Rule-based omics mining reveals antimicrobial macrocyclic peptides against drug-resistant clinical isolates.

Antimicrobial resistance remains a significant global threat, driving up mortality rates worldwide. Ribosomally synthesized and post-translationally modified peptides have emerged as a promising source of novel peptide antibiotics due to their diverse chemical structures. Here, we report the discovery of new aminovinyl-(methyl)cysteine (Avi(Me)Cys)-containing peptide antibiotics through a synergistic approach combining biosynthetic rule-based omics mining and heterologous expression. We first bioinformatically identify 1172 RiPP biosynthetic gene clusters (BGCs) responsible for Avi(Me)Cys-containing peptides formation from a vast pool of over 50,000 bacterial genomes. Subsequently, we successfully establish the connection between three identified BGCs and the biosynthesis of five peptide antibiotics via biosynthetic rule-guided metabolic analysis. Notably, we discover a class V lanthipeptide, massatide A, which displays excellent activity against gram-positive pathogens, including drug-resistant clinical isolates like linezolid-resistant S. aureus and methicillin-resistant S. aureus, with a minimum inhibitory concentration of 0.25 µg/mL. The remarkable performance of massatide A in an animal infection model, coupled with a relatively low risk of resistance and favorable safety profile, positions it as a promising candidate for antibiotic development. Our study highlights the potential of Avi(Me)Cys-containing peptides in expanding the arsenal of antibiotics against multi-drug-resistant bacteria, offering promising drug leads in the ongoing battle against infectious diseases.

Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts.

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.

Newly Isolated Limosilactobacillus reuteri B1/1 Modulates the Expression of Cytokines and Antimicrobial Proteins in a Porcine ex Vivo Model.

BACKGROUND: The epithelia of the intestine perform various functions, playing a crucial role in providing a physical barrier and an innate immune defense against infections. By generating a "three-dimensional" (3D) model of cell co-cultures using the IPEC-J2 cell line and porcine blood monocyte-derived macrophages (MDMs), we are getting closer to mimicking the porcine intestine ex vivo.Methods: The effect of Limosilactobacillus reuteri B1/1 and Limosilactobacillus fermentum CCM 7158 (indicator strain) on the relative gene expression of interleukins (IL-1ß, IL-6, IL-8, IL-18 and IL-10), genes encoding receptors for TLR4 and TLR2, tight junction proteins such as claudin-1 (CLDN1), occludin (OCLN) and important antimicrobial proteins such as lumican (LUM) and olfactomedin-4 (OLMF-4) was monitored in this model. RESULTS: The results obtained from this pilot study point to the immunomodulatory potential of newly isolated L. reuteri B1/1, as it was able to suppress the enhanced pro-inflammatory response to lipopolysaccharide (LPS) challenge in both cell types. L. reuteri B1/1 was even able to up-regulate the mRNA levels of genes encoding antimicrobial proteins LUM and OLFM-4 and to increase tight junction (TJ)-related genes CLDN1 and OCLN, which were significantly down-regulated in LPS-induced IPEC-J2 cells. Conversely, L. fermentum CCM 7158, chosen as an indicator lactic acid bacteria (LAB) strain, increased the mRNA levels of the investigated pro-inflammatory cytokines (IL-18, IL-6, and IL-1ß) in MDMs when LPS was simultaneously applied to basally deposited macrophages. Although L. fermentum CCM 7158 induced the production of pro-inflammatory cytokines, synchronous up-regulation of the anti-inflammatory cytokine IL-10 was detected in both LAB strains used in both cell cultures. CONCLUSIONS: The obtained results suggest that the recently isolated LAB strain L. reuteri B1/1 has the potential to alleviate epithelial disruption caused by LPS and to influence the production of antimicrobial molecules by enterocytes.

The involvement of VEGF and VEGFR in bacterial recognition and regulation of antimicrobial peptides in Eriocheir sinensis.

Vascular endothelial growth factor (VEGF) and VEGF reporter (VEGFR) are essential molecules in VEGF signalling pathway. Although the functions of VEGF and VEGFR have been well reported in vertebrates, their functions are still poorly understood in invertebrates. In this study, the open reading frame sequences of EsVEGF1 and EsVEGFR4 were cloned from Eriocheir sinensis, and their corresponding proteins shared typical structure characteristics with their counterparts in other species. EsVEGF1 were predominantly expressed in hepatopancreas and muscle while EsVEGFR4 mainly expressed in hemocytes and intestine. The expression levels of EsVEGF1 in hemocytes were rapidly induced by Staphylococcus aureus and Vibrio parahaemolyticus, and it also increased rapidly in hepatopancreas after being challenged with V. parahaemolyticus. The expression levels of EsVEGFR4 only increased in hepatopancreas of crabs injected with S. aureus. The extracellular immunoglobulin domain of EsVEGFR4 could bind with Gram-negative and Gram-positive bacteria as well as lipopolysaccharide and peptidoglycan. EsVEGF1 could act as the ligand for EsVEGFR4 and Toll-like receptor and regulate the expression of crustins and lysozyme with a tissue-specific manner, while have no regulatory function on that of anti-lipopolysaccharide factors. This study will provide new insights into the immune defense mechanisms mediated by VEGF and VEGFR in crustaceans.

Mycobacterium tuberculosis suppresses host antimicrobial peptides by dehydrogenating L-alanine.

Antimicrobial peptides (AMPs), ancient scavengers of bacteria, are very poorly induced in macrophages infected by Mycobacterium tuberculosis (M. tuberculosis), but the underlying mechanism remains unknown. Here, we report that L-alanine interacts with PRSS1 and unfreezes the inhibitory effect of PRSS1 on the activation of NF-κB pathway to induce the expression of AMPs, but mycobacterial alanine dehydrogenase (Ald) Rv2780 hydrolyzes L-alanine and reduces the level of L-alanine in macrophages, thereby suppressing the expression of AMPs to facilitate survival of mycobacteria. Mechanistically, PRSS1 associates with TAK1 and disruptes the formation of TAK1/TAB1 complex to inhibit TAK1-mediated activation of NF-κB pathway, but interaction of L-alanine with PRSS1, disables PRSS1-mediated impairment on TAK1/TAB1 complex formation, thereby triggering the activation of NF-κB pathway to induce expression of AMPs. Moreover, deletion of antimicrobial peptide gene ß-defensin 4 (Defb4) impairs the virulence by Rv2780 during infection in mice. Both L-alanine and the Rv2780 inhibitor, GWP-042, exhibits excellent inhibitory activity against M. tuberculosis infection in vivo. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses its own alanine dehydrogenase to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.

Multimodal binding and inhibition of bacterial ribosomes by the antimicrobial peptides Api137 and Api88.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

Can mesenchymal stem/stromal cells and their secretomes combat bacterial persisters?

The increasing number of life-threatening infections caused by persister bacteria is associated with various issues, including antimicrobial resistance and biofilm formation. Infections due to persister cells are often difficult to suppress without the use of last-resort antibiotics. Throughout the world, bacterial persistence and resistance create an unmet clinical demand for the exploration of newly introduced therapeutic approaches. Mesenchymal stem / stromal cells (MSCs) have an antimicrobial activity to protect against bacterial infections, including those caused by bacterial persisters. MSCs have substantial potential to secrete antimicrobial peptides (AMPs), including cathelicidin, beta-defensins, lipocalin-2, hepcidin, indoleamine 2,3-dioxygenase (IDO), cysteine proteases, and inducible nitric oxide synthases (iNOS). MSCs possess the potential to contribute to innate immunity by regulating the immune response. Recently, MSCs and their secreted components have been reported to improve antimicrobial activity. Bactericidal activity by MSCs and their secretomes has been shown to be mediated in part by the secretion of AMPs. Even though they were discovered more than 80 years ago, therapeutic options for persisters are restricted, and there is an urgent need for alternative treatment regimens. Hence, this review intends to critically assess the current literature on the effects of MSCs and their secretomes on persister bacteria. MSCs and their secretome-based therapies could be preferred as an up-and-coming approach to reinforce the antimicrobial efficiency in persister infections.

Mesenchymal stem cells: A promising antimicrobial therapy in veterinary medicine.

Growing antimicrobial resistance (AMR) is a threat to human and animal populations citing the limited available options. Alternative antimicrobial options or functional enhancement of currently available antimicrobials remains only options. One of the potential options seems stem cells especially the mesenchymal stem cells (MSCs) that show antimicrobial properties. These cells additionally have pro-healing effects that may plausibly improve healing outcomes. MSCs antimicrobial actions are mediated either through direct cell-cell contact or their secretome that enhances innate immune mediated antimicrobial activities. These cells synergistically enhance efficacy of currently available antimicrobials especially against the biofilms. Reciprocal action from antimicrobials on the MSCs functionality remains poorly understood. Currently, the main limitation with MSCs based therapy is their limited efficacy. This demands further understanding and can be enhanced through biotechnological interventions. One of the interventional options is the 'priming' to enhance MSCs resistance and specific expression potential. The available literature shows potential antimicrobial actions of MSCs both ex vivo as well as in vivo. The studies on veterinary species are very promising although limited by number and extensiveness in details for their utility as standard therapeutic agents. The current review aims to discuss the role of animals in AMR and the potential antimicrobial actions of MSCs in veterinary medicine. The review also discusses the limitations in their utilization as standard therapeutics.

Deubiquitinase cylindromatosis (CYLD) regulates antibacterial immunity and apoptosis in Chinese mitten crab (Eriocheir sinensis).

Ubiquitination and deubiquitination of target proteins is an important mechanism for cells to rapidly respond to changes in the external environment. The deubiquitinase, cylindromatosis (CYLD), is a tumor suppressor protein. CYLD from Drosophila melanogaster participates in the antimicrobial immune response. In vertebrates, CYLD also regulates bacterial-induced apoptosis. However, whether CYLD can regulate the bacterial-induced innate immune response in crustaceans is unknown. In the present study, we reported the identification and cloning of CYLD in Chinese mitten crab, Eriocheir sinensis. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that EsCYLD was widely expressed in all the examined tissues and was upregulated in the hemolymph after Vibrio parahaemolyticus challenge. Knockdown of EsCYLD in hemocytes promoted the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation and increased the expression of corresponding antimicrobial peptides. In vivo, silencing of EsCYLD promoted the removal of bacteria from the crabs and enhanced their survival. In addition, interfering with EsCYLD expression inhibited apoptosis of crab hemocytes caused by V. parahaemolyticus stimulation. In summary, our findings revealed that EsCYLD negatively regulates the nuclear translocation of Relish to affect the expression of corresponding antimicrobial peptides and regulates the apoptosis of crab hemocytes, thus indirectly participating in the innate immunity of E. sinensis.

Helical structure motifs made searchable for functional peptide design.

The systematic design of functional peptides has technological and therapeutic applications. However, there is a need for pattern-based search engines that help locate desired functional motifs in primary sequences regardless of their evolutionary conservation. Existing databases such as The Protein Secondary Structure database (PSS) no longer serves the community, while the Dictionary of Protein Secondary Structure (DSSP) annotates the secondary structures when tertiary structures of proteins are provided. Here, we extract 1.7 million helices from the PDB and compile them into a database (Therapeutic Peptide Design database; TP-DB) that allows queries of compounded patterns to facilitate the identification of sequence motifs of helical structures. We show how TP-DB helps us identify a known purification-tag-specific antibody that can be repurposed into a diagnostic kit for Helicobacter pylori. We also show how the database can be used to design a new antimicrobial peptide that shows better Candida albicans clearance and lower hemolysis than its template homologs. Finally, we demonstrate how TP-DB can suggest point mutations in helical peptide blockers to prevent a targeted tumorigenic protein-protein interaction. TP-DB is made available at http://dyn.life.nthu.edu.tw/design/ .

Effect of hydrophobicity on distinct anticancer mechanism of antimicrobial peptide chensinin-1b and its lipoanalog PA-C1b in breast cancer cells.

Chensinin-1b and its lipoanalogs demonstrate different anticancer activities against selected cancer cells, and the anticancer activity of PA-C1b is improved up to 3-fold compared with that of the parent peptide chensinin-1b. However, detailing the mechanism of action of these peptides is required to better understand the structure-function relationship. In this study, chensinin-1b and PA-C1b were selected as the representative peptides to investigate the mode of action in cancer cells. The results indicated that the boundary of the cell membrane was broken when the cells were treated with chensinin-1b, while that of cells treated with PA-C1b remained intact based on morphological changes. Apoptosis assays indicated that PA-C1b induced MCF-7 cancer cell apoptosis, while chensinin-1b mainly damaged the cell membrane. MCF-7 cancer cells treated with the peptides induced the loss of mitochondrial membrane potential, and cytochrome c was released from mitochondria, but PA-C1b enhanced ROS generation. Additionally, PA-C1b uptake occurred via an energy-dependent pathway and was inhibited by selected endocytosis inhibitors. Furthermore, treatment of MCF-7 cells with PA-C1b suppressed Bcl-2 mRNA levels and increased Bax mRNA levels, upregulated the expression of the proapoptotic protein Bax and downregulated the expression of the antiapoptotic protein Bcl-2. These results indicate that the anticancer mechanism of AMPs may be considerably affected by only a slight difference in the hydrophobicity of the two peptides; and such a study may facilitate the design of novel peptide-based anticancer agents.

PANoptosis: A New Insight Into Oral Infectious Diseases.

The oral microbiome, one of the most complex and intensive microbial ecosystems in the human body, comprises bacteria, archaea, fungi, protozoa, and viruses. Dysbiosis of the oral microbiome is the initiating factor that leads to oral infectious diseases. Infection is a sophisticated biological process involving interplay between the pathogen and the host, which often leads to activation of programmed cell death. Studies suggest that pyroptosis, apoptosis, and necroptosis are involved in multiple oral infectious diseases. Further understanding of crosstalk between cell death pathways has led to pyroptosis, apoptosis, and necroptosis being integrated into a single term: PANoptosis. PANoptosis is a multifaceted agent of the immune response that has important pathophysiological relevance to infectious diseases, autoimmunity, and cancer. As such, it plays an important role in innate immune cells that detect and eliminate intracellular pathogens. In addition to the classical model of influenza virus-infected and Yersinia-infected macrophages, other studies have expanded the scope of PANoptosis to include other microorganisms, as well as potential roles in cell types other than macrophages. In this review, we will summarize the pathophysiological mechanisms underlying inflammation and tissue destruction caused by oral pathogens. We present an overview of different pathogens that may induce activation of PANoptosis, along with the functional consequences of PANoptosis in the context of oral infectious diseases. To advance our understanding of immunology, we also explore the strategies used by microbes that enable immune evasion and replication within host cells. Improved understanding of the interplay between the host and pathogen through PANoptosis will direct development of therapeutic strategies that target oral infectious diseases.

Esc peptides as novel potentiators of defective cystic fibrosis transmembrane conductance regulator: an unprecedented property of antimicrobial peptides.

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.

Brevinin-2GHk, a Peptide Derived from the Skin of Fejervarya limnocharis, Inhibits Zika Virus Infection by Disrupting Viral Integrity.

Several years have passed since the Zika virus (ZIKV) pandemic reoccurred in 2015-2016. However, there is still a lack of proved protective vaccines or effective drugs against ZIKV. The peptide brevinin-2GHk (BR2GK), pertaining to the brevinin-2 family of antimicrobial peptides, has been reported to exhibit only weak antibacterial activity, and its antiviral effects have not been investigated. Thus, we analyzed the effect of BR2GK on ZIKV infection. BR2GK showed significant inhibitory activity in the early and middle stages of ZIKV infection, with negligible cytotoxicity. Furthermore, BR2GK was suggested to bind with ZIKV E protein and disrupt the integrity of the envelope, thus directly inactivating ZIKV. In addition, BR2GK can also penetrate the cell membrane, which may contribute to inhibition of the middle stage of ZIKV infection. BR2GK blocked ZIKV E protein expression with an IC50 of 3.408 ± 0.738 µΜ. In summary, BR2GK was found to be a multi-functional candidate and a potential lead compound for further development of anti-ZIKV drugs.

Antitumor Activity and Mechanism of Action of Hormonotoxin, an LHRH Analog Conjugated to Dermaseptin-B2, a Multifunctional Antimicrobial Peptide.

Prostate cancer is the most common cancer in men. For patients with advanced or metastatic prostate cancer, available treatments can slow down its progression but cannot cure it. The development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2, a natural multifunctional antimicrobial peptide isolated from Amazonian frog skin, has been reported to possess antitumor activity. To improve its pharmacological properties and to decrease its peripheral toxicity and lethality we developed a hormonotoxin molecule composed of dermaseptin-B2 combined with d-Lys6-LHRH to target the LHRH receptor. This hormonotoxin has a significant antiproliferative effect on the PC3 tumor cell line, with an IC50 value close to that of dermaseptin-B2. Its antitumor activity has been confirmed in vivo in a xenograft mouse model with PC3 tumors and appears to be better tolerated than dermaseptin-B2. Biophysical experiments showed that the addition of LHRH to dermaseptin-B2 did not alter its secondary structure or biological activity. The combination of different experimental approaches indicated that this hormonotoxin induces cell death by an apoptotic mechanism instead of necrosis, as observed for dermaseptin-B2. These results could explain the lower toxicity observed for this hormonotoxin compared to dermaseptin-B2 and may represent a promising targeting approach for cancer therapy.

Short Peptides Make a Big Difference: The Role of Botany-Derived AMPs in Disease Control and Protection of Human Health.

Botany-derived antimicrobial peptides (BAMPs), a class of small, cysteine-rich peptides produced in plants, are an important component of the plant immune system. Both in vivo and in vitro experiments have demonstrated their powerful antimicrobial activity. Besides in plants, BAMPs have cross-kingdom applications in human health, with toxic and/or inhibitory effects against a variety of tumor cells and viruses. With their diverse molecular structures, broad-spectrum antimicrobial activity, multiple mechanisms of action, and low cytotoxicity, BAMPs provide ideal backbones for drug design, and are potential candidates for plant protection and disease treatment. Lots of original research has elucidated the properties and antimicrobial mechanisms of BAMPs, and characterized their surface receptors and in vivo targets in pathogens. In this paper, we review and introduce five kinds of representative BAMPs belonging to the pathogenesis-related protein family, dissect their antifungal, antiviral, and anticancer mechanisms, and forecast their prospects in agriculture and global human health. Through the deeper understanding of BAMPs, we provide novel insights for their applications in broad-spectrum and durable plant disease prevention and control, and an outlook on the use of BAMPs in anticancer and antiviral drug design.

Cationic Peptides and Their Cu(II) and Ni(II) Complexes: Coordination and Biological Characteristics.

Antimicrobial peptides are a promising group of compounds used for the treatment of infections. In some cases, metal ions are essential to activate these molecules. Examples of metalloantibiotics are, for instance, bleomycin and dermcidin. This study is focused on three new pseudopeptides with potential biological activity. The coordination behavior of all ligands with Cu(II) and Ni(II) ions has been examined. Various analytical methods such as potentiometric titration, UV-Vis and CD spectroscopies, and mass spectrometry were used. All compounds are convenient chelators for metal ion-binding. Two of the ligands tested have histidine residues. Surprisingly, imidazole nitrogen is not involved in the coordination of the metal ion. The N-terminal amino group, Dab side chains, and amide nitrogen atoms of the peptide bonds coordinated Cu(II) and Ni(II) in all the complexes formed. The cytotoxicity of three pseudopeptides and their complexes was evaluated. Moreover, their other model allowed for assessing the attenuation of LPS-induced cytotoxicity and anti-inflammatory activities were also evaluated, the results of which revealed to be very promising.

New Role for Radical SAM Enzymes in the Biosynthesis of Thio(seleno)oxazole RiPP Natural Products.

Ribosomally synthesized post-translationally modified peptides (RiPPs) are ubiquitous and represent a structurally diverse class of natural products. The ribosomally encoded precursor polypeptides are often extensively modified post-translationally by enzymes that are encoded by coclustered genes. Radical S-adenosyl-l-methionine (SAM) enzymes catalyze numerous chemically challenging transformations. In RiPP biosynthetic pathways, these transformations include the formation of C-H, C-C, C-S, and C-O linkages. In this paper, we show that the Geobacter lovleyi sbtM gene encodes a radical SAM protein, SbtM, which catalyzes the cyclization of a Cys/SeCys residue in a minimal peptide substrate. Biochemical studies of this transformation support a mechanism involving H-atom abstraction at the C-3 of the substrate Cys to initiate the chemistry. Several possible cyclization products were considered. The collective biochemical, spectroscopic, mass spectral, and computational observations point to a thiooxazole as the product of the SbtM-catalyzed modification. To our knowledge, this is the first example of a radical SAM enzyme that catalyzes a transformation involving a SeCys-containing peptide and represents a new paradigm for formation of oxazole-containing RiPP natural products.

Topological Dissection of Proteomic Changes Linked to the Limbic Stage of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia worldwide. In AD, neurodegeneration spreads throughout different areas of the central nervous system (CNS) in a gradual and predictable pattern, causing progressive memory decline and cognitive impairment. Deposition of neurofibrillary tangles (NFTs) in specific CNS regions correlates with the severity of AD and constitutes the basis for disease classification into different Braak stages (I-VI). Early clinical symptoms are typically associated with stages III-IV (i.e., limbic stages) when the involvement of the hippocampus begins. Histopathological changes in AD have been linked to brain proteome alterations, including aberrant posttranslational modifications (PTMs) such as the hyperphosphorylation of Tau. Most proteomic studies to date have focused on AD progression across different stages of the disease, by targeting one specific brain area at a time. However, in AD vulnerable regions, stage-specific proteomic alterations, including changes in PTM status occur in parallel and remain poorly characterized. Here, we conducted proteomic, phosphoproteomic, and acetylomic analyses of human postmortem tissue samples from AD (Braak stage III-IV, n=11) and control brains (n=12), covering all anatomical areas affected during the limbic stage of the disease (total hippocampus, CA1, entorhinal and perirhinal cortices). Overall, ~6000 proteins, ~9000 unique phosphopeptides and 221 acetylated peptides were accurately quantified across all tissues. Our results reveal significant proteome changes in AD brains compared to controls. Among others, we have observed the dysregulation of pathways related to the adaptive and innate immune responses, including several altered antimicrobial peptides (AMPs). Notably, some of these changes were restricted to specific anatomical areas, while others altered according to disease progression across the regions studied. Our data highlights the molecular heterogeneity of AD and the relevance of neuroinflammation as a major player in AD pathology. Data are available via ProteomeXchange with identifier PXD027173.

Evaluation of antimicrobial and anticancer activities of three peptides identified from the skin secretion of Hylarana latouchii.

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.