical bundle and form the greater interaction with the native lipids and SMA polymer and may represent a more dynamic region of the protein. This work shows the promise of using an SMA approach for single particle cryo-EM studies to provide sub-nm structures., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The identity of organelles in the endomembrane system of any eukaryotic cell critically depends on the correctly localized Rab GTPase, which binds effectors and thus promotes membrane remodeling or fusion. However, it is still unresolved which factors are required and therefore define the localization of the correct fusion machinery. Using SNARE-decorated proteoliposomes that cannot fuse on their own, we now demonstrate that full fusion activity can be achieved by just four soluble factors: a soluble SNARE (Vam7), a guanine nucleotide exchange factor (GEF, Mon1-Ccz1), a Rab-GDP dissociation inhibitor (GDI) complex (prenylated Ypt7-GDI), and a Rab effector complex (HOPS). Our findings reveal that the GEF Mon1-Ccz1 is necessary and sufficient for stabilizing prenylated Ypt7 on membranes. HOPS binding to Ypt7-GTP then drives SNARE-mediated fusion, which is fully GTP-dependent. We conclude that an entire fusion cascade can be controlled by a GEF., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active K v 1.1 and K v 1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using K v 1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of K v 1.1 and K v 1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ∼20 Å above the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ∼50-Å isoprenoid chain. However, how ubiquinone/ubiquinol exchange occurs on catalytically relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly charged region at isoprenoids 4-7. We demonstrate that ubiquinol-10 dissociation is not rate determining and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion., Competing Interests: The authors declare no conflict of interest.

EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux., Competing Interests: The authors declare no conflict of interest.

Atomic force microscopy (AFM) is one of the most commonly used scanning probe microscopy techniques for nanoscale imaging and characterization of lipid-based particles. However, obtaining images of such particles using AFM is still a challenge. The present study extends the capabilities of AFM to the characterization of proteoliposomes, a special class of liposomes composed of lipids and proteins, mimicking matrix vesicles (MVs) involved in the biomineralization process. To this end, proteoliposomes were synthesized, composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), with inserted tissue-nonspecific alkaline phosphatase (TNAP) and/or annexin V (AnxA5), both characteristic proteins of osteoblast-derived MVs. We then aimed to study how TNAP and AnxA5 insertion affects the proteoliposomes' membrane properties and, in turn, interactions with type II collagen, thus mimicking early MV activity during biomineralization. AFM images of these proteoliposomes, acquired in dynamic mode, revealed the presence of surface protrusions with distinct viscoelasticity, thus suggesting that the presence of the proteins induced local changes in membrane fluidity. Surface protrusions were measurable in TNAP-proteoliposomes but barely detectable in AnxA5-proteoliposomes. More complex surface structures were observed for proteoliposomes harboring both TNAP and AnxA5 concomitantly, resulting in a lower affinity for type II collagen fibers compared to proteoliposomes harboring AnxA5 alone. The present study achieved the topographic analysis of lipid vesicles by direct visualization of structural changes, resulting from protein incorporation, without the need for fluorescent probes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The outer membrane of heterotrophic Gram-negative bacteria plays the role of a selective permeability barrier that prevents the influx of toxic compounds while allowing the nonspecific passage of small hydrophilic nutrients through porin channels. Compared with heterotrophic Gram-negative bacteria, the outer membrane properties of cyanobacteria, which are Gram-negative photoautotrophs, are not clearly understood. In this study, using small carbohydrates, amino acids, and inorganic ions as permeation probes, we determined the outer membrane permeability of Synechocystis sp. strain PCC 6803 in intact cells and in proteoliposomes reconstituted with outer membrane proteins. The permeability of this cyanobacterium was >20-fold lower than that of Escherichia coli The predominant outer membrane proteins Slr1841, Slr1908, and Slr0042 were not permeable to organic nutrients and allowed only the passage of inorganic ions. Only the less abundant outer membrane protein Slr1270, a homolog of the E. coli export channel TolC, was permeable to organic solutes. The activity of Slr1270 as a channel was verified in a recombinant Slr1270-producing E. coli outer membrane. The lack of putative porins and the low outer membrane permeability appear to suit the cyanobacterial autotrophic lifestyle; the highly impermeable outer membrane would be advantageous to cellular survival by protecting the cell from toxic compounds, especially when the cellular physiology is not dependent on the uptake of organic nutrients. IMPORTANCE Because the outer membrane of Gram-negative bacteria affects the flux rates for various substances into and out of the cell, its permeability is closely associated with cellular physiology. The outer membrane properties of cyanobacteria, which are photoautotrophic Gram-negative bacteria, are not clearly understood. Here, we examined the outer membrane of Synechocystis sp. strain PCC 6803. We revealed that it is relatively permeable to inorganic ions but is markedly less permeable to organic nutrients, with >20-fold lower permeability than the outer membrane of Escherichia coli Such permeability appears to fit the cyanobacterial lifestyle, in which the diffusion pathway for inorganic solutes may suffice to sustain the autotrophic physiology, illustrating a link between outer membrane permeability and the cellular lifestyle., (Copyright © 2017 American Society for Microbiology.)

Membrane pores can significantly alter not only the permeation dynamics of biological membranes but also their elasticity. Large membrane pores able to transport macromolecular contents represent an interesting model to test theoretical predictions that assign active-like (non-equilibrium) behavior to the permeability contributions to the enhanced membrane fluctuations existing in permeable membranes [Maneville et al. Phys. Rev. Lett. 82, 4356 (1999)]. Such high-amplitude active contributions arise from the forced transport of solvent and solutes through the open pores, which becomes even dominant at large permeability. In this paper, we present a detailed experimental analysis of the active shape fluctuations that appear in highly permeable lipid vesicles with large macromolecular pores inserted in the lipid membrane, which are a consequence of transport permeability events occurred in an osmotic gradient. The experimental results are found in quantitative agreement with theory, showing a remarkable dependence with the density of membrane pores and giving account of mechanical compliances and permeability rates that are compatible with the large size of the membrane pore considered. The presence of individual permeation events has been detected in the fluctuation time-series, from which a stochastic distribution of the permeation events compatible with a shot-noise has been deduced. The non-equilibrium character of the membrane fluctuations in a permeation field, even if the membrane pores are mere passive transporters, is clearly demonstrated. Finally, a bio-nano-technology outlook of the proposed synthetic concept is given on the context of prospective uses as active membrane DNA-pores exploitable in gen-delivery applications based on lipid vesicles., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP 139-151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP 139-151 and cyclic (139-151) (L 144 , R 147 ) PLP 139-151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP 139-151 epitope as well as non peptide mimetics that rises as an ultimate goal.

Virtually all mitochondrial matrix proteins and a considerable number of inner membrane proteins carry a positively charged, N-terminal presequence and are imported by the TIM23 complex (presequence translocase) located in the inner mitochondrial membrane. The voltage-regulated Tim23 channel constitutes the actual protein-import pore wide enough to allow the passage of polypeptides with a secondary structure. In this study, we identify amino acids important for the cation selectivity of Tim23. Structure based mutants show that selectivity is provided by highly conserved, pore-lining amino acids. Mutations of these amino acid residues lead to reduced selectivity properties, reduced protein import capacity and they render the Tim23 channel insensitive to substrates. We thus show that the cation selectivity of the Tim23 channel is a key feature for substrate recognition and efficient protein import.

Bovine NK-lysins, which are functionally and structurally similar to human granulysin and porcine NK-lysin, are predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Although antimicrobial activity of bovine NK-lysin has been assessed for several bacterial pathogens, not all the important bacterial pathogens that are involved in the bovine respiratory disease complex have been studied. Therefore the objective of the present study was to evaluate the antimicrobial activity of bovine NK-lysin-derived peptides on bovine respiratory pathogen Histophilus somni. Four, 30-mer peptides corresponding to the functional region of NK-lysin helices 2 and 3 were synthesized and assessed for antibacterial activity on four bovine pneumonic H. somni isolates. Although there were some differences in the efficiency of bactericidal activity among the NK-lysin peptides at lower concentrations (2-5 μM), all four peptides effectively killed most H. somni isolates at higher concentrations (10-30 μM) as determined by a bacterial killing assay. Confocal microscopic and flow cytometric analysis of Live/Dead Baclight stained H. somni (which were preincubated with NK-lysin peptides) were consistent with the killing assay findings and suggest NK-lysin peptides are bactericidal for H. somni. Among the four peptides, NK2A-derived peptide consistently showed the highest antimicrobial activity against all four H. somni isolates. Electron microscopic examination of H. somni following incubation with NK-lysin revealed extensive cell membrane damage, protrusions of outer membranes, and cytoplasmic content leakage. Taken together, the findings from this study clearly demonstrate the antimicrobial activity of all four bovine NK-lysin-derived peptides against bovine H. somni isolates.

Proteo-lipobeads (PLBs) are investigated as cell-free model systems to encapsulate membrane proteins such as ion channels and transporters. PLBs are based on nickel nitrile tri-acetic acid (Ni-NTA)-functionalized agarose beads, onto which membrane proteins (MP) are bound via histidine(his)-tag. Composite beads thus obtained (subsequently called proteobeads) are dialyzed in the presence of lipid micelles to form PLBs. As an example we employed cytochrome c oxidase from P. denitrificans with a his-tag fused to the C-terminus of subunitI. In this orientation the P side of CcO faces the outside of the PLB and hence protons are released to the outer aqueous phase, when electron transfer is initiated by light excitation of Ru complexes. Proton release kinetics was probed by fluorescence microscopy using the pH-sensitive sensor molecule fluorescein DHPE inserted into the lipid layer. In order to monitor the generation of membrane potentials we performed a FLIPR assay on the CcO embedded in PLBs using the FRET pair CC2-DMPE/DiSBAC2(3). The combined results show that PLBs can be used as a model system designed to quantify the kinetic parameters of membrane proteins. In addition, the FLIPR assay demonstrates the feasibility of PLBs for high throughput screening applications., (Copyright © 2017 Elsevier Inc. All rights reserved.)

At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans -SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis -SNARE complexes.

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Chicken NK-lysin (cNK-lysin), the chicken homologue of human granulysin, is a cationic amphiphilic antimicrobial peptide (AMP) that is produced by cytotoxic T cells and natural killer cells. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core α-helical region of cNK-lysin, have antimicrobial activity against apicomplexan parasites such as Eimeria spp., via membrane disruption. In addition to the antimicrobial activity of AMPs, the immunomodulatory activity of AMPs mediated by their interactions with host cells is increasingly recognized. Thus, in this study, we investigated whether cNK-lysin derived peptides modulate the immune response in the chicken macrophage cell line HD11 and in chicken primary monocytes by evaluating the induction of chemokines, anti-inflammatory properties, and activation of signalling pathways. cNK-2 induced the expression of CCL4, CCL5 and interleukin(IL)-1β in HD11 cells and CCL4 and CCL5 in primary monocytes. We also determined that cNK-2 suppresses the lipopolysaccharide-induced inflammatory response by abrogating IL-1β expression. The immunomodulatory activity of cNK-2 involves the mitogen-activated protein kinases-mediated signalling pathway, including p38, extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinases, as well as the internalization of cNK-2 into the cells. These results indicate that cNK-2 is a potential novel immunomodulating agent rather than an antimicrobial agent.

Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum is particularly intriguing, as it comprises morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation, but how exactly they mediate network formation and whether they are all required are unclear. Here we reconstitute a dynamic tubular membrane network with purified endoplasmic reticulum proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p (refs 6, 7) and the curvature-stabilizing protein Yop1p (refs 8, 9) from Saccharomyces cerevisiae form a tubular network upon addition of GTP. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favours the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon and receptor expression-enhancing proteins (REEPs) from different species. Atlastin, the vertebrate orthologue of Sey1p, forms a GTP-hydrolysis-dependent network on its own, serving as both a fusion and curvature-stabilizing protein. Our results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly.