Transmembrane Peptide Research Articles

Penetrating-peptide-mediated non-invasive Axitinib delivery for anti-neovascularisation.

The unique physiological makeup of the eye limits the use of small-molecule drugs for treating the posterior segment of the eye. Nevertheless, transmembrane-peptide-mediated non-invasive drug delivery can serve as an ideal treatment strategy, as it is capable of delivering small-molecule drugs across the membrane in the form of eye drops, thereby achieving the effective treatment of neovascularisation in the posterior cavity. In this study, we screened and compared the posterior segment distribution of two poly(ethylene glycol)-distearoylphosphatidylethanolamine carriers modified using targeting-peptides. Thereafter, a transmembrane peptide (i.e., PENE) with a greater ability of transmembrane delivery was selected for delivering the anti-vascular drug (i.e., Axitinib) to the posterior segment of the eye. Using two different mouse models with fundus neovascular diseases, the complete non-invasive delivery of Axitinib to the posterior segment of the eye was confirmed using the targeted system; the designed eye drops (i.e., PENE-nanoparticles) could achieve drug distribution to the retina and veins of the eye as well as good drug permeability for renewal. Moreover, using the eye-drop treatment, neovascularisation was substantially reduced, demonstrating the high efficacy of this drug delivery system. This study, which combines nanodrug-loading technology and the transmembrane delivery of penetrating-peptides to achieve the goal of the non-invasive delivery of small-molecule drugs through the dense blood vessels of the sclera, shows wide applicability and considerably expands the use of ocular drugs. Thus, this study is expected to help develop a more acceptable drug administration strategy for the drug treatment of the posterior segment of the eye.

Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Analysis of GSH1-GSH3 Genes by Comprehensive Use of Bioinformatics Method in Tomato

GSH is a ubiquitous gene family in animals and plants, which plays a key regulatory role in plant growth, development and stress response. In this study, three GSH genes are identified in the Phytozome database, and bioinformatics analysis of physicochemical properties, structural domains, promoter cis-acting elements and phosphorylation sites is performed. The results show that the numbers of introns and exons of tomato GSH1-GSH3 are quite different. Promoters cis-acting elements are analyzed in GSH gene promoters, which account for a large proportion of light-responsive, hormone-responsive and protein synthesis elements. Transcription factors based on different tissues group data analysis reveal that the expression of GSH genes is different in different tissue development processes. The highest expression sites of GSH1-GSH3 are in 1-cm fruit, GSH2 currant tomato fruit and roots five days after veraison. According to physicochemical properties, transmembrane structure and signal peptide, the study finds that GSH proteins are all hydrophilic proteins and do not have a transmembrane structure and signal peptides. Based on the data analysis of protein phosphorylation sites and glycosylation sites, it is found that the number of the two sites differ significantly between GSH proteins. By means of prediction of secondary and tertiary structures in proteins, it finds that the three GSH proteins have four secondary structures: α-helix, extended chain, β-turn and random coil, but the proportions are different, and the tertiary structures of the three are quite different. Based on the analysis of protein phylogenetic tree, it is believed that tomato and potato are closely related. According to the protein interaction network, it points out that there is a strong link between GSH1 and GSH3. Through the analysis of tomato GSH family system, this study is expected to provide a theoretical basis for further research on the biological function of GSH gene.

Lateral Interaction of Cylindrical Transmembrane Peptides in a One-Dimensional Approximation

Lateral Interaction of Cylindrical Transmembrane Peptides in a One-Dimensional Approximation

Preparation and pharmacokinetics of glycyrrhetinic acid and cell transmembrane peptides modified with liposomes for liver targeted-delivery

The article presents a hepatocellular carcinoma cell surface-specific ligand glycyrrhetinic acid (GA) and cell-penetrating peptide (TAT) with good cell membrane penetration to modify the anti-tumor drug pingyangmycin (PYM) liver delivery system, which achieve targeted delivery of drugs and improve anti-tumor efficiency. In this study, we synthesized the pingyangmycin liposome modified by glycyrrhetinic acid and cell penetrating peptide(GA-TAT-PYM-L) and evaluated the anti-tumor effect of GA-TAT-PYM-L in vitro. Using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromidecell proliferation method, GA-TAT-PYM-L had a stronger inhibitory effect on HepG2 cells than the free drug PYM at the same concentration. Acridine orange-ethidium bromide staining assays showed that GA-TAT-PYM-L had stronger apoptosis promotion effects on HepG2 cells in comparison to PYM. Pharmacokinetic studies indicated that, compared with PYM, GA-TAT-PYM-L enhanced mean residence time (MRT0–∞) and area under curve (AUC0–∞) by about 2.79-fold and 2.45-fold. The T 1/2 was prolonged to 140.23 ± 14.13 min. Tissue distribution results showed that the PYM concentrations in livers from the GA-TAT-PYM-L group were always higher than other tissues at each monitoring period after 5 min, indicating that GA-TAT-PYM-L can achieve liver targeting.

Conformational dynamics of the membrane enzyme LspA upon antibiotic and substrate binding

Lipoprotein signal peptidase (LspA) is an aspartyl protease that cleaves the transmembrane helix signal peptide of lipoproteins as part of the lipoprotein-processing pathway. Members of this pathway are excellent targets for the development of antibiotic therapeutics because they are essential in Gram-negative bacteria, are important for virulence in Gram-positive bacteria, and may not develop antibiotic resistance. Here, we report the conformational dynamics of LspA in the apo state and bound to the antibiotic globomycin determined using molecular dynamics simulations and electron paramagnetic resonance. The periplasmic helix fluctuates on the nanosecond timescale and samples unique conformations in the different states. In the apo state, the dominant conformation is the most closed and occludes the charged active site from the lipid bilayer. With antibiotic bound there are multiple binding modes with the dominant conformation of the periplasmic helix in a more open conformation. The different conformations observed in both bound and apo states indicate a flexible and adaptable active site, which explains how LspA accommodates and processes such a variety of substrates.

Development of Tumor‐selective Transmembrane Peptides to Promote the Activity of the Receptor Protein Tyrosine Phosphatase J by Disrupting its Homodimerization

Receptor Protein Tyrosine Phosphatases (RPTPs) are one of the most important regulators of receptor tyrosine kinases (RTKs) and therefore play a crucial role in mammalian signal transduction. But our still‐incomplete understanding of the structural aspects of their regulation and the lack of selective agonists have hampered efforts to understand their roles in downstream signaling regulation, and to pursue them as potential therapeutic targets. However, the reported ability of RPTP homodimerization to antagonize their catalytic activity presents potential opportunities to develop unique strategies to modulate the activity of RPTPs.We recently reported (Block et al., JBC, 2019; doi:10.1074/jbc.RA119.010229) that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ; also known as DEP1) is regulated by specific transmembrane (TM) residues, and that these interactions are essential in regulating its enzymatic activity and substrate access in cells.Building on these new insights and in response to the lack of selective agonists, we present here the design and testing of a novel class of tumor‐selective peptide capable of binding to the TM domain of PTPRJ and disrupting its homodimerization. By doing so, it promotes PTPRJ TM‐mediated access to EGFR, reduces the phosphorylation of EGFR (a known substrate) and other downstream signaling effectors, and antagonizes EGFR‐driven cancer cell phenotypes.This class of peptide represents a novel allosteric and a possibly orthogonal way to target the activity of PTPRJ and RTKs phosphorylation. It could therefore be used not only as probes to tease out PTPRJ regulating mechanisms but also for therapeutic purposes via the attenuation of signaling by dysregulated RTKs.

PD53-10 IDENTIFYING BLADDER UROTHELIAL CARCINOMAS USING FLUORESCENT SIGNAL INTENSITY OF PH LOW INSERTING PEPTIDES

You have accessJournal of UrologyCME1 May 2022PD53-10 IDENTIFYING BLADDER UROTHELIAL CARCINOMAS USING FLUORESCENT SIGNAL INTENSITY OF PH LOW INSERTING PEPTIDES Borivoj Golijanin, Michael DuPont, Ali Amin, Anna Moshnikova, Ohad Kott, Yana Reshetnyak, Oleg Andreev, and Dragan Golijanin Borivoj GolijaninBorivoj Golijanin More articles by this author , Michael DuPontMichael DuPont More articles by this author , Ali AminAli Amin More articles by this author , Anna MoshnikovaAnna Moshnikova More articles by this author , Ohad KottOhad Kott More articles by this author , Yana ReshetnyakYana Reshetnyak More articles by this author , Oleg AndreevOleg Andreev More articles by this author , and Dragan GolijaninDragan Golijanin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002630.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urothelial carcinomas (UC) are heterogenous malignancy with an acidic microenvironment. pH low insertion peptides (pHLIPs) are a class of pH specific transmembrane peptides that target the acidic microenvironment of cancer cells. pHLIP variant-3 (Var3-pHLIP) was conjugated to a near infrared fluorescent (NIRF) dye to evaluate the specificity and sensitivity as a UC targeting molecular probe. METHODS: After incubation for 15 minutes with Var3-pHLIP conjugated to indocyaninie green (ICG) or IRDye® 800CW, 38 ex-vivo bladder specimens from patients undergoing robotic assisted laparoscopic radical cystectomy for bladder cancer were placed under NIRF capable laser to excite the fluorophore. Peak signal intensity and the corrected mean intensity of malignant and nonmalignant cases were analyzed. The number of lesions visible under NIRF was compared with the number identified under white light. Paired-samples t-test was used to analyzed these data. Expression of NIRF signal and identification of lesions under white light by urologic oncology pathologist were compared to histopathology for specificity and sensitivity calculations. RESULTS: Of 58 lesions processed for histopathology, 47 (81%) were seen under white light and 57 (98.3%) were seen with Var3-pHLIP, representing an improved diagnosis by 17.3% (p=0.003). In NIRF, Var3-pHLIP demonstrated an average peak signal intensity of 116.4 relative fluorescent units (RFU) in malignant cases, and nonmalignant cases demonstrated an average of 44.3 RFU (p <0.001). Corrected average signal intensity of malignant cases demonstrated an average of 52.9 RFU/μm2 and nonmalignant cases demonstrated an average of 25 RFU/μm2 (p <0.001). Var3-pHLIP demonstrated 98% sensitivity and 100% specificity in identification of UC. CONCLUSIONS: NIRF of Var3-pHLIP enhanced visualization and improved diagnosis of UC. Categorizing into the borader categories of malignant and nonmalignant, the unique Var3-pHLIP NIRF signal can be used to differentiate the two groups on both peak signal intensity and mean intensity per unit area of lesion. NIRF of Var3-pHLIP identified UC irrespective of subtype, previous treatment, and stage. All CIS cases missed by white light cystoscopy were diagnosed using Var3-pHLIP NIRF imaging. Additional work into digital, automated analysis of Var3-pHLIP NIRF signal and development of a fluorescent signal analysis capable cystoscope can lead to diagnosis of bladder cancer at the time of cystoscopy. Source of Funding: This study was funded by internal departmental funds allocated to DG, OA, and YK © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e913 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Borivoj Golijanin More articles by this author Michael DuPont More articles by this author Ali Amin More articles by this author Anna Moshnikova More articles by this author Ohad Kott More articles by this author Yana Reshetnyak More articles by this author Oleg Andreev More articles by this author Dragan Golijanin More articles by this author Expand All Advertisement PDF DownloadLoading ...

In Situ Assembly of Transmembrane Proteins from Expressed and Synthetic Components in Giant Unilamellar Vesicles.

Reconstituting functional transmembrane (TM) proteins into model membranes is challenging due to the difficulty of expressing hydrophobic TM domains, which often require stabilizing detergents that can perturb protein structure and function. Recent model systems solve this problem by linking the soluble domains of membrane proteins to lipids, using noncovalent conjugation. Herein, we test an alternative solution involving the in vitro assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic giant unilamellar vesicle (GUV) membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. We show that the extracellular domain of programmed cell death protein 1, a mammalian immune checkpoint receptor, retains its ligand-binding function at a membrane interface after ligation to a synthetic TM peptide in GUVs, facilitating the study of receptor-ligand interactions.

Structural basis for the assembly and quinone transport mechanisms of the dimeric photosynthetic RC\u2013LH1 supercomplex

The reaction center (RC) and light-harvesting complex 1 (LH1) form a RC–LH1 core supercomplex that is vital for the primary reactions of photosynthesis in purple phototrophic bacteria. Some species possess the dimeric RC–LH1 complex with a transmembrane polypeptide PufX, representing the largest photosynthetic complex in anoxygenic phototrophs. However, the details of the architecture and assembly mechanism of the RC–LH1 dimer are unclear. Here we report seven cryo-electron microscopy (cryo-EM) structures of RC–LH1 supercomplexes from Rhodobacter sphaeroides. Our structures reveal that two PufX polypeptides are positioned in the center of the S-shaped RC–LH1 dimer, interlocking association between the components and mediating RC–LH1 dimerization. Moreover, we identify another transmembrane peptide, designated PufY, which is located between the RC and LH1 subunits near the LH1 opening. PufY binds a quinone molecule and prevents LH1 subunits from completely encircling the RC, creating a channel for quinone/quinol exchange. Genetic mutagenesis, cryo-EM structures, and computational simulations provide a mechanistic understanding of the assembly and electron transport pathways of the RC–LH1 dimer and elucidate the roles of individual components in ensuring the structural and functional integrity of the photosynthetic supercomplex.

Cloning and functional analysis of &amp;lt;italic&amp;gt;ScGA20ox1&amp;lt;/italic&amp;gt; gibberellin oxidase gene in sugarcane

<p id="C3">GA20-oxidase (GA20ox) is a key rate-limiting enzyme in the synthesis of gibberellic acid (GA), but the biological function and expression pattern of gene <italic>GA20ox1 </italic>in sugarcane remain unknown. In this study, we cloned GA20-oxidase gene (<italic>ScGA20ox1</italic>) from the sugarcane variety ROC22 sequence by RT-PCR and RACE, which had a total length of 1574 bp and a complete open reading frame (ORF) of 1125 bp that encoded 375 amino acids. The ScGA20ox1 protein is a hydrophilic protein with a molecular weight of 42.3 kD, an iscelectric point (pI) of 5.95, and no transmembrane structure or signal peptide. Real-time fluorescence quantitative analysis revealed that <italic>ScGA20ox1</italic> had the highest expression levels in stems, medium in the leaves, and the lowest in roots of sugarcane seedlings. Drought stress, low temperature stress, and GA₃ treatment change the expression patterns of <italic>ScGA20ox1</italic> in different tissues. <italic>ScGA20ox1</italic> was transformed into Arabidopsis via Agrobacterium-mediated transformation. Transgenic <italic>Arabidopsis thaliana</italic> plants with overexpressed <italic>ScGA20ox1</italic> were obtained from the transformed <italic>Arabidopsis thaliana via</italic> Agrobacterium-mediated transformation, which showed a phenotypic variation of plant height and internode length increase. In conclusion, <italic>ScGA20ox1</italic> was involved in sugarcane’s growth as a crucial regulator in response to abiotic stress and the phenotypic variation of overexpressed transgenic <italic>Arabidopsis thaliana</italic> changed, which would lay a theoretical basis for future in-depth studies on <italic>ScGA20ox1</italic>’s biological function and analysis of the molecular mechanism of sugarcane plant shape regulation.

Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy

Mitochondrial replacement therapy (MRT) has been used to prevent maternal transmission of disease-causing mutations in mitochondrial DNA (mtDNA). However, because MRT requires nuclear transfer, it carries the risk of mtDNA carryover and hence of the reversion of mtDNA to pathogenic levels owing to selective replication and genetic drift. Here we show in HeLa cells, mouse embryos and human embryos that mtDNA heteroplasmy can be reduced by pre-labelling the mitochondrial outer membrane of a donor zygote via microinjection with an mRNA coding for a transmembrane peptide fused to an autophagy receptor, to induce the degradation of the labelled mitochondria via forced mitophagy. Forced mitophagy reduced mtDNA carryover in newly reconstructed embryos after MRT, and had negligible effects on the growth curve, reproduction, exercise capacity and other behavioural characteristics of the offspring mice. The induction of forced mitophagy to degrade undesired donor mtDNA may increase the clinical feasibility of MRT and could be extended to other nuclear transfer techniques.

Comparative molecular dynamics simulation studies of realistic eukaryotic, prokaryotic, and archaeal membranes

The OPM (Orientation of Proteins in Membranes) database currently holds, classifies, and annotates around 14,000 three-dimensional structures of transmembrane and peripheral membrane proteins and peptides positioned with respect to membrane boundaries. Developing uniform templates of biological membranes with realistic native-like lipid composition would provide a convenient and accurate platform for researchers who simulate membrane-associated proteins and peptides in natural environments. In this study, for the first time, we performed the standardized molecular dynamic simulations and comparative analysis of structures and properties of 18 native-like membrane models.

A pH-responsive transmembrane peptide acts as an inhibitor of T cell receptor activation

The T cell receptor cluster of differentiation 3 (TCR-CD3) is a membrane protein receptor complex present at the surface of T cells. TCR-CD3 complex is composed of eight transmembrane subunits (αβ-εγ-εδ-ζζ). Dysfunction of TCR-CD3 activation causes autoimmune and inflammatory diseases. No effective treatment targeting TCR-CD3 has been developed so far. Of note, the microenvironment of autoimmune and inflammatory diseases is acidic. We took advantage of this property for the rational design of a targeted peptide that comprised the sequence of the transmembrane domain of the ζ subunit.

De novo design of peptide nanopores with β-barrel structure synthesized by cell-free expression

Biological nanopores have been applied to single-molecule detection and recently commercialized as detectors for a nanopore DNA sequencer. To extend the nanopores to more versatile sensing such as amino acid sequencing, the construction of the tailored size and structure of the nanopore is essential. Among several strategies such as engineering proteins or developing solid-state nanopores, the de novo design of amino acid sequence will be promising to create target-specific nanopores.We have previously designed the de novo β-hairpin transmembrane peptide (SVG28, 28 residues) which assembles to form a β-barrel nanopore.

Hydrophobic Mismatch Controls the Mode of Membrane-Mediated Interactions of Transmembrane Peptides.

Various cellular processes require the concerted cooperative action of proteins. The possibility for such synchronization implies the occurrence of specific long-range interactions between the involved protein participants. Bilayer lipid membranes can mediate protein–protein interactions via relatively long-range elastic deformations induced by the incorporated proteins. We considered the interactions between transmembrane peptides mediated by elastic deformations using the framework of the theory of elasticity of lipid membranes. An effective peptide shape was assumed to be cylindrical, hourglass-like, or barrel-like. The interaction potentials were obtained for membranes of different thicknesses and elastic rigidities. Cylindrically shaped peptides manifest almost neutral average interactions—they attract each other at short distances and repel at large ones, independently of membrane thickness or rigidity. The hourglass-like peptides repel each other in thin bilayers and strongly attract each other in thicker bilayers. On the contrary, the barrel-like peptides repel each other in thick bilayers and attract each other in thinner membranes. These results potentially provide possible mechanisms of control for the mode of protein–protein interactions in membrane domains with different bilayer thicknesses.

Disrupting GPCR Complexes with Smart Drug-like Peptides.

G protein-coupled receptors (GPCRs) are a superfamily of proteins classically described as monomeric transmembrane (TM) receptors. However, increasing evidence indicates that many GPCRs form higher-order assemblies made up of monomers pertaining to identical (homo) or to various (hetero) receptors. The formation and structure of these oligomers, their physiological role and possible therapeutic applications raise a variety of issues that are currently being actively explored. In this context, synthetic peptides derived from TM domains stand out as powerful tools that can be predictably targeted to disrupt GPCR oligomers, especially at the interface level, eventually impairing their action. However, despite such potential, TM-derived, GPCR-disrupting peptides often suffer from inadequate pharmacokinetic properties, such as low bioavailability, a short half-life or rapid clearance, which put into question their therapeutic relevance and promise. In this review, we provide a comprehensive overview of GPCR complexes, with an emphasis on current studies using GPCR-disrupting peptides mimicking TM domains involved in multimerization, and we also highlight recent strategies used to achieve drug-like versions of such TM peptide candidates for therapeutic application.

A proof-of-concept study of the secondary structure of influenza A, B M2 and MERS- and SARS-CoV E transmembrane peptides using folding molecular dynamics simulations in a membrane mimetic solvent.

Here, we have carried out a proof-of-concept molecular dynamics (MD) simulation with adaptive tempering in a membrane mimetic environment to study the folding of single-pass membrane peptides. We tested the influenza A M2 viroporin, influenza B M2 viroporin, and protein E from coronaviruses MERS-Cov-2 and SARS-CoV-2 peptides with known experimental secondary structures in membrane bilayers. The two influenza-derived peptides are significantly different in the peptide sequence and secondary structure and more polar than the two coronavirus-derived peptides. Through a total of more than 50 μs of simulation time that could be accomplished in trifluoroethanol (TFE), as a membrane model, we characterized comparatively the folding behavior, helical stability, and helical propensity of these transmembrane peptides that match perfectly their experimental secondary structures, and we identified common motifs that reflect their quaternary organization and known (or not) biochemical function. We showed that BM2 is organized into two structurally distinct parts: a significantly more stable N-terminal half, and a fast-converting C-terminal half that continuously folds and unfolds between α-helical structures and non-canonical structures, which are mostly turns. In AM2, both the N-terminal half and C-terminal half are very flexible. In contrast, the two coronavirus-derived transmembrane peptides are much more stable and fast helix-formers when compared with the influenza ones. In particular, the SARS-derived peptide E appears to be the fastest and most stable helix-former of all the four viral peptides studied, with a helical structure that persists almost without disruption for the whole of its 10 μs simulation. By comparing the results with experimental observations, we benchmarked TFE in studying the conformation of membrane and hydrophobic peptides. This work provided accurate results suggesting a methodology to run long MD simulations and predict structural properties of biologically important membrane peptides.

High-yield production and purification of the fusion pH-responsive peptide GST-pHLIP in &lt;i&gt;Escherichia coli&lt;/i&gt; BL21

&lt;abstract&gt; &lt;p&gt;The pH Low Insertion Peptide (pHLIP) has versatile applications in several diseases due to its differential behavior at slightly different pH values. pHLIP is an unstructured and peripheral membrane-associated peptide at neutral pH and an α-helical transmembrane peptide at acidic values. Similar to what happened to insulin and growth hormone, pHLIP´s expanding applications require high-yield production to further scale-up its usefulness. To date, synthesis of the pHLIP has not been reported in a prokaryotic platform, mainly relying on solid-phase synthesis. Bacterial production arises as an option for high-amount peptide generation and larger pHLIP fusion protein-synthesis; however, cell-based pH-responsive peptide production could be challenging due to intracellular peptide interactions or degradation due to unstructured conformations. An &lt;italic&gt;Escherichia coli&lt;/italic&gt; (E. coli)-BL21 cell culture was induced with Isopropyl ß-D-1-thiogalactopyranoside (IPTG) in order to produce a Glutathione S-transferase-pHLIP (GST-pHLIP) fusion construct. Purification was done with Glutathione (GSH)-decorated magnetic beads using 4 ml of the induced cell culture. The production was quantified with Bradford reagent and characterized with SDS-PAGE and Western blot, contrasting Bradford results with densitometry analysis to obtain production approximate absolute values. A purified approximate total yield of ~26 µg with an apparent GSH-bead saturation and a total production of ~82 µg was obtained. Our Western Blot assay confirmed the presence of the GST-pHLIP construct in all the IPTG-induced fractions. Conclusion: A high-yield pHLIP production irrespective of its membrane affinity in acidic environments or its unstructured nature was achieved. Our study could be useful to scale up pHLIP synthesis for future applications.&lt;/p&gt; &lt;/abstract&gt;

Regulation of Antimicrobial Peptide Activity via Tuning Deformation Fields by Membrane-Deforming Inclusions.

Antimicrobial peptides (AMPs) are considered prospective antibiotics. Some AMPs fight bacteria via cooperative formation of pores in their plasma membranes. Most AMPs at their working concentrations can induce lysis of eukaryotic cells as well. Gramicidin A (gA) is a peptide, the transmembrane dimers of which form cation-selective channels in membranes. It is highly toxic for mammalians as being majorly hydrophobic gA incorporates and induces leakage of both bacterial and eukaryotic cell membranes. Both pore-forming AMPs and gA deform the membrane. Here we suggest a possible way to reduce the working concentrations of AMPs at the expense of application of highly-selective amplifiers of AMP activity in target membranes. The amplifiers should alter the deformation fields in the membrane in a way favoring the membrane-permeabilizing states. We developed the statistical model that allows describing the effect of membrane-deforming inclusions on the equilibrium between AMP monomers and cooperative membrane-permeabilizing structures. On the example of gA monomer-dimer equilibrium, the model predicts that amphipathic peptides and short transmembrane peptides playing the role of the membrane-deforming inclusions, even in low concentration can substantially increase the lifetime and average number of gA channels.