Proline-rich Peptides Research Articles

Ca(2+) -complex stability of GAPAGPLIVPY peptide in gas and aqueous phase, investigated by affinity capillary electrophoresis and molecular dynamics simulations and compared to mass spectrometric results.

Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann etal. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.

Affinity adsorbents for proline-rich peptide sequences: a new role for WW domains

The WW domain derived from human Yes-associated protein (hYAP65_WW) recognizes proline-rich peptides.

Proline-Rich Peptide Mimics Effects of Enamel Matrix Derivative on Rat Oral Mucosa Incisional Wound Healing.

Proline-rich peptides have been shown to promote periodontal regeneration. However, their effect on soft tissue wound healing has not yet been investigated. The aim of this study is to evaluate the effect of enamel matrix derivative (EMD), tyrosine-rich amelogenin peptide (TRAP), and a synthetic proline-rich peptide (P2) on acute wound healing after a full-thickness flap procedure in an incisional rat model. This experimental study has a split-mouth, randomized, placebo-controlled design. Test and control wounds were created on the palatal mucosa of 54 Sprague-Dawley rats. Wounds were histologically processed, and reepithelialization, leukocyte infiltration, and angiogenesis were assessed at days 1, 3, and 7 post-surgery. EMD and P2 significantly promoted early wound closure at day 1 (P <0.001 and P = 0.004, respectively). EMD maintained a significant acceleration of reepithelialization at day 3 (P = 0.004). Wounds treated by EMD and P2 showed increased angiogenesis during the first 3 days of healing (P = 0.03 and 0.001, respectively). Leukocyte infiltration was decreased in EMD-treated wounds at day 1 (P = 0.03), and P2 and TRAP induced a similar effect at days 3 (P = 0.002 and P <0.0001, respectively) and 7 (P = 0.005 and P <0.001). EMD and P2 promoted reepithelialization and neovascularization in full-thickness surgical wounds on rat oral mucosa.

Antiviral Cystine Knot α-Amylase Inhibitors from Alstonia scholaris

Cystine knot α-amylase inhibitors are cysteine-rich, proline-rich peptides found in the Amaranthaceae and Apocynaceae plant species. They are characterized by a pseudocyclic backbone with two to four prolines and three disulfides arranged in a knotted motif. Similar to other knottins, cystine knot α-amylase inhibitors are highly resistant to degradation by heat and protease treatments. Thus far, only the α-amylase inhibition activity has been described for members of this family. Here, we show that cystine knot α-amylase inhibitors named alstotides discovered from the Alstonia scholaris plant of the Apocynaceae family display antiviral activity. The alstotides (As1-As4) were characterized by both proteomic and genomic methods. All four alsotides are novel, heat-stable and enzyme-stable and contain 30 residues. NMR determination of As1 and As4 structures reveals their conserved structural fold and the presence of one or more cis-proline bonds, characteristics shared by other cystine knot α-amylase inhibitors. Genomic analysis showed that they contain a three-domain precursor, an arrangement common to other knottins. We also showed that alstotides are antiviral and cell-permeable to inhibit the early phase of infectious bronchitis virus and Dengue infection, in addition to their ability to inhibit α-amylase. Taken together, our results expand membership of cystine knot α-amylase inhibitors in the Apocynaceae family and their bioactivity, functional promiscuity that could be exploited as leads in developing therapeutics.

Abstract B190: Allosteric modulation of Src family kinases via SH3 domain displacement

Abstract The Src family of non-receptor protein-tyrosine kinases (SFKs) plays a crucial role in cancer progression by affecting multiple signaling pathways linked to tumor cell adhesion, invasion and survival. Recent work from our group and others has shown that individual Src-family members may have unique and in some cases opposing roles in the regulation of cell growth and differentiation. Discovery and development of SFK isoform-selective inhibitors and agonists is therefore essential for the most effective therapeutic targeting of this ubiquitous kinase family. Current SFK inhibitors target the ATP-binding site of the kinase domain or an allosteric pocket nearby. However, the highly conserved nature of the active site across the Src kinase family limits identification of isoform-selective compounds. One possible solution to this problem involves small molecule modulation of SFK activity via the regulatory SH3 and SH2 domains. SFK activity is tightly regulated by intramolecular interactions of the SH3 domain with the SH2:kinase linker and the SH2 domain with the phosphorylated C-terminal tail. Small molecules that either enhance or disrupt these interactions are anticipated to result in kinase inhibition or activation, respectively. Recently, we reported that binding of a short proline-rich peptide to the SH3 domains of several Src-family members is sufficient to induce kinase activation in vitro. This peptide, known as VSL12 (VSLARRPLPLP), was postulated to disrupt the regulatory SH3:linker interaction in the downregulated kinase. In the present study, we used VSL12 as probe to better understand the structural basis of kinase activation by SH3 displacement. We first compared the affinity and kinetics of the VSL12 peptide interaction with SFK SH3 domains vs. the near full-length kinase proteins using surface plasmon resonance. We found that VSL12 bound to the isolated SH3 domains of Src, Hck, Lyn and Fyn with similar micromolar potencies. Surprisingly, the KD values for VSL12 binding to the corresponding near-full-length kinases were in the low nanomolar range, suggesting that the peptide makes additional contacts with residues outside of the SH3 domain that result in the stable, active complex. This idea was tested further using differential scanning fluorimetry to explore the thermal stability of the protein:peptide complexes. Addition of VSL12 enhanced the thermal stability of a recombinant Src SH3-SH2-linker protein, consistent with increased molecular order. However, VSL12 decreased the thermal stability of near full-length c-Src, which may reflect conformational changes in the kinase domain that accompany kinase activation. Addition of the kinase domain inhibitor dasatinib restored thermal stability of the Src:VSL12 complex. Complexes of the Src SH3-SH2-linker protein with the VSL12 peptide readily formed crystals that diffract X-rays to 2.6 Å resolution, providing further support for the stabilization of the complex by VSL12 binding. Determining the structures of the Src SH3-SH2-linker protein and the near-full-length kinase in complex with VSL12 will provide fresh insight as to the mechanism of c-Src activation by SH3 domain displacement and aid in the design of potent and selective non-peptide allosteric modulators. Citation Format: Heather L. Rust, Jamie A. Moroco, John J. Alvarado, John J. Engen, Thomas E. Smithgall. Allosteric modulation of Src family kinases via SH3 domain displacement. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B190.

Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin.

Prolyl endopeptidases are key enzymes in the digestion of proline-rich proteins. Fungal extracts rich in prolyl endopeptidases produced by a species such as Aspergillus oryzae used in food fermentation would be of particular interest for the development of an oral enzyme therapy product in patients affected by intolerance to gluten. Two major A. oryzae secreted prolyl endopeptidases of the MEROPS S28 peptidase family, AoS28A and AoS28B, were identified when this fungus was grown at acidic pH in a medium containing soy meal protein or wheat gliadin as the sole source of nitrogen. AoS28B was produced by 12 reference A. oryzae strains used in food fermentation. AoS28A was secreted by six of these 12 strains. This protease is the orthologue of the previously characterized Aspergillus fumigatus (AfuS28) and Aspergillus niger (AN-PEP) prolyl endopeptidases which are encoded by genes with a similar intron-exon structure. Large amounts of secreted AoS28A and AoS28B were obtained by gene overexpression in A. oryzae. AoS28A and AoS28B are endoproteases able to cleave N-terminally blocked proline substrates. Both enzymes very efficiently digested the proline-rich 33-mer of gliadin, the most representative immunotoxic peptide deriving from gliadin, with some differences in terms of specificity and optimal pH. Digestion of the gliadin peptide in short peptides with both enzymes was found to occur from its N terminus.

Oncocin Onc72 is efficacious against antibiotic-susceptible Klebsiella pneumoniae ATCC 43816 in a murine thigh infection model.

Oncocins and apidaecins are short proline-rich antimicrobial peptides (PrAMPs) representing novel antibiotic drug lead compounds that kill bacteria after internalization and inhibition of intracellular targets (e.g. 70S ribosome and DnaK). Oncocin Onc72 is highly active against Gram-negative bacteria in vitro and in vivo protecting mice in systemic infection models with Escherichia coli and KPC-producing Klebsiella pneumoniae. Here we studied its efficacy in a murine thigh infection model using meropenem as antibiotic comparator that had a 44-fold higher molar in vitro activity than Onc72. Male CD1 mice were rendered neutropenic using cyclophosphamide for four days before intramuscular infection with K. pneumoniae ATCC 43816. After 75 min oncocin Onc72 or the antibiotic comparator meropenem were administered subcutaneously with 100 mg (43 µmol) and 25 mg (65 µmol) per kg of body weight, respectively, six times every 75 min. Onc72 and meropenem administered subcutaneously reduced the thigh tissue burden of K. pneumoniae ATCC 43816 in neutropenic mice significantly by 4.14 and 4.65 a log10 cfu/g, respectively. The bacterial counts were ∼0.5 and ∼1 log10 below the pre-treatment burden, respectively, indicating bactericidal effects for both compounds. Thus, Onc72 was as efficacious as meropenem in vivo despite its much lower in vitro activity determined according to CLSI standard antimicrobial activity tests.

Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system

Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step.

Short Proline-Rich Antimicrobial Peptides Inhibit Either the Bacterial 70S Ribosome or the Assembly of its Large 50S Subunit.

Short proline-rich antimicrobial peptides (PrAMPs) are a promising class of antibiotics that use novel mechanisms, thus offering the potential to overcome the health threat of multiresistant pathogens. The peptides bind to the bacterial 70S ribosome and can inhibit protein translation. We report that PrAMPs can be divided into two classes, with each class binding to a different site, and thus use different lethal mechanisms. Oncocin-type peptides inhibit protein translation in Escherichia coli by binding to the exit tunnel of the 70S ribosome with half maximal inhibitory concentrations (IC50 values) of around 2 to 6 μmol L(-1), whereas apidaecin-type peptides block the assembly of the large (50S) subunit of the ribosome, resulting in similar IC50 values. The revealed mechanisms should allow the design of new antibiotics to overcome current bacterial resistance mechanisms.

Effect of distal sugar and interglycosidic linkage of disaccharides on the activity of proline rich antimicrobial glycopeptides.

The effect of glycosylation on protein structure and function depends on a variety of intrinsic factors including glycan chain length. We have analyzed the effect of distal sugar and interglycosidic linkage of disaccharides on the properties of proline-rich antimicrobial glycopeptides, formaecin I and drosocin. Their glycosylated analogs-bearing lactose, maltose and cellobiose, as a glycan side chain on their conserved threonine residue, were synthesized where these disaccharides possess identical proximal sugar and vary in the nature of distal sugar and/or interglycosidic linkage. The structural and functional properties of these disaccharide-containing formaecin I and drosocin analogs were compared with their corresponding monoglycosylated forms, β-D-glucosyl-formaecin I and β-D-glucosyl-drosocin, respectively. We observed neither major secondary structural alterations studied by circular dichroism nor substantial differences in the toxicity with mammalian cells among all of these analogs. The comparative analyses of antibacterial activities of these analogs of formaecin I and drosocin displayed that β-D-maltosyl-formaecin I and β-D-maltosyl-drosocin were more potent than that of respective β-D-Glc-analog, β-D-cellobiosyl-analog and β-D-lactosyl-analog. Despite the differences in their antibacterial activity, all the analogs exhibited comparable binding affinity to DnaK that has been reported as one of the targets for proline-rich class of antibacterial peptides. The comparative-quantitative internalization studies of differentially active analogs revealed the differences in their uptake into bacterial cells. Our results exhibit that the sugar chain length as well as interglycosidic linkage of disaccharide may influence the antibacterial activity of glycosylated analogs of proline-rich antimicrobial peptides and the magnitude of variation in antibacterial activity depends on the peptide sequence.

Antibacterial activity and therapeutic efficacy of Fl-P(R)P(R)P(L)-5, a cationic amphiphilic polyproline helix, in a mouse model of staphylococcal skin infection.

The antibacterial activities and therapeutic efficacy of the cationic, unnatural proline-rich peptide Fl-PRPRPL-5 were evaluated against multidrug-resistant Staphylococcus aureus in a mouse model of skin infection. Fl-PRPRPL-5 showed potent activity against all clinical isolates of S. aureus tested, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA, respectively). Fl-PRPRPL-5 was also superior in clearing established in vitro biofilms of S. aureus and Staphylococcus epidermidis, compared with the established antimicrobials mupirocin and vancomycin. Additionally, topical treatment of an MRSA-infected wound with Fl-PRPRPL-5 enhanced wound closure and significantly reduced bacterial load. Finally, 0.5% Fl-PRPRPL-5 significantly reduced the levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) in wounds induced by MRSA skin infection. In conclusion, the results of this study suggest the potential application of Fl-PRPRPL-5 in the treatment of staphylococcal skin infections.

Identification of a Novel Proline-Rich Antimicrobial Peptide from Brassica napus.

Proline-rich antimicrobial peptides (PR-AMPs) are a group of cationic host defense peptides that are characterized by a high content of proline residues. Up to now, they have been reported in some insects, vertebrate and invertebrate animals, but are not found in plants. In this study, we performed an in silico screening of antimicrobial peptides, which led to discovery of a Brassica napus gene encoding a novel PR-AMP. This gene encodes a 35-amino acid peptide with 13 proline residues, designated BnPRP1. BnPRP1 has 40.5% identity with a known proline-rich antimicrobial peptide SP-B from the pig. BnPRP1 was artificially synthetized and cloned into the prokaryotic expression vector pET30a/His-EDDIE-GFP. Recombinant BnPRP1 was produced in Escherichia coli and has a predicted molecular mass of 3.8 kDa. Analysis of its activity demonstrated that BnPRP1 exhibited strong antimicrobial activity against Gram-positive bacterium, Gram-negative bacterium, yeast and also had strong antifungal activity against several pathogenic fungi, such as Sclerotinia sclerotiorum, Mucor sp., Magnaporthe oryzae and Botrytis cinerea. Circular dichroism (CD) revealed the main secondary structure of BnPRP1 was the random coil. BnPRP1 gene expression detected by qRT-PCR is responsive to pathogen inoculation. At 48 hours after S. sclerotiorum inoculation, the expression of BnPRP1 increased significantly in the susceptible lines while slight decrease occurred in resistant lines. These suggested that BnPRP1 might play a role in the plant defense response against S. sclerotiorum. BnPRP1 isolated from B. napus was the first PR-AMP member that was characterized in plants, and its homology sequences were found in some other Brassicaceae plants by the genome sequences analysis. Compared with the known PR-AMPs, BnPRP1 has the different primary sequences and antimicrobial activity. Above all, this study gives a chance to cast a new light on further understanding about the AMPs’ mechanism and application.

Structure-activity relationship study using peptide arrays to optimize Api137 for an increased antimicrobial activity against Pseudomonas aeruginosa

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa has a low susceptibility to common antibiotics. Additionally, around 15% of all clinical isolates bear acquired resistance genes. Thus, the development of new antibiotics to combat this pathogen in pneumonia, urinary tract infections, and bacteremia, represents an urgent task. The activity spectrum of the proline-rich antimicrobial peptide apidaecin 1b, originally isolated from honeybees (Apis mellifera), was extended in previous studies to further human pathogens including P. aeruginosa. However, the in vitro activity of the optimized peptide Api137 is limited to diluted medium conditions. Thus, we synthesized 323 analogs of Api137 on cellulose membranes using the SPOT strategy by substituting each residue individually by 19 other amino acids or deleting the residue. The peptides were deprotected with trifluoroacetic acid and cleaved with aqueous trimethylamine as C-terminal acids providing around 30 μg crude peptide per spot. This amount allowed determining the minimal inhibitory concentrations in a microdilution broth assay. The most promising substitutions were selected to synthesize 44 doubly and triply substituted Api137 analogs on the membrane. The 19 best peptides were synthesized at a larger scale and purified. Eight triply substituted Api137 analogs were up to 16-fold more active against P. aeruginosa at high medium concentrations without losing activities against Klebsiella pneumoniae and Acinetobacter baumannii and only slightly against Escherichia coli. The eight most active Api137 analogs were non-hemolytic to human erythrocytes and non-toxic to HeLa cells.

Optimization of oncocin for antibacterial activity using a SPOT synthesis approach: extending the pathogen spectrum to Staphylococcus aureus.

The identification of lead molecules against multidrug-resistant bacteria ensuing the development of novel antimicrobial drugs is an urgent task. Proline-rich antimicrobial peptides are highly active in vitro and in vivo, but only against a few Gram-negative human pathogens, with rather weak activities against Pseudomonas aeruginosa and Staphylococcus aureus. This reduced level of efficacy could be related to inadequate uptake mechanisms or structural differences of the intracellular target proteins, i.e., the 70S ribosome or chaperone DnaK. Here we synthesized peptide arrays on cellulose membranes using cleavable linkers to release the free individual peptides for further antimicrobial tests. Thus, a library of singly substituted oncocin analogs was produced by replacing each residue by all other 19 canonical amino acids yielding a set of 361 individual peptides to be evaluated against a luminescent P. aeruginosa strain. Thirteen substitutions appeared promising and their improved antibacterial activities were confirmed for different bacteria after larger scale synthesis of these analogs. By combining two favorable substitutions into one peptide, we finally obtained an oncocin analog that was ten times more active against P. aeruginosa and even 100-fold more active against S. aureus than the original oncocin, providing minimal inhibitory concentrations of 4-8 and 0.5 µg/mL, respectively.

Multimerization of a Proline-Rich Antimicrobial Peptide, Chex-Arg20, Alters Its Mechanism of Interaction with the Escherichia coli Membrane

A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization.

Characterization of the cell penetrating properties of a human salivary proline-rich peptide

Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins.Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

Polyvinyl alcohol nanofiber formulation of the designer antimicrobial peptide APO sterilizes Acinetobacter baumannii-infected skin wounds in mice.

Native and designer cationic antimicrobial peptides are increasingly acknowledged as host defense molecules rather than true antimicrobials. Due to their ability to activate the innate immune system, these structures are used to treat uninfected and bacterially-infected wounds, including those harboring Acinetobacter baumannii. Previously we documented that when administered intramuscularly or topically in liquid formulations, the proline-rich host defense peptide dimer A3-APO accelerates uninfected wound re-epithelization and eliminates systemic and local A. baumannii, methicillin-resistant Staphylococcus aureus and other pathogen load from infected lesions better than conventional antibiotics. In the current study we sought to produce and characterize a novel delivery system, suitable for immediate and convenient application in non-hospital environments. The APO monomer was incorporated into polyvinyl alcohol nanofibers and the complex was polymerized into a solid patch dressing. Mice were subjected to skin abrasion where the wounds were either left uninfected or were inoculated with a near lethal dose of multidrug resistant A. baumannii strain. Analyzed after 3 days, APO monomer-containing patches improved wound appearance significantly better than polymer patches without antibiotics. When compared to colistin, the APO patches accelerated wound healing, and statistically significantly reduced wound size and wound bacterial load. The in vivo antimicrobial effect was more extensive than after intramuscular administration of the peptide drug, by using only one tenth of the active pharmaceutical ingredient. These data suggest that the APO monomer-impregnated nanofiber dressing can be developed as an economical first-line treatment option to skin injuries in general and battlefield burn and blast injuries in particular.

Characterization of WWP1 protein expression in skeletal muscle of muscular dystrophy chickens.

A missense mutation in the gene encoding WWP1 was identified as the most promising candidate responsible for chicken muscular dystrophy (MD) by genetic linkage analysis. WWP1 is a HECT-type E3 ubiquitin protein ligase composed of 922 amino acids, which contains 4 tandem WW domains that interact with the proline-rich peptide motifs of target proteins. The missense mutation changes arginine 441 that is located in the centre of the WW domains into glutamine (R441Q), which potentially affects the function of the WWP1 protein. Here, we show that WWP1 is detected as ∼130-kDa protein that localizes to various structures, such as the plasma membrane (sarcolemma), sarcoplasmic reticulum, mitochondria and nucleus, in normal chicken skeletal muscle. However, in MD chickens, the mutant WWP1 protein was markedly degraded and was absent in the sarcolemma. These changes were also observed in the muscles of chickens in early pre-pathological states. Moreover, in vitro expression analysis showed significant degradation of mutant, but not wild-type WWP1, specifically in myogenic cells. Altogether, our data revealed that the R441Q missense mutation in the WWP1 protein causes degradation and loss of the sarcolemmal localization of WWP1, which may play a role in the pathogenesis of chicken MD.

Immunogenicity and pharmacokinetics of short, proline-rich antimicrobial peptides.

The potential of proline-rich antimicrobial peptides (PrAMPs) to treat multidrug-resistant Gram-negative pathogens has been intensively investigated. They are efficacious at low doses in infection models and well tolerated in healthy mice at high doses. PrAMPs Onc72 and Api88 were nonimmunogenic in mice unless conjugated to a carrier protein. Monoclonal IgG1/IgG2b antibodies produced by hybridoma cells were mapped to different Onc72 regions and combined in a sandwich-ELISA in a pharmacokinetic study. Onc72 was detected at concentrations up to 32 µg/ml in murine blood after administering 20 mg/kg and reached several organs within 10 min. Both PrAMPs were not immunogenic and Onc72 concentrations in blood were well above the minimal inhibitory concentrations for Enterobacteriaceae further confirming their potential as novel antibiotics.

Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.