Basic Peptides Research Articles

Understanding the physiological role of endogenous allosteric modulators at the muscarinic acetylcholine receptors

Allosteric binding sites on G protein-coupled receptor (GPCR) can be targeted by synthetic or natural (endogenous) molecules (van der Westhuizen et al., 2015). However, the (patho)physiological role(s) of many endogenous allosteric modulators remain poorly understood. One interesting example is major basic protein (MBP), a highly basic peptide that acts as a negative allosteric modulator (NAM) of acetylcholine (ACh) at airway M2 muscarinic acetylcholine receptors (mAChR; Jacoby et al., 1993). We hypothesized that, in addition to MBP, other endogenous basic peptides, including the antimicrobial, LL-37, involved in chemotaxis, maturation of immune cells and apoptosis (Kahlenberg et al., 2013) could also interact allosterically with the M2 mAChRs and have major physiological impacts. In this study, we aim to characterise the pharmacological properties and the putative (patho)physiological roles of LL-37 at mAChRs. Using IMR-32, a native cell line endogenously expressing human M2 mAChRs and mouse tissues predominantly expressing mouse M2 mAChRs, we performed [3H]NMS radioligand binding and [35S]GTPgS turnover as a functional measure of receptor activation, to assess the allosteric effect of LL-37.

Understanding the physiological role of endogenous allosteric modulators at the muscarinic acetylcholine receptors

Allosteric binding sites on G protein-coupled receptor (GPCR) can be targeted by synthetic or natural (endogenous) molecules (van der Westhuizen et al., 2015). However, the (patho)physiological role(s) of many endogenous allosteric modulators remain poorly understood. One interesting example is major basic protein (MBP), a highly basic peptide that acts as a negative allosteric modulator (NAM) of acetylcholine (ACh) at airway M2 muscarinic acetylcholine receptors (mAChR; Jacoby et al., 1993). We hypothesized that, in addition to MBP, other endogenous basic peptides, including the antimicrobial, LL-37, involved in chemotaxis, maturation of immune cells and apoptosis (Kahlenberg et al., 2013) could also interact allosterically with the M2 mAChRs and have major physiological impacts. In this study, we aim to characterise the pharmacological properties and the putative (patho)physiological roles of LL-37 at mAChRs. Using IMR-32, a native cell line endogenously expressing human M2 mAChRs and mouse tissues predominantly expressing mouse M2 mAChRs, we performed [3H]NMS radioligand binding and [35S]GTPgS turnover as a functional measure of receptor activation, to assess the allosteric effect of LL-37.

Microwave-Assisted Synthesis and Immunological Evaluation of Self-Assembling Peptide Vaccines.

Self-assembling peptides spontaneously associate into functional supramolecular scaffolds, which have found numerous biomedical applications. These molecular assemblies have applications in nerve regeneration, wound healing, and both prophylactic and therapeutic vaccination. They can also be useful tools for proliferation assays, sustained culture of difficult cell lines, or activation of cell lines for immunoassays. This protocol will describe the basic peptide synthesis and purification of model self-assembling peptide immunogen and methods for vaccinating mice, collecting lymph nodes, and stimulating cells ex vivo.

Utilization of blood by-products: An in silico and experimental combined study for BSA usage

In order to exploit industrial discards, protein enzymatic hydrolysis is a currently popular methodology for obtaining bioactive peptides. However, once released, most promising peptides have to be selected from the mixture. In this work, the suitability of pepsin (EC 3.4.23.1) to hydrolyse serum albumin in order to obtain bioactive peptides was assessed. Then, a suitable process to obtain best separation of bioactive peptides was evaluated, using polyethersulfone membranes at different pH values. Serum albumin was easily hydrolysed by pepsin, reaching a DH value of the 65.64 ± 1.57% of the maximum possible. A 23.25% of the identified peptides possessed high bioactivity scores (greater than 0.5), and one of them had reported bioactivity (LLL). Charge mechanisms always predominated over the sieve effect, and best transmission was accomplished at pH values close to the peptides isoelectric points. Basic and neutral peptides with the highest scores were always the most transmitted. Membrane material had greater influence than NMWCO in determining peptide transmission. In order to obtain purified fractions rich in peptides with high bioactivity scores from serum albumin, polyethersulfone membranes (applicable to industrial scale) of 5 kDa MWCO should be used at basic pH values after pepsin digestion.

Metal-Dependent DNA Recognition and Cell Internalization of Designed, Basic Peptides.

A fragment of the DNA basic region (br) of the GCN4 bZIP transcription factor has been modified to include two His residues at designed i and i+4 positions of its N-terminus. The resulting monomeric peptide (brHis2) does not bind to its consensus target DNA site (5′-GTCAT-3′). However, addition of Pd(en)Cl2 (en, ethylenediamine) promotes a high-affinity interaction with exquisite selectivity for this sequence. The peptide–DNA complex is disassembled by addition of a slight excess of a palladium chelator, and the interaction can be reversibly switched multiple times by playing with controlled amounts of either the metal complex or the chelator. Importantly, while the peptide brHis2 fails to translocate across cell membranes on its own, addition of the palladium reagent induces an efficient cell internalization of this peptide. In short, we report (1) a designed, short peptide that displays highly selective, major groove DNA binding, (2) a reversible, metal-dependent DNA interaction, and (3) a metal-promoted cell internalization of this basic peptide.

Peptide-Binding Nanoparticle Materials with Tailored Recognition sites for Basic Peptides.

Peptides rich in basic residues such as lysine and arginine play important roles in biology such as bacterial defense and cell penetration. Although peptide-binding materials with high sequence-specificity have broad potential applications, the diverse functionalities of peptide side chains make their molecular recognition extremely difficult. By covalently capturing micelles of a doubly cross-linkable surfactant with solubilized peptide templates, we prepared water-soluble molecularly imprinted nanoparticles with high sequence-specificity for basic peptides. The nanoparticles interact with the side chains of lysine and arginine through hydrogen bonds strengthened by the nonpolar environment of the micelle. They have hydrophobic pockets in their core complementary to the hydrophobic side chains in size and shape. These recognition sites allowed the micelles to bind basic biological peptides strongly in water, with tens to hundreds of nanomolar in binding affinity.

Design, Synthesis, and Anticancer Activities of Cyclometalated Tris(2‐phenylpyridine)iridium(III) Complexes with Cationic Peptides at the 4′‐Position of the 2‐Phenylpyridine Ligand

We previously reported the design and synthesis of amphiphilic Ir complex–cationic peptide hybrids (2a–2f), which contain basic peptide sequences such as KKGG (K = lysine, G = glycine) at the 5′‐positions (para position with respect to the C–Ir bond) of three 2‐(4′‐tolyl)pyridine (tpy) ligands. Among them, 2c–2e induced the necrosis‐like cell death of Jurkat cells through a calcium‐dependent pathway, possibly involving a Ca2+–calmodulin (CaM) complex. Herein, we report the synthesis of amphiphilic Ir(ppy)3 complexes (ppy = 2‐phenylpyridine) containing the KKGG sequence at the 4′‐position of the ppy moiety (4a–4d) to examine the effect of the position of the cationic peptide sequence on the cytotoxicities of the complexes against Jurkat cells. The results of 3‐(4,5‐dimethly‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assays and a mechanistic study indicate that 4b and 4c, which contain C6 and C8 linkers, induce cell death through a calcium‐dependent pathway accompanied by membrane disruption in a manner similar to that of 2c–2e but with smaller half‐maximal effective concentration (EC50) values than those of 2c–2e. The results of the photoaffinity labeling of Jurkat cells with 5b containing a photoreactive 3‐trifluoromethyl‐3‐phenyldiazirine (TFPD) unit and co‐staining experiments with specific probes for intracellular organelles suggest that 4b and 4c bind to Ca2+–CaM and are localized in the mitochondria during cell death.

RNase H As Gene Modifier, Driver of Evolution and Antiviral Defense.

Retroviral infections are ‘mini-symbiotic’ events supplying recipient cells with sequences for viral replication, including the reverse transcriptase (RT) and ribonuclease H (RNase H). These proteins and other viral or cellular sequences can provide novel cellular functions including immune defense mechanisms. Their high error rate renders RT-RNases H drivers of evolutionary innovation. Integrated retroviruses and the related transposable elements (TEs) have existed for at least 150 million years, constitute up to 80% of eukaryotic genomes and are also present in prokaryotes. Endogenous retroviruses regulate host genes, have provided novel genes including the syncytins that mediate maternal-fetal immune tolerance and can be experimentally rendered infectious again. The RT and the RNase H are among the most ancient and abundant protein folds. RNases H may have evolved from ribozymes, related to viroids, early in the RNA world, forming ribosomes, RNA replicases and polymerases. Basic RNA-binding peptides enhance ribozyme catalysis. RT and ribozymes or RNases H are present today in bacterial group II introns, the precedents of TEs. Thousands of unique RTs and RNases H are present in eukaryotes, bacteria, and viruses. These enzymes mediate viral and cellular replication and antiviral defense in eukaryotes and prokaryotes, splicing, R-loop resolvation, DNA repair. RNase H-like activities are also required for the activity of small regulatory RNAs. The retroviral replication components share striking similarities with the RNA-induced silencing complex (RISC), the prokaryotic CRISPR-Cas machinery, eukaryotic V(D)J recombination and interferon systems. Viruses supply antiviral defense tools to cellular organisms. TEs are the evolutionary origin of siRNA and miRNA genes that, through RISC, counteract detrimental activities of TEs and chromosomal instability. Moreover, piRNAs, implicated in transgenerational inheritance, suppress TEs in germ cells. Thus, virtually all known immune defense mechanisms against viruses, phages, TEs, and extracellular pathogens require RNase H-like enzymes. Analogous to the prokaryotic CRISPR-Cas anti-phage defense possibly originating from TEs termed casposons, endogenized retroviruses ERVs and amplified TEs can be regarded as related forms of inheritable immunity in eukaryotes. This survey suggests that RNase H-like activities of retroviruses, TEs, and phages, have built up innate and adaptive immune systems throughout all domains of life.

P012 Structure directed identification of human metabolite binding in the P4 pocket of MHC class-II DRB1∗15:01 and DRB1∗15:03 and implications for multiple sclerosis risk

Aim Robust models of HLA mediated drug hypersensitivity demonstrate that binding of small molecules in the peptide-binding groove may result in changes in conformation or register shift of bound peptide, leading to an altered immune response. We performed virtual screening of the complete Human Metabolite Database (HMDB) for docking to the multiple sclerosis (MS) susceptible DRB1 ∗ 15:01 allele and compared the results to the closely related yet non-susceptible DRB1 ∗ 15:03 allele; and assessed the potential impact on binding of human myelin basic peptide (MBP). Methods The crystal structure of DRB1∗15:01 was obtained from the protein data bank under accession number 1BX2. An in silico DRB1 ∗ 15:03 structure was generated from the known structure of DRB1 ∗ 15:01 by mutating residue Tyr 30 > His 30 . The target proteins ( DRB1 ∗ 15:01 and DRB1 ∗ 15:03 ) were prepared with N-acetyl-D-glucosamine (NAG) for docking with human metabolites using UCSF Chimera (v1.11.2). We conducted independent screenings to probe each pocket of the peptide binding groove for both proteins using HMDB library v3.6, which consists of 41993 human metabolites. Results We observed higher energy scores for metabolite binding to DRB1 ∗ 15:01 than DRB1 ∗ 15:03. We found that 78 and 82 metabolites bind strictly to DRB1 ∗ 15:01 or DRB1 ∗ 15:03 respectively, while 79 metabolites bind to both structures. Structural comparison of docked metabolites with DRB1 ∗ 15:01 and DRB1 ∗ 15:03 complexed with MBP revealed that Phenylalanine MBP 92 allows binding of metabolites in the P4 pocket of DRB1 ∗ 15:01 but Valine MBP 89 abrogates metabolite binding in the P1 pocket. We observed differences in the energy score for the binding of the same metabolites in the P4 pockets of DRB1 ∗ 15:01 and DRB1 ∗ 15:03 or different metabolites in the P4 pockets of DRB1 ∗ 15:01 and DRB1 ∗ 15:03 suggesting stronger binding to DRB1 ∗ 15:01. Further, several of these metabolites have been previously associated with MS. Conclusions Our study confirmed that specific, disease-associated human metabolites bind effectively with the most polymorphic P4 pockets of DRB1 ∗ 15:01, the primary MS susceptible allele in most populations. Our results suggest that endogenous human metabolites bound in specific pockets of HLA may be immunomodulatory and implicated in autoimmune disease.

Mast cell degranulation via MRGPRX2 by isolated human albumin fragments

BackgroundMast cells are important modulators of the human immune system via their release of several inflammatory mediators and proteases. The release can be activated by different pathways: the classical immunoglobulin E-dependent pathway and by the non-immunological immunoglobulin E-independent pathway. MAS-related G protein-coupled receptor X2 (MRGPRX2) is expressed in mast cells and it is one of the endogenous receptor responsible for the IgE-independent activation of human mast cell. The MRGPRX2 is classified as orphan receptor and unlike most GPCRs, the MRGPRX2 recognizes a wide range of basic molecules. Thus, there still might be several unknown ligands for the receptor. MethodsMRGPRX2 activating peptides were isolated from human plasma using consecutive HPLC purification steps. The isolation process was monitored with MRGPRX2 transfected HEK 293 cells. The isolated peptides were sequenced by MS and synthetized. The synthetic peptides were used to determine degranulation of the human LAD 2 mast cell line by measuring β-hexosaminidase release. ResultsThree endogenous MRGPRX2 activating peptides were isolated from human plasma. These peptides are identified as fragments of albumin. The isolated fragments activate MRGPRX2 and degranulate MRGPRX2 expressing LAD 2 cells in dose-dependent manner. ConclusionsThe isolated basic peptides generated from human albumin are able to degranulate mast cells via the MRGPRX2. General significanceThese endogenous albumin fragments, cleaved from albumin by mast cell secreted proteases, provide a possible pathway for self-perpetuating mast cell dependent inflammation.

A cell-based high-throughput screening assay system for inhibitor compounds of antigen presentation by HLA class II molecule

A number of autoimmune diseases are associated with the genotypes of human leukocyte antigen class II (HLA), some of which present peptides derived from self-proteins, resulting in clonal expansion of self-reactive T cells. Therefore, selective inhibition of self-peptide loading onto such disease-associated HLA could ameliorate the diseases. To effectively identify such compounds, in this study, we established, for the first time, a cell- and 96-well microplate-based high-throughput screening system for inhibitors of antigen presentation. A panel of DRB1 genes plus DRA*01:01 gene were expressed in HEK293T cells and in 3T3 cells, and their binding with biotinylated known self-antigen peptides was measured by flow cytometry. HLA-DR1 (DRB1*01:01) and DR15 (DRB1*15:01) showed a high affinity with myelin basic protein peptide (MBP83-98). Therefore, in 96-well plate wells, MBP83-99 was allowed to bind to DR1 or DR15 on 3T3 cells in competition with a test compound, and the HLA-bound peptide was detected by streptavidin-conjugated β-galactosidase, thereby identifying inhibitor compounds for rheumatoid arthritis or multiple sclerosis. Our assay system has a potential for broad applications, including designing peptide vaccines.

Enhancement of nose-to-brain delivery of hydrophilic macromolecules with stearate- or polyethylene glycol-modified arginine-rich peptide

Recently, nose-to-brain delivery is a highly versatile route, which, in combination with novel drugs being developed for treating intractable CNS diseases, is a promising approach for the treatment of disorders. Furthermore, nano-sized drug carriers may improve nose-to-brain drug delivery by their capability to increase the transmucosal penetration of the drugs across nasal mucosal tissue barrier. However, there is still not enough information regarding mechanism of absorption pathway from nasal cavity to brain using nanocarriers. In this study, to investigate the nose-to-brain transport pathway using nanocarriers, the distribution in whole brain, nasal mucosa, and trigeminal nerve after intranasal administration of two kinds of nanocarriers which have hydrophobic or hydrophilic moiety. We used CHHRRRRHHC peptide (CH2R4H2C) as basic peptide carriers, and modified with stearic acid (STR) as a hydrophobic moiety (STR-CH2R4H2C) or polyethylene glycol (PEG)-based block copolymer (PEG-PCL) as hydrophilic moiety (PEG-PCL-CH2R4H2C). The nose-to-brain drug delivery can be improved by using STR-CH2R4H2C and PEG-PCL-CH2R4H2C as carriers. Specifically, hydrophobic STR-CH2R4H2C is more suitable for the transport of drugs targeting the forebrain, while PEG-PCL-modified CH2R4H2C is more suitable for transporting drugs targeting the hindbrain or whole brain tissue. In conclusion, the results of this study support the possibility that drug delivery pathways can be controlled depending on the properties of different carrier complexes.

Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli.

Glycinin basic peptide (GBP) is an antibacterial ingredient that occurs naturally in the basic parts of soybean glycinin. The antibacterial actions of GBP against Escherichia coli ATCC 8739 were investigated in this study. The minimum inhibitory concentration of GBP against E. coli was 200 μg/mL. The exposure of E. coli cells to GBP induced significant cell damage and inactivated intracellular esterases (stressed and dead cells, 70.9% ± 0.04 for 200 μg/mL of GBP and 91.9% ± 0.06 for 400 μg/mL of GBP), as determined through dual staining in flow cytometry. GBP resulted in the exposure of phosphatidylserine in E. coli cells. The analyses of flow cytometry-manifested GBP treatment led to the shrinkage of the cell surface and the complication of cell granularity. The observations in transmission electron microscopy demonstrated that 400 μg/mL of GBP severely disrupted the membrane integrity, resulting in ruptures or pores in the membrane, outflows of intracellular contents, or aggregation of the cytoplasm. Release of alkaline phosphatase, lipopolysaccharide, and reducing sugar further verified that the membrane damage was due to GBP. In addition, GBP treatment changed the helicity and base staking of DNA, as determined by circular dichroism spectroscopy. These results showed that GBP had strong antibacterial activity against E. coli via membrane damage and DNA perturbation. Additionally, GBP exhibited no cytotoxicity on the viability of human embryonic kidney cells. Thus, GBP may be a promising candidate as a natural antibacterial agent.

Expression of Recombinant Antibody Fragment, Anti BNP-SCFV on the Periplasm of Escherichia Coli for the Detection of Heart Failure

Basic natriuretic peptide (BNP) is a polypeptide hormone consist of 32 amino acids that secreted by the heart ventricle to respond the excessive stretching of heart muscle cells. BNP can be used as prognostic marker for patients with heart failure. The presence of BNP in blood can be detected by BNP antibody, which is anti BNP-single chain variable fragment (Anti BNP-SCFV). The antibody is a combination of polypeptides between varying region on the heavy chain (VH) and the light chain (VL) of immunoglobulin. Anti BNP-SCFV will bind to BNP through the antigen-antibody interaction. Concentration of BNP in a patient’s blood can be detected through the interaction of BNP with Anti BNP-SCFV using immunosensor method. Production of recombinant Anti BNP-SCFV in Escherichia coli as host is reported in the present study. Anti BNP-SCFV was expressed in fusion form with OmpC signal peptide that direct the protein to a periplasmic space. Expression was performed under RhaBad promoter as control using L-rhamnose as inducer. SDS-PAGE characterization showed consistent band at 28 kDa, which was assumed as Anti BNP-SCFV. The optimum expression was found at four hours after induction with 4 mM inducer. Anti BNP-SCFV was secreted from the cell as characterized by the presence of the protein on periplasmic membrane and extracellular fraction.

PSA3, a Protein on the Stromal Face of the Thylakoid Membrane, Promotes Photosystem I Accumulation in Cooperation with the Assembly Factor PYG7.

PSI is a large protein-pigment complex located in the thylakoid membrane in cyanobacteria, plants, and algae. Although the structure and components of PSI are well characterized, mechanisms that orchestrate its assembly are poorly understood. In this study, we discovered a novel nucleus-encoded protein, Photosystem I Assembly3 (PSA3), that is required for PSI accumulation. PSA3 is conserved among green photosynthetic eukaryotes but is lacking in cyanobacteria. Mutations in the psa3 gene cause the specific loss of PSI in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Ribosome profiling and pulse-labeling analyses showed that chloroplast- encoded PSI subunits are synthesized at normal rates in psa3 mutants, indicating that PSA3 is involved in the biogenesis of PSI at a posttranslational step. PSA3 resides on the stromal face of the thylakoid membrane, where it is found in a complex that is slightly smaller than PSI. Structural predictions suggest that PSA3 binds a basic peptide in a manner that is sensitive to the oxidation state of Cys pairs flanking the predicted peptide binding groove. PSA3 and the previously described PSI biogenesis factor PYG7 interact in yeast two-hybrid and bimolecular fluorescence complementation assays, and they are found in thylakoid membrane complexes of similar size. These and other results indicate that PSA3 cooperates with PYG7 to promote the stable assembly of PSI, and that the PsaC subunit is likely to be the primary target of their action.

PH triggered re-assembly of nanosphere to nanofiber: The role of peptide conformational change for enhanced cancer therapy

pH-triggered conformational change and subsequent re-assembly of nanostructures provide a new strategy in nanomedicine for controlled drug release and enhanced therapy. Here, we reported the development of a novel pH-responsive nano-assembly as a drug carrier from peptide amphiphile (PA) consisting of mimicking peptide and stearic acid moieties. The mimicking peptide is a basic 17-amino acid peptide derived from antennapedia homeodomain, and undergoes a conformational transition of the secondary structure from β-sheet at pH7.4 to α-helix at pH5.0. Such transition therefore leads to simultaneous evolution of the self-assembled structure of PA from nanosphere to nanofiber, promotes assemblies retention and then release drugs in the cytoplasm of tumor cell. In vitro studies showed that the doxorubicin (Dox)-loaded PA nanoparticle (PA@Dox) could be uptaken efficiently by the cell due to the membrane penetrating capability of the mimicking peptide and subsequently the released Dox further induce apoptosis of murine colon carcinoma CT26 (MCCC) cell. In a mouse xenograft model of MCCC, administration of PA@Dox via lateral tail vein injection could remarkably retard the tumor growth. The overall results suggested that the PA-based nanocarriers adopting the novel strategy of pH-triggered secondary structural change could enhance therapeutic efficacy and be used as a promising platform for potential development of new generation of drug carriers for cancer therapy.

MRGPRX2 receptor activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities.

Abstract Mast cells play key roles in allergy, anaphylaxis and pathogen/toxin defense. They contain cytoplasmic granules with a wide spectrum of pleotropic mediators (e.g. histamine, tryptase, serotonin) that are released upon activation. Mast cell degranulation (MCD) occurs upon clustering of the IgE receptor in presence of antigen or via non-IgE mediated mechanisms, one of which is by means of the Mas-related G protein coupled receptor (MRGPRX2). MRGPRX2 is a non-selective, low affinity binding receptor that responds to many basic biogenic amines and peptides. Consequently, MCD is an important potential safety liability for peptide therapeutics. To improve screening of peptides for this liability, we assessed a MRGPRX2-CHO cell-based receptor activation assay using positive (n=30) and negative (n=29) control peptides, which were previously characterized for MCD liability in CD34+ bone marrow-derived human mast cell cultures. MRGPRX2-CHO cells were engineered to selectively activate beta-arrestin upon receptor stimulation. In addition to beta-arrestin, Ca2+ was also measured to determine the potential of ligand bias (selective engagement of G-protein dependent or independent pathways). The MRGPRX2-CHO receptor assay had a sensitivity of 97% (false negative 3%) and specificity of 93% (false positive 7%) against the outcome in human mast cell cultures. No clear evidence of ligand bias was observed. Because MRGPRX2 is overexpressed in CHO cells, the assay may slightly over predict, and does not inform on other mechanisms of MCD not mediated by MRGPRX2. Assessing MRGPRX2 receptor activation provides a rapid, high-throughput and economical mechanism-based approach for detecting peptide-mediated human MCD liabilities.

A Basic ApoE-Based Peptide Mediator to Deliver Proteins across the Blood-Brain Barrier: Long-Term Efficacy, Toxicity, and Mechanism

We have investigated delivery of protein therapeutics from the bloodstream into the brain using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal disease due to deficiencies in tripeptidyl peptidase 1 (TPP1). Supraphysiological levels of TPP1 are delivered to the mouse brain byacute intravenous injection when co-administered with K16ApoE, a peptide that in trans mediates passage across the blood-brain barrier (BBB). Chronic treatment of LINCL mice with TPP1 and K16ApoE extended the lifespan from 126 to>294days, diminished pathology, and slowed locomotor dysfunction. K16ApoE enhanced uptake of a fixable biotin tracer by brain endothelial cells in a dose-dependent manner, suggesting that its mechanism involves stimulation of endocytosis. Pharmacokinetic experiments indicated that K16ApoE functions without disrupting the BBB, with minimal effects on overall clearance or uptake by the liver and kidney. K16ApoE has a narrow therapeutic index, with toxicity manifested as lethargy and/or death in mice. To address this, we evaluated variant peptides but found that efficacy and toxicity are associated, suggesting that desired and adverse effects are mechanistically related. Toxicity currently precludes direct clinical application of peptide-mediated delivery in its present form but it remains a useful approach to proof-of-principle studies for biologic therapies to the brain in animal models.

A novel cecropin‐like peptide from black soldier fly, Hermetia illucens: Isolation, structural and functional characterization

AbstractCecropins are basic antibacterial peptides that have potent antimicrobial activities. We induced and purified a novel antimicrobial peptide exhibiting activity against Gram‐negative bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel cecropin‐like peptide 1 (CLP1) was purified using solid‐phase extraction and reverse‐phase chromatography. The purified CLP1 demonstrated a molecular weight of 4,840 Da, as determined by matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF). From analysis of CLP1 by N‐terminal amino acid sequencing using Edman degradation, combined with MALDI‐TOF and rapid amplification of cDNA ends‐polymerase chain reaction (RACE‐PCR), the amino acid sequence of the mature peptide was determined to be GWRKRVFKPVEKFGQRVRDAGVQGIAIAQQGANVLATARGGP PQQG. In NCBI BLAST, the amino acid sequence of CLP1 was found to be 60 % identical to the Drosophila melanogaster cecropin C. In silico analysis revealed that CLP1 was suggested to be part of the cecropin superfamily of AMPs characterized as cationic, linear, α‐helical, and amphipathic polypeptides. Analysis of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) showed that CLP1 exerted antibacterial effects against Gram‐negative bacteria. The expression of CLP1 transcripts in several tissues after bacterial challenge was measured by quantitative real‐time PCR. CLP1 expression was negligible throughout the body before immunization, and was mostly evident in the fat body after immunization.

Recombinant expression of Intrepicalcin from the scorpion Vaejovis intrepidus and its effect on skeletal ryanodine receptors

BackgroundScorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. MethodsRecombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site. Results[3H]Ryanodine binding, used as a functional index of RyR activity, revealed that recombinant Intrepicalcin activates skeletal RyR (RyR1) dose-dependently with Kd=17.4±4.0nM. Intrepicalcin significantly augments the bell-shaped [Ca2+]-[3H]ryanodine binding curve at all [Ca2+] ranges, as is characteristic of the calcins. In single channel recordings, Intrepicalcin induces the appearance of a subconductance state in RyR1 with a fractional value ∼55% of the full conductance state, very close to that of Vejocalcin. Furthermore, Intrepicalcin stimulates Ca2+ release at an initial dose=45.3±2.5nM, and depletes ~50% of Ca2+ load from skeletal sarcoplasmic reticulum vesicles. ConclusionsWe conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. General significanceThis is the first calcin heterologously expressed in the periplasma of Escherichia coli BL21-DE3, shown to be pharmacologically effective, thus paving the way for the generation of Intrepicalcin variants that are required for structure-function relationship studies of calcins and RyRs.