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The amphipathic a-helical peptide KIA14 [(KIAGKIA)(2)-NH2] was studied in membranes using circular dichroism and solid-state NMR spectroscopy to obtain global as well as local structural information. By analyzing H-2 NMR data from 10 analogues of KIA14 that were selectively labeled with Ala-d(3), those positions that are properly folded into a helix could be determined within the membrane-bound peptide. The N-terminus was found to be unraveled, whereas positions 4-14 formed an ideal helix all the way to the C-terminus. The helicity did not change when Gly residues were replaced by Ala-d3 but was reduced when Ile was replaced, indicating that large hydrophobic residues are required for membrane binding and helix formation. The reduced helicity was strongly correlated with a decrease in peptide-induced leakage from lipid vesicles. The orientation of the short KIA14 peptide was assessed in several lipid systems and compared with that of the longer KIA21 sequence [(KIAGKIA)(3)-NH2]. In 1,2-dioleoylsn-glycero-3-phosphatidylcholine, both peptides are aligned flat on the membrane surface, whereas in 1,2-dimyristoyl-sn-glycero3-phosphatidylcholine (DMPC)/1-myristoy1-2-hydroxy-sn-glycero-3-phosphatidylcholine (lyso-MPC) both are inserted into the membrane in an upright orientation. These two types of lipid systems had been selected for their strongly negative and positive spontaneous curvature, respectively. We propose that in these cases, the peptide orientation is largely determined by the lipid properties. On the other hand, in plain DMPC and 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine, which have only a slight positive curvature, a marked difference in orientation is evident: the short KIA14 lies almost flat on the membrane surface, whereas the longer KIA21 is more tilted. We thus propose that out of the lipid systems tested here, DMPC (with hardly any curvature) is the least biased,lipid system in which peptide orientation and realignment can be studied, allowing to compare and discriminate the intrinsic effects of the properties of the peptides as such.

Rationale: In cardiomyocytes, Na V 1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective: The objective of this study was to test whether Na V 1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1 Δ314-315 or Kir2.1 R44A/R46A with wild-type (WT) Na V 1.5 WT in heterologous cells reduced inward sodium current compared with Na V 1.5 WT alone or coexpressed with Kir2.1 WT . In cell surface biotinylation experiments, expression of Kir2.1 Δ314-315 reduced Na V 1.5 channel surface expression. Glycosylation analysis suggested that Na V 1.5 WT and Kir2.1 WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of Na V 1.5 WT , and vice versa, whereas coexpression with Kir2.1 Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1 Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with Na V 1.5 WT and Kir2.1 WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1 WT , Na V 1.5 WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that Na V 1.5 WT channels interact with AP1 through the Na V 1.5 Y1810 residue, suggesting that, like for Kir2.1 WT , AP1 can mark Na V 1.5 channels for incorporation into clathrin-coated vesicles at the trans -Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions: The Na V 1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na V 1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

A series of novel aromatic primary sulfonamides decorated with diversely substituted 1,2,4-oxadiazole periphery groups has been prepared using a parallel chemistry approach. The compounds displayed a potent inhibition of cytosolic hCA II and membrane-bound hCA IX isoforms. Due to a different cellular localization of the two target enzymes, the compounds can be viewed as selective inhibition tools for either isoform, depending on the cellular permeability profile. The SAR findings revealed in this study has been well rationalized by docking simulation of the key compounds against the crystal structures of the relevant hCA isoforms.

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.

Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability. Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence. CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity. The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time. In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. Disclosures Allen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

A comparative study of the properties of membrane‐bound polyphenol oxidase (mPPO) from three apple cultivars, namely Red Fuji (FJ), Granny Smith (GS) and Golden Delicious (GD), was carried out for the first time. Data indicate that mPPOs from three cultivars exhibit significantly different properties. GS mPPO had the strongest affinity to catechol, but FJ mPPO had the highest maximum velocity. Red Fuji (FJ) mPPO had the significantly higher activity than those of GD and GS mPPOs. Red Fuji (FJ) mPPO had the highest activity at pH 8.00, while GD and GS mPPOs at 4.50 and 7.50–8.00, respectively. Red Fuji (FJ) mPPO was more stable than GD and GS mPPOs over the pH range of 5.0–8.5. The optimal temperature for GS mPPO was within 70–75 °C, which is higher than those for mPPOs from FJ and GD. Thermal inactivation of the three mPPOs followed a first‐order kinetic model with different inactivation kinetic parameters.

The characterization of soluble polyphenol oxidase (sPPO) and membrane-bound (mPPO) from ‘Lijiang snow’ peach was compared, and the inactivation effects by thermal treatment ranging from 45 to 85 °C and high-pressure processing at 550 MPa for 10–50 min were investigated. sPPO and mPPO both showed optimum activity at pH 7.0, while another optimum pH at 5.0 was observed for mPPO. The optimum temperatures for sPPO and mPPO were 30 °C and 50 °C, respectively. The optimum substrate of sPPO was caffeic acid, while mPPO showed the highest activity against 4-methylcatechol. All inhibitors showed poorer inhibitory effects on mPPO compared with sPPO. According to kinetic parameters, sPPO and mPPO were well described by the first-order model and the fractional-conversion model, respectively. The activity of sPPO was activated after HPP at 550 MPa for 20–50 min, while mPPO was more stable under the treatment conditions investigated.

Background Solvatochromic dyes have demonstrated themselves to be useful probes in the molecular-level study of homeopathic potencies. The range of dyes available, and their broad division into two main electronic structural groups, is permitting a detailed investigation of the fundamental nature of potencies. The immobilisation of solvatochromic dyes on transparent membranes takes these investigations to a new level of investigative precision. Aims To attach as many different solvatochromic dyes as possible to triacetylcellulose membranes, and where successful, to observe in detail the spectroscopic changes that occur on exposure to potencies. Immobilisation of dyes onto membranes de-couples the initial dye-potency interaction from later aggregation processes, thereby allowing a host of questions to be asked about what exactly potencies are doing to solvatochromic dyes and in turn indicating what the identities of potencies are likely to be. Methodology A number of methods have been explored for covalently linking dyes to membranes, which at the same time maintain the integrity of the solvatochromic chromophore. Immobilised dyes, in contrast to dyes in solution, behave differently, which means water-insoluble dyes for instance, can be used under a range of solution conditions that were not possible previously. Results and discussion Results from three positively solvatochromic dyes show that potencies cause a bathochromic shift in their spectra, confirming what has been seen previously with encapsulated dyes but with greater clarity. Results can be explained by invoking a polarising effect of potencies and differential stabilisation of the dyes’ ground and excited electronic states. The kinetics of the dye-potency interaction indicate the effect of potency reaches a maximum and then declines. Conclusion Immobilisation of solvatochromic dyes presents a significant step forward in the ability to investigate homeopathic potencies at the physico-chemical level.

Here, we review the recent progress in the characterisation and utilisation of coacervates as protocell models in origin of life studies. We provide evidence that coacervation could have played a unique role during the origin of life based on its ability to form from a range of different prebiotically relevant molecules; partition solutes and maintain the solute’s partition coefficient during enzymatic reactions; support and alter RNA catalysis and readily deform its shape. We discuss how these properties could have been important for the formation of the first membrane bound cells; supporting RNA-peptide evolution and primitive metabolism; and in replicating and proliferating by growth and division processes.

G protein-coupled receptors (GPCRs) are important targets for development of drugs for the treatment of many diseases. However, crystal structures are available for only a small fraction of these membrane bound proteins. Accurate homology models will provide opportunities for effective drug design targeting GPCRs. Recently, several serotonin receptor crystal structures were solved and needed to be evaluated as potential templates. In the first part of this work different measures of similarity in template selection were explored and methods for homology modelling, docking and refinement of aminergic GPCR-ligand complexes were developed and evaluated by comparing models of the D3-R/eticlopride complex with the crystal structure. Homology models of the three α1 adrenergic receptor subtypes and of a serotonin receptor subtype were then constructed using these methods These models were evaluated by docking a range of antagonists into them.

Research spanning almost 50 years has highlighted unique characteristics and irreplaceable list of diverse functions performed by peroxisomes in various model systems. Peroxisomes are single membrane bound highly dynamic organelles ubiquitous to most eukaryotic cells. Proliferation by division of pre-existing organelles and the role of endoplasmic reticulum in the biogenesis of these organelles is now well established. The earliest identified conserved functions of peroxisomes are β-oxidation of fatty acids and reactive oxygen species metabolism. Several studies over the last few decades have reported the importance of this organelle and its numerous cell type, tissue and environment-dependent functions. Their role in several aspects of human health and disease is now under investigation. Studies related to peroxisome biology and functions are now also extended to diverse model systems like Drosophila melanogaster, trypanosomatids, etc. Peroxisomes also intricately collaborate and carry out these functions together with several other organelles in a cell. In this review, we aim to present an overview of our current knowledge of the repertoire of functions of peroxisomes in various model systems.

Tagetes is one of the well established and well researched plant materials that are extensively used owing to their enumerable beneficial properties. Extraction of its bioactive components is well established in terms of organic solvent based extractions. However, organic solvent based extractions limit the practical applicability of the extracts and its practical implementation. In this regard, water based extraction strikes a high note. For the first time, the sonication assisted extraction of Tagetes has been demonstrated and the methodology validated for its superior extraction success compared to the conventional hot water extraction method. The results confirm the use of ultrasonication to lead to accelerated and extremely significant (2–3 order increase) extraction of total phenolics, antioxidants and flavanoids, leading to enhanced antimicrobial activity. The flowers held the highest bioactive molecules. FESEM based imaging studies revealed membrane bound damage as the reason behind the bactericidal activity. The future implementation of ultrasonication technology for accelerated ultrafast extraction of herbal and tea infusion formulations is highly recommended.

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter Thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca2+. D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4.0. In addition, the enzyme oxidized D-fructose to 5-keto-D-fructose, D-psicose to 5-keto-D-psicose, including the other polyols such as, glycerol, D-ribitol, D-arabitol, and D-sorbitol. Thus, D-pentonate 4-dehydrogenase was found to be identical with glycerol dehydrogenase (GLDH), a major polyol dehydrogenase in Gluconobacter species. The reaction versatility of quinoprotein GLDH was notified in this study., Facultad de Ciencias Exactas, Centro de Investigación y Desarrollo en Fermentaciones Industriales

// Soochi Kim 1, 2 , HyeRan Gwak 2, 3 , Hee Seung Kim 2, 4 , Boyun Kim 2, 5 , Danny N. Dhanasekaran 6 , Yong Sang Song 1, 2, 3, 4 1 Interdisciplinary Program in Cancer Biology, College of Medicine, Seoul National University, Seoul, Republic of Korea 2 Cancer Research Institute, Seoul National University, Seoul, Republic of Korea 3 Biomodulation, Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea 4 Department of Obstetrics and Gynecology, Seoul National University, Seoul, Republic of Korea 5 Nano System Institute, Seoul National University, Seoul, Korea 6 Stephenson Cancer Center, University of Oklahoma Health Sciences Center, USA Correspondence to: Yong Sang Song, email: yssong@snu.ac.kr Keywords: ascites, migration, invasion, IL-6R, ovarian cancer Received: April 18, 2016 Accepted: October 11, 2016 Published: November 4, 2016 ABSTRACT Transcoelomic route is the most common and the earliest route of metastasis, causing the ascites formation in advanced epithelial ovarian cancer (EOC). We demonstrated that interleukin 6 (IL-6) is enriched in the malignant ascites from patients with ovarian cancer, which enhanced invasive properties of EOC cells. Interestingly, the expression of IL-6R on cell membrane of EOC cells correlated with ascites-induced invasion. Selective knockdown of IL-6R or inhibition with IL-6 neutralizing antibody, suppressed the stimulatory effects of ascites on EOC invasion. Moreover, the ascites treatment induced the phosphorylation of JAK2-STAT3 and use of selective inhibitors of JAK2 and STAT3, blocked the expression of epithelial-mesenchymal transition related proteins in parallel with the suppression of EOC invasion. Thus, IL-6/IL-6R mediated JAK2-STAT3 signaling pathway could be a promising therapeutic target for anticancer therapy in ovarian cancer patients with ascites.

This study describes the isolation and purification of a protein complex with -ATPase activity and sensitivity to GABAAergic ligands from rat brain plasma membranes. The ATPase complex was enriched...

Marine mussels (Mytilus edulis) fabricate byssal threads, high-performance biopolymeric fibers, which exhibit exceptional toughness and self-healing capacity. These properties are associated with collagenous proteins in the fibrous thread core known as preCols that self-organize into a hierarchical semicrystalline structure. Threads assemble individually in a bottom-up process lasting just minutes via secretion of membrane bound vesicles filled with preCols. However, very little is understood about the details and dynamics of this assembly process. Here, we explore the hypothesis that preCols are stored within the vesicles in a liquid crystalline phase, which contributes to fiber assembly by preordering molecules. To achieve this, a protocol was developed for extracting and isolating intact preCol secretory vesicles in high yield and purity. Vesicles were characterized and were manipulated in vitro, clearly indicating the dynamic liquid crystalline nature of the proteins within. Moreover, mechanical shear...

Presenilins 1 and 2 (PS1 or PS2) are main genetic risk factors of familial Alzheimer’s disease (AD) that produce the β-amyloid (Aβ) peptides and also have important stand-alone functions related to, e.g. calcium signaling. Most work so far has focused on PS1, but humans carry both PS1 and PS2, and mutations in both cause AD. Here, we develop a computational model of PS2 in the membrane to address the question how pathogenic PS2 mutations affect the membrane-embedded protein. The models are based on cryo-electron microscopy structures of PS1 translated to PS2, augmented with missing residues and a complete all-atom membrane–water system, and equilibrated using three independent 500-ns simulations of molecular dynamics with a structure-balanced force field. We show that the nine-transmembrane channel structure is substantially controlled by major dynamics in the hydrophilic loop bridging TM6 and TM7, which functions as a ‘plug’ in the PS2 membrane channel. TM2, TM6, TM7 and TM9 flexibility controls the size of this channel. We find that most pathogenic PS2 mutations significantly reduce stability relative to random mutations, using a statistical ANOVA test with all possible mutations in the affected sites as a control. The associated loss of compactness may also impair calcium affinity. Remarkably, similar properties of the open state are known to impair the binding of substrates to γ-secretase, and we thus argue that the two mechanisms could be functionally related. Communicated by Ramaswamy H. Sarma

In the present work, we analyzed how external factors can modulate the efficiency of epigallocatechin‑3‑O‑gallate (EGCG) inhibition of a membrane-bound isoform of the acetylcholinesterase. Increasing the ionic strength but not the osmolarity of the bulk medium proved to be an important factor. In addition, we verified a clear correlation between the inhibitory activity with the order degree of the membranes by using cholesterol-partially depleted red blood cell ghosts. These two factors i.e. high salt concentration in the bulk medium and less viscous membranes, allow a deeper insertion of the EGCG into the lipid bilayer, thus leading to a greater inhibition of AChE. As a corollary, we propose that any treatment or process that leads to a slight decrease in cholesterol content in the membranes can efficiently enhance the inhibitory activity of EGCG, which can have important consequences in all the pathologies where the inhibition of AChE is recommended. Fil: Salazar, Paula Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Dupuy, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: de Athayde Moncovo Collado, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.

Membrane proteins control and shape membrane trafficking processes. The role of protein structure in shaping cellular membranes is well established. However, a significant fraction of membrane proteins is disordered or contains long disordered regions. It becomes more and more clear that these disordered regions contribute to the function of membrane proteins. While the fold of a structured protein is essential for its function, being disordered seems to be a crucial feature of membrane bound intrinsically disordered proteins and protein regions. Here we outline the motifs that encode function in disordered proteins and discuss how these functional motifs enable disordered proteins to modulate membrane properties. These changes in membrane properties facilitate and regulate membrane trafficking processes which are highly abundant in eukaryotes.

Emerging evidence suggests that CD83, a dendritic cells (DCs) maturation marker in humans and mice, may prossess immunomodulatory capacities. Although porcine CD83 shares ∼75% sequence homology with its human counterpart, whether it functions as an immunoregulatory molecule remains unknown. To investigate porcine CD83 function, we deleted it in porcine DCs by RNA intereference. Results show that membrane-bound CD83 (mCD83) promotes DC-mediated T cell proliferation and cytokine production, thus confirming its immunoregulatory capacity. Intriguingly, porcine soluble CD83 (sCD83) treatment instead led to inhibition of DC-mediated T cell activation. Moreover, porcine sCD83 also inhibited differentiation of prepheral blood mononuclear cells (PBMCs) into DCs. These results collectively indicate that in addition to being a DC maturation maker, both membrane bound and souble porcine CD83 serve as immunoregulatory molecules with opposite effects on DC-mediated T cell activation and DC differentiation.

ESCRT-III is an evolutionarily conserved membrane remodeling machinery that, with the Vps4 ATPase, forms filaments able to cut biological membranes from the cytosolic side. This activity of ESCRT-III is essential for the final stage of cell division in archaea and in many eukaryotes, the formation of vesicles, the creation of exosomes, the release of viruses such as HIV-1 and Ebola, and for the repair and sealing of cellular membranes. While there has been recent rapid progress in describing the biochemical and cell biology details of different ESCRT-III functions, we lack an understanding of the physical mechanism involved in ESCRT-III-mediated membrane remodelling. Here, through the development of coarse-grained molecular dynamic simulations, we present a minimal model that captures the ability of ESCRT-III to induce experimentally reported cases of ESCRT-III driven membrane sculpting, including the formation of cones and tubules, and membrane scission. This model suggests a universal physical mechanism of action, that differs from that of other cytoskeletal elements, whereby a change in the twist of membrane bound ESCRT-III filaments drives transitions between a flat spiral and a 3D helix to induce membrane deformation and scission. We expect the mechanistic principles revealed here to be useful in manipulating ESCRT-III-driven processes in cells and in guiding the engineering of synthetic membrane-sculpting systems.

Numerous biosynthetic pathways have been shown to assemble at the surface of cellular membranes into efficient dynamic supramolecular assemblies termed metabolons. In response to environmental stimuli, metabolons assemble on-demand making them highly dynamic and fragile. This transient nature has previously hampered isolation and molecular characterization of dynamic metabolons. In contrast to conventional detergents, which tend to disrupt weak protein-protein interactions and remove lipids, the competence of a styrene maleic acid copolymer to carve out discrete lipid nanodisc from membranes offers immense potential for isolation of intact protein assemblies. Here, we present a method to extract the entire membrane-bound dhurrin pathway directly from microsomal fractions of the cereal Sorghum bicolor. This detergent-free nanodisc approach may be generally transposed for isolation of entire plant biosynthetic metabolons. This method provides a simple practical toolkit for the study of membrane protein complexes.

Solid-state 19F-NMR is a valuable tool in structural studies of membrane-associated peptides. Progress in this area became possible after successful design and synthesis of the fluorine-contained amino acids suitable to substitute natural amino acid residues in the peptides without compromising their structure and properties. These amino acids (labels) are incorporated into the molecules of the studied peptides and provide structural constraints for the labeled peptides embedded into oriented lipid bilayers under quasinatural conditions. The chapter overviews the literature on the design, synthesis, and validation of the labels. Recent examples of the label applications to study membrane-active peptides are given.

The bioactive and anti-inflammatory role of human milk components has been recognized; active milk components include soluble forms of Toll-like receptors (TLRs). Preterm babies are more susceptible to infections and may succumb to necrotizing enterocolitis (NEC), a gastrointestinal disease which is exacerbated by an excessive inflammatory response after TLR activation. Here, we investigated the presence of Toll-like receptors TLR1/2/4/6 in colostrum and mature milk of women who delivered before (preterm) or after (term) 37 weeks of gestational age, integrating classical immune-related techniques with proteomic LC-MS/MS analysis. We have detected immunoreactivity for TLRs mostly in preterm samples, even for TLR1 and TLR6, until now not described in human milk. We demonstrated the presence of only TLR2 in the milk fat globule membrane, while the immunoreactivity of TLR1/4/6 was ascribed to crossreaction with some interesting milk proteins sharing leucine-rich repeat domains. These results will provide new insights into the definition of the role of TLRs in intestinal immune regulation of the newborns.

Sequestration of a transcription factor in a cellular membrane and releasing it on demand is an additional layer of gene regulation that is considered a rapid mode to reprogram a gene expression cascade when a plasma membrane stress signal is perceived. Better understanding of the dynamic exchange of membrane-bound transcription factors (MTFs) during biotic stress requires the development of a simple, efficient, and quick assay system. Here we report an Agrobacterium-based transient transformation method to assay the localization of fluorescent protein-tagged MTFs in tomato leaf epidermal peels that are subsequently infected with a pathogenic fungus. Essentially, our method mimics natural infection and facilitates the realistic monitoring of MTF movement during activation of a signaling event.

Unculturable bacteria are those bacteria which proliferate in their native habitat but unable to grow or thrive in the normal laboratory media and conditions. The molecular techniques have revealed the significance of these uncultured bacteria in terms of their functional diversity and potential to produce secondary metabolites. To achieve these benefits, scientists have attempted to isolate and cultivate unculturable bacteria in the laboratory using transwell plates, optical tweezers, laser microdissection, microbioreactors, and diffusions bioreactors. However, these techniques are still inadequate to resolve the difficulties of cultivating unculturable bacteria. Therefore, it is essential to develop new cultivation method that enables growth of diverse range of bacteria in the laboratory conditions. Diffusion bioreactor is a membrane bound chamber which allows microbes to proliferate in their native environment by providing the excess to naturally occurring nutrients and signaling compounds. This paper presents efficient and reliable protocol to construct a diffusion bioreactor and its utilization to isolate and cultivate unculturable soil bacteria in laboratory.

Here we present the simple and effective method for obtaining clones of NK cells, sorted according to a given set of surface markers. The lifespan of clones grown by this method can reach 14 weeks and cloning efficiency is up to 50%, depending on the original NK cell subset. Obtained clones are functionally active; they are able to lyse target cells and produce IFNγ. The principle of method includes single cell sorting and further clonal expansion in the presence of IL-2 and gene-modified K562 feeder cells. Our method allows to expand the progeny of a single NK cell up to 10-20×106 NK cells suitable to study phenotype, proliferative and functional activity of a certain NK cell clone. The work was supported by Russian Science Foundation, grant #16-15-00309.

A pattern of NK cell heterogeneity in each individual determines proliferative and functional responses of NK cells to activating stimuli. Obtaining the progeny of a single cell by cloning original population is one of the ways to study the NK cell heterogeneity. In this work, we used single cell sorting into a plate and stimulation by IL-2 and gene-modified K562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21) that led to generation of phenotypically confirmed and functionally active NK cell clones. We applied two models of clone cultivation, which differently affected their phenotype, lifespan and functional activity. The first model, which included weekly restimulation of clones with K562-mbIL21 and IL-2, resulted in the generation of relatively short-lived (5-7 weeks) clones of highly activated NK cells. HLA-DR expression in the expanded NK cells correlated strongly with IFN-γ production. The second model, in which NK cells were restimulated mainly with IL-2 alone, produced long-lived clones (8-14 weeks) that expanded up to 107 cells with lower ability to produce IFN-γ. Our method is applicable for studying variability in phenotype, proliferative and functional activity of the certain NK cell progeny in response to the stimulation, which may help in selecting NK cells best suited for clinical use.