Enhancing the Antimicrobial Properties of Peptides through Cell-Penetrating Peptide Conjugation: A Comprehensive Assessment.

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus, at concentrations ranging from 3 to 12 µM, exhibited enhanced antimicrobial activity against B. cereus. TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa, at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli (E. coli) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus, MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development.

Exposure to High Dosage of Gold Nanoparticles Accelerates Growth Rate by Modulating Ribosomal Protein Expression.

Gold nanoparticles (AuNPs) have been utilized in various biomedical applications including diagnostics and drug delivery. However, the cellular and metabolic responses of cells to these particles remain poorly characterized. In this study, we used bacteria (Escherichia coli and Bacillus subtilis) and a fungus (Saccharomyces cerevisiae) as model organisms to investigate the cellular and metabolic effects of exposure to different concentrations of citrate-capped spherical AuNPs with diameters of 5 and 10 nm. In different growth media, the synthesized AuNPs displayed stability and microorganisms exhibited uniform levels of uptake. Exposure to a high concentration of AuNPs (1012 particles) resulted in a reduced cell division time and a 2-fold increase in cell density in both bacteria and fungus. The exposed cells exhibited a decrease in average cell size and an increase in the expression of FtsZ protein (cell division marker), further supporting an accelerated growth rate. Notably, exposure to such a high concentration of AuNPs did not induce DNA damage, envelope stress, or a general stress response in bacteria. Differential whole proteome analysis revealed modulation of ribosomal protein expression upon exposure to AuNPs in both E. coli and S. cerevisiae. Interestingly, the accelerated growth observed upon exposure to AuNPs was sensitive to sub-minimum inhibitory concentration (sub-MIC) concentration of drugs that specifically target ribosome assembly and recycling. Based upon these findings, we hypothesize that exposure to high concentrations of AuNPs induces stress on the translation machinery. This leads to an increase in the protein synthesis rate by modulating ribosome assembly, which results in the rapid proliferation of cells.

Identification of newly synthetized proteins by mass spectrometry to understand palmitate-induced early cellular changes in pancreatic islets.

Obesity and lipid metabolism dysregulation are often associated with insulin resistance, and can lead to type 2 diabetes. However, mechanisms linking insulin resistance, high levels of plasma free fatty acids (FFA), and ß cell failure remain unclear. The aim of this work was to search for proteins whose synthesis was modified by a short exposure to FFA. This could help in the future to identify molecular mechanisms underlying islet dysfunction in the presence of FFA. Therefore, we assessed by mass spectrometry de novo protein synthesis of freshly isolated rat islets after palmitate short exposure. Quantitative proteome and secretome analyses were performed by combining metabolic incorporation of azidohomoalanine (AHA) and pulse labeling with stable isotope labeling by amino acids in cell culture (SILAC). We showed that pancreatic islets, in response to 4-h exposure to palmitate, increased the synthesis of ribosomal proteins and proteins of the cytoskeleton, and increased their secretion of proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. First, these results show that de novo protein quantification analysis by LC-MS/MS is a useful method to investigate cellular modifications induced by FFA on pancreatic islets. Also, these results show that short exposure to palmitate increases the expression of ribosomal proteins and proteins involved in insulin secretion, and it remains to be determined if these effects are responsible or linked to the harmful effect of palmitate on ß cells.NEW & NOTEWORTHY These results show that pancreatic rat islets cultured with palmitate mainly increase synthesis of ribosomal proteins and some proteins of the cytoskeleton. They also show a significant increase of secreted proteins involved in insulin synthesis and insulin secretion, as well as insulin itself. These data provide information to understand the mechanisms of ß cell failure induced by lipotoxicity via the identification of all newly synthesized proteins in islets in response to short-term exposure to palmitate.

18ß-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells.

BACKGROUND: Gastric carcinoma (GC) is a common gastrointestinal malignancy worldwide. Based on the cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. AIM: To explore the mechanism by which 18ß-glycyrrhetinic acid (18ß-GRA) regulates mitochondrial ribosomal protein L35 (MRPL35) related signal proteins to inhibit the proliferation of GC cells. METHODS: Cell counting kit-8 assay was used to detect the effects of 18ß-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823. The apoptosis and cell cycle were assessed by flow cytometry. Cell invasion and migration were evaluated by Transwell assay, and cell scratch test was used to detect cell migration. Furthermore, a tumor model was established by hypodermic injection of 2.5 × 106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18ß-GRA on GC cell proliferation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect MRPL35 expression in the engrafted tumors in mice. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry to screen for differentially expressed proteins (DEPs) extracted from GC cells and control cells after 18ß-GRA intervention. A detailed bioinformatics analysis of these DEPs was performed, including Gene Ontology annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and so on. Moreover, STRING database (https://string-db.org/) was used to predict protein-protein interaction (PPI) relationships and Western blot was used to detect the expression of proteins of interest in GC cells. RESULTS: The results indicated that 18ß-GRA could inhibit the proliferation of GC cells in a dose- and time-dependent manner. It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase. The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose, and the migration and invasiveness of GC cells were inhibited. The results of animal experiments showed that 18ß-GRA could inhibit tumor formation in BALB/c nude mice, and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice. Using TMT technology, 609 DEPs, among which 335 were up-regulated and 274 were down-regulated, were identified in 18ß-GRA intervention compared with control. We found that the intervention of 18ß-GRA in GC cells involved many important biological processes and signaling pathways, such as cellular processes, biological regulation, and TP53 signaling pathway. Notably, after the drug intervention, MRPL35 expression was significantly down-regulated (P = 0.000247), TP53 expression was up-regulated (P = 0.02676), and BCL2L1 was down-regulated (P = 0.01699). Combined with the Retrieval of Interacting Genes/Proteins database, we analyzed the relationship between MRPL35, TP53, and BCL2L1 signaling proteins, and we found that COPS5, BAX, and BAD proteins can form a PPI network with MRPL35, TP53, and BCL2L1. Western blot analysis confirmed the intervention effect of 18ß-GRA on GC cells, MRPL35, TP53, and BCL2L1 showed dose-dependent up/down-regulation, and the expression of COPS5, BAX, and BAD also increased/decreased with the change of 18ß-GRA concentration. CONCLUSION: 18ß-GRA can inhibit the proliferation of GC cells by regulating MRPL35, COPS5, TP53, BCL2L1, BAX, and BAD.

Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients.

BACKGROUND: Activation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease-related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients. METHODS: The effects of SMase on force production and intracellular Ca2+ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III-IV, ejection fraction 25 ± 1.0%, peak VO2 14.4 ± 0.6 mL × kg × min) and healthy age-matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO2 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure. RESULTS: Sphingomyelinase reduced muscle fibre force production (-30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca2+ release (P < 0.05) and reducing myofibrillar Ca2+ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up-regulated and down-regulated, respectively, at a FDR of 1%. Gene-set enrichment analysis identified 'proteasome degradation' as an up-regulated pathway (average fold-change 1.1, P = 0.008), while the pathway 'cytoplasmic ribosomal proteins' (average fold-change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold-change 0.8, P = 0.0002) where identified as down-regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age-matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF-receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow-up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease. CONCLUSIONS: The present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure-related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease-related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Is It Possible to Create Antimicrobial Peptides Based on the Amyloidogenic Sequence of Ribosomal S1 Protein of P. aeruginosa?

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.

Identification of ribosomal protein L30 as an uncharacterized antimicrobial protein.

Several ribosomal proteins have been shown to adopt for an antimicrobial function as antimicrobial proteins (AMPs). However, information as such is rather limited and their mode of action remains ill-defined. Here we demonstrated that amphioxus RPL30, BjRPL30, was a previously uncharacterized AMP, which was not only capable of binding Gram-negative and Gram-positive bacteria via interaction with LPS, LTA and PGN but also capable of killing the bacteria. We also showed that the residues positioned at 2-46 formed the core region for the antimicrobial activity of BjRPL30. Notably, both the hydrophobic ratio and net charge as well as 3D structures of the residues corresponding to BjRPL302-27 and BjRPL3023-46 from both eukaryotic and prokaryotic RPL30 proteins were closely similar to those of BjRPL302-27 and BjRPL3023-46, suggesting the antibacterial activity of RPL30 was highly conserved. This was further corroborated by the fact that the synthesized counterparts human RPL5-30 and RPL26-49 also had antibacterial activity. We show that the recombinant protein BjRPL30 executes antimicrobial function in vitro by a kind of membranolytic action including interaction with bacterial membrane through LPS, LTA and PGN as well as induction of membrane depolarization. Finally, we found that neither BjRPL30 nor its truncated form BjRPL302-27 and BjRPL3023-46 had hemolytic activity towards human red blood cells, making them promising lead molecules for the design of novel AMPs against bacteria. Altogether, these indicated that RPL30 is a member of AMP which has ancient origin and is highly conserve throughout evolution.

Ribosomal protein S3-derived repair domain peptides regulate UV-induced matrix metalloproteinase-1.

Ultraviolet (UV) radiation is a major factor that causes wrinkle formation by affecting the collagen level in the skin. Here, we show that a short peptide (A8) derived from the repair domain of the ribosomal protein S3 (rpS3) reduces UV irradiation-induced increase in matrix metalloproteinase-1 (MMP-1) and prevents collagen degradation by reducing the activation of the mitogen-activated protein kinase (MAPK) signaling proteins (extracellular signal-regulated kinase [ERK], p38, and c-Jun N-terminal kinases [JNK]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in cells. Furthermore, A8 also prevents the increase in the levels of inflammatory modulators such as tumor necrosis factor-alpha (TNF-α) or interleukin-6 (IL-6) in UV-irradiated cells. Collectively, our study suggests that the A8 peptide, derived from yeast or human, has anti-photoaging potential as it prevents UV-induced wrinkle formation.

Landornamides: Antiviral Ornithine-Containing Ribosomal Peptides Discovered through Genome Mining.

Proteusins are a family of bacterial ribosomal peptides that largely remain hypothetical genome-predicted metabolites. The only known members are the polytheonamide-type cytotoxins, which have complex structures due to numerous unusual posttranslational modifications (PTMs). Cyanobacteria contain large numbers of putative proteusin loci. To investigate their chemical and pharmacological potential beyond polytheonamide-type compounds, we characterized landornamide A, the product of the silent osp gene cluster from Kamptonema sp. PCC 6506. Pathway reconstruction in E. coli revealed a peptide combining lanthionines, d-residues, and, unusually, two ornithines introduced by the arginase-like enzyme OspR. Landornamide A inhibited lymphocytic choriomeningitis virus infection in mouse cells, thus making it one of the few known anti-arenaviral compounds. These data support proteusins as a rich resource of chemical scaffolds, new maturation enzymes, and bioactivities.

Foot-and-Mouth Disease Virus Capsid Protein VP1 Interacts with Host Ribosomal Protein SA To Maintain Activation of the MAPK Signal Pathway and Promote Virus Replication.

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a highly contagious, economically important viral disease. The structural protein VP1 plays significant roles during FMDV infection. Here, we identified that VP1 interacted with host ribosomal protein SA (RPSA). RPSA is a viral receptor for dengue virus and classical swine fever virus infections. However, the incubation of susceptible cells using the anti-RPSA antibodies did not block the infection of FMDV. Overexpression of porcine RPSA in the insusceptible cells could not trigger FMDV infection, suggesting that RPSA was not responsible for FMDV entry and infection. On the contrary, we found that overexpression of RPSA suppressed FMDV replication, and knockdown of RPSA enhanced FMDV replication. We further determined that FMDV infection activated the mitogen-activated protein kinase (MAPK) pathway and demonstrated that MAPK pathway activation was critically important for FMDV replication. RPSA negatively regulated MAPK pathway activation during FMDV infection and displayed an antiviral function. FMDV VP1 interacted with RPSA to abrogate the RPSA-mediated suppressive role in MAPK pathway activation. Together, our study indicated that MAPK pathway activation was required for FMDV replication and that host RPSA played a negatively regulatory role on MAPK pathway activation to suppress FMDV replication. FMDV VP1 bound to RPSA to promote viral replication by repressing RPSA-mediated function and maintaining the activation of MAPK signal pathway.IMPORTANCE Identification of virus-cell interactions is essential for making strategies to limit virus replication and refine the models of virus replication. This study demonstrated that FMDV utilized the MAPK pathway for viral replication. The host RPSA protein inhibited FMDV replication by suppressing the activation of the MAPK pathway during FMDV infection. FMDV VP1 bound to RPSA to repress the RPSA-mediated regulatory effect on MAPK pathway activation. This study revealed an important implication of the MAPK pathway for FMDV infection and identified a novel mechanism by which FMDV VP1 has evolved to interact with RPSA and maintain the activation of the MAPK pathway, elucidating new information regarding the signal reprogramming of host cells by FMDV.

Peptide Occurring in Enterobacteriaceae Triggers Streptococcus pneumoniae Cell Death.

Non-encapsulated Streptococcus pneumoniae often possess two genes, aliB-like ORF 1 and aliB-like ORF 2, in place of capsule genes. AliB-like ORF 1 is thought to encode a substrate binding protein of an ABC transporter which binds peptide SETTFGRDFN, found in 50S ribosomal subunit protein L4 of Enterobacteriaceae. Here, we investigated the effect of binding of AliB-like ORF 1 peptide on the transcriptome and proteome of non-encapsulated pneumococci. We found upregulation of gene expression of a metacaspase and a gene encoding N-acetylmuramoyl-L-alanine amidase, both of which are proposed to be involved in programmed cell death in prokaryotic cells. Proteome profiling indicated upregulation of transcriptional regulators and downregulation of metabolism-associated genes. Exposure to the peptide specifically triggered death in pneumococci which express AliB-like ORF 1, with the bacteria having an apoptotic appearance by electron microscopy. We propose that binding of the AliB-like ORF 1 peptide ligand by the pneumococcus signals a challenging environment with hostile bacterial species leading to death of a proportion of the pneumococcal population.

Characterization of the retina-induced relaxation in mice.

PURPOSE: The retinal relaxing factor (RRF) is a continuously released factor from the retina that causes vasorelaxation, the identity and potential role in physiology of which remain largely unknown. Experiments were performed to find out whether the RRF-induced relaxation is influenced by serotonin, glutamate, L-cysteine, the cytochrome P450 pathway, the cyclooxygenase pathway, or oxidative stress. In addition, the sensitivity of retinal and non-retinal arteries towards the RRF was compared. METHODS: In vitro tension measurements were performed on isolated mouse femoral or bovine retinal arteries to study the vasorelaxing effect of the RRF, induced by mouse or bovine retinas. RESULTS: The presence of serotonin, glutamate, or L-cysteine did not alter the RRF-induced relaxation. Increasing oxidative stress by hydroquinone and diethyldithiocarbamic acid sodium salt enhanced the RRF response. Inhibition of the cytochrome P450 or the cyclooxygenase pathway did not cause any alteration. Surprisingly, the RRF-induced relaxation was enhanced by the presence of flufenamic acid or carbenoxolone. Furthermore, bringing retinal tissue in close contact with retinal or non-retinal arteries induced comparable relaxations. CONCLUSIONS: Serotonin, glutamate, L-cysteine, the cytochrome P450, and the cyclooxygenase pathway do not influence the RRF-induced relaxation and the RRF-induced relaxation seems to be resistant to oxidative stress. The mechanism responsible for the enhanced RRF-induced relaxation in the presence of flufenamic acid or carbenoxolone remains elusive and the RRF does not show more effectivity on retinal arteries.

Enhanced anticancer effect of MAP30-S3 by cyclosproin A through endosomal escape.

Cyclosporin A (CsA) is a calcium antagonist and can enhance the efficacy of some protein drugs, but its mechanism remains unknown. In this study, MAP30, a ribosome-inactivating protein reported to have apoptotic effects on cancer cells, was fused with S3, an epidermal growth factor receptor (EGFR)-targeting peptide. In addition, CsA was used to investigate whether it can further promote the apoptotic effects of S3 fused MAP30 (MAP30-S3). Our result showed that the internalization of FITC-labeled MAP30-S3 was increased significantly by S3 in HeLa cells. Unexpectedly, MAP30-S3 only showed a minor decrease in the viability of EGFR-overexpressing cancer cells, including HeLa, SMMC-7721, and MGC803 (IC50>5 µmol/l). However, 2 µmol/l CsA significantly increased the cytotoxicity of MAP30-S3, especially for HeLa cells (IC50=40.3 nmol/l). In comparison, CsA did not further decrease the cytotoxicity of MAP30-S3 on MRC-5, an EGFR low-expressing cell line from normal lung tissue, indicating that CsA did not affect the cancer-targeting specificity of MAP30-S3. Our results also showed that CsA further increased the apoptotic activity of MAP30-S3 in HeLa cells. CsA could promote the endosomal escape of FITC-MAP30-S3 with a diffused pattern in the cytoplasm. Five endocytic inhibitors were used to investigate the cellular uptake mechanism of MAP30-S3, and the results showed that the endosomal escape-enhancing effect of CsA on MAP30-S3 may be associated with the clathrin-dependent endocytic pathways. Our study suggested that CsA could be a novel endosomal escape enhancer to potentiate the intracellular release of anticancer protein drugs, resulting in their improved therapeutic efficacy.

Purification and characterization of ribosomal proteins L27 and L30 having antimicrobial activity produced by the Lactobacillus salivarius SGL 03.

AIMS: The aim of this study was to investigate the antimicrobial potential of proteins secreted by a new strain of Lactobacillus salivarius. METHODS AND RESULTS: The secretome of L. salivarius SGL 03 strain was analysed by gel-assisted fractionation and MS/MS to identify low-molecular-mass proteins. This strategy allowed us to identify 10 secreted proteins. Then, a combination of heterologous expression and agar well diffusion was used to characterize them as to their antimicrobial activity, mechanisms of action and stability. Our findings indicate that L27 and L30 proteins of the 50S ribosomal subunit have antimicrobial activity against Streptococcus pyogenes, Streptococcus uberis and Enterococcus faecium. In addition, both proteins are bactericidal against S. pyogenes and maintain their antimicrobial activity after different protease treatments, at acidic pH, after heat treatment, and if stored in a refrigerated ambient at least at 4°C. CONCLUSIONS: The overall results demonstrated that the L27 and L30 ribosomal proteins are of interest as new antimicrobial molecules to prevent the growth of S. pyogenes, S. uberis and E. faecium. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide the first insight into the extra-ribosomal activity of L27 and L30 secreted proteins of L. salivarius. This study demonstrated the capacity of L. salivarius SGL 03 to produce antimicrobial molecules and suggested this strain as a promising probiotic candidate.

Antimicrobial effect of the 60S ribosomal protein L29 (cgRPL29), purified from the gill of pacific oyster, Crassostrea gigas.

We purified an ∼6.4-kDa antimicrobial peptide from an acidified gill extract of the Pacific oyster, Crassostrea gigas, by cation-exchange and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide was composed of 54 amino acids and had a molecular weight of 6484.6 Da. Comparison of the amino acid sequence and molecular weight with those of other known proteins or peptides revealed that the peptide had high identity with the 60S ribosomal protein L29, and so was named cgRPL29. The full-length cgRPL29 cDNA of the Pacific oyster comprised 325-bp, including a 5'-untranslated region (UTR) of 100-bp, a 3'-UTR of 57-bp, and an open reading frame of 168-bp encoding 55 amino acids, with a Met residue at the N-terminus. The cgRPL29 mRNA tissue distribution suggested that it is constitutively expressed in a non-tissue-specific manner. Secondary structural prediction and homology modeling indicated cgRPL29 have an unordered structure containing two partial α-helical regions. This is to our knowledge the first report of the antimicrobial effect of the 60S ribosomal protein L29 from marine invertebrates.

The roles of a ribosomal protein S19 polymer in a mouse model of carrageenan-induced acute pleurisy.

C5-deficient mice usually present moderate neutrophil activation during the initiation phase of acute inflammation. Conversely, C5a receptor (C5aR)-deficient mice show unusually excessive activation of neutrophils. We identified the ribosomal protein S19 (RP S19) polymer, which is cross-linked at Lys122 and Gln137 by transglutaminases in apoptotic neutrophils, as a second C5aR ligand during the resolution phase of acute inflammation. The RP S19 polymer promotes apoptosis via the neutrophil C5aR and phagocytosis via the macrophage C5aR. To confirm the roles of the RP S19 polymer, we employed a carrageenan-induced acute pleurisy mouse model using C57BL/6J mice with a knock-in of the Gln137Glu mutant RP S19 gene and replaced the RP S19 polymer with either an S-tagged C5a/RP S19 recombinant protein or the RP S19122-145 peptide monomer and dimer (as functional C5aR agonists/antagonists) and the RP S19122-145 peptide trimer (as a functional C5aR antagonist). Neutrophils and macrophages were still present in the thoracic cavities of the knock-in mice at 24h and 7days after carrageenan injection, respectively. Knock-in mice showed structural organization and severe hemorrhaging from the surrounding small vessels of the alveolar walls in the lung parenchyma. In contrast to the RP S19122-145 peptide monomer and trimer, the simultaneous presence of S-tagged C5a/RP S19 and the RP S19122-145 peptide dimer completely improved the physiological and pathological acute inflammatory cues. The RP S19 polymer, especially the dimer, appears to play a role at the resolution phase of carrageenan-induced acute pleurisy in C57BL/6J model mice.

Functional and Structural Characterization of FAU Gene/Protein from Marine Sponge Suberites domuncula.

Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed (FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with "higher" metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis.

Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 µM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent.